Bacterial ribosome peptidyl transferase center (PTC) of the 50S subunit. [2]
Bacterial ribosome peptidyl transferase center (PTC) of the 50S subunit. [3]

Lefamulin acetate (0–1 mg/L) exhibits inhibitory activity against M. genitalium, N. gonorrhoeae, and C. trachomatis[2].
Lefamulin acetate has MIC values of ≤0.008 μg/mL, indicating strong activity against all strains of M. pneumoniae[3].

---

Lefamulin acetate (BC-3781 acetate) demonstrated potent activity against Chlamydia trachomatis (15 serovars) with MIC50/90 of 0.02/0.04 mg/L. Activity against Mycoplasma genitalium (6 strains, including multidrug-resistant) showed an MIC range of 0.002 to 0.063 mg/L. Against Neisseria gonorrhoeae (24 clinical isolates), MIC50/90 was 0.12/0.5 mg/L. Activity was comparable to doxycycline, moxifloxacin, and clarithromycin against C. trachomatis, and was unchanged regardless of resistance to fluoroquinolones, tetracyclines, or penicillins in N. gonorrhoeae. [2]
Lefamulin acetate (BC-3781 acetate) was highly active against all 60 Mycoplasma pneumoniae strains tested (18 macrolide-susceptible, 42 macrolide-resistant) with all MICs ≤ 0.008 μg/mL. MIC50/90 for macrolide-susceptible strains were ≤0.001/≤0.001 μg/mL and for macrolide-resistant strains were 0.002/0.002 μg/mL. The MBC was within 2 dilutions of the MIC, indicating a bactericidal effect. [3]
In a lipopolysaccharide (LPS)-induced lung neutrophilia mouse model, pre-treatment with lefamulin acetate (BC-3781 acetate) (10-140 mg/kg SC) resulted in dose-dependent reduction of BALF neutrophil counts. LPS-induced pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, GM-CSF), chemokines (CXCL-1, CXCL-2, CCL-2), and MMP-9 levels were significantly and dose-dependently reduced in mouse lung tissue. [4]
In vitro, in J774.2 mouse macrophages and human PBMCs, lefamulin acetate (BC-3781 acetate) (0.03 to 10 μM) had little to no effect on LPS-induced cytokine/chemokine/MMP-9 levels. It also had no effect on IL-8-induced human neutrophil chemotaxis at concentrations of 0.03 to 30 μM. [4]
In vitro, in J774.2 mouse macrophages and human PBMCs, lefamulin acetate (BC-3781 acetate) (0.03 to 10 μM) had little to no effect on LPS-induced cytokine/chemokine/MMP-9 levels. It also had no effect on IL-8-induced human neutrophil chemotaxis at concentrations of 0.03 to 30 μM. [5]

Lefamulin (10-140 mg/kg, s.c.) acetate inhibits inflammation in a mouse model of lung neutrophilia caused by lipopolysaccharide [4].
Lefamulin (1.25-160 mg/kg, s.c.) acetate exhibits antimicrobial activity in lung infection-challenged mice challenged with S. pneumoniae or S. aureus[5].

---

In a mouse model of LPS-induced lung neutrophilia, single subcutaneous (SC) doses of lefamulin acetate (BC-3781 acetate) (10, 30, 100 mg/kg) administered 30 minutes before LPS challenge significantly reduced total cell and neutrophil counts in bronchoalveolar lavage fluid (BALF) at 4 hours post-challenge. The reduction was comparable to or more potent than azithromycin (10-100 mg/kg SC) and dexamethasone (0.5/1 mg/kg oral/IP). [4]
In the same model, lefamulin acetate (BC-3781 acetate) (10, 30, 100 mg/kg SC) significantly reduced LPS-induced levels of TNF-α, IL-6, GM-CSF, CXCL-1, CXCL-2, CCL-2, and MMP-9 in lung homogenates. The effects were comparable to or more potent than dexamethasone (1 mg/kg IP) or azithromycin. Significant reduction of IL-1β was observed only at the highest dose (100 mg/kg). [4]
In a mouse model of LPS-induced lung neutrophilia, single subcutaneous (SC) doses of lefamulin acetate (BC-3781 acetate) (10, 30, 100 mg/kg) administered 30 minutes before LPS challenge significantly reduced total cell and neutrophil counts in bronchoalveolar lavage fluid (BALF) at 4 hours post-challenge. The reduction was comparable to or more potent than azithromycin (10-100 mg/kg SC) and dexamethasone (0.5/1 mg/kg oral/IP). [5]
In the same model, lefamulin acetate (BC-3781 acetate) (10, 30, 100 mg/kg SC) significantly reduced LPS-induced levels of TNF-α, IL-6, GM-CSF, CXCL-1, CXCL-2, CCL-2, and MMP-9 in lung homogenates. The effects were comparable to or more potent than dexamethasone (1 mg/kg IP) or azithromycin. Significant reduction of IL-1β was observed only at the highest dose (100 mg/kg). [5]

Cell Line: C. trachomatis, N. gonorrhoeae, and M. genitalium
Concentration: 0-1 mg/L
Incubation Time:
Result: demonstrated inhibition of bacterial activity at MIC50s of 0.02 mg/L, 0.063 mg/L, and 0.12 mg/L, in that order.

---

For Chlamydia trachomatis: MICs were determined on monolayers of McCoy cells or HeLa 229 cells infected with C. trachomatis (10² to 10³ inclusion forming units [IFU] per 3×10⁵ cells) on glass coverslips. Drug concentrations ranged from 2.56 to 0.0003 mg/L. After incubation at 35°C (5% CO₂) for 48 to 72 h, cells were fixed in methanol and inclusions stained with an alcoholic iodine solution. The MIC was defined as the lowest antibiotic concentration with no inclusions observed. [2]
For Mycoplasma genitalium: Susceptibility testing was performed using Vero cell culture and a quantitative real-time PCR method. [2]
For Mycoplasma pneumoniae: MIC testing was performed using the broth microdilution technique in SP4 broth. The inoculum was ~10⁴ to 10⁵ CFU/mL. MICs were recorded as the lowest concentration inhibiting color change at the time the growth control well changed from pink to yellow. The MBC was determined by pipetting 30 μL of fluid from wells showing no color change into 2.97 mL of broth to dilute the antibiotic, followed by prolonged incubation. The MBC was the lowest concentration with no evidence of color change. [3]
For chemotaxis assay: Human neutrophils were isolated from buffy coat. Cells (5.55×10⁶ cells/mL) were pre-incubated with lefamulin acetate (BC-3781 acetate) (0.03 to 30 μM) for 30 min at RT. Then, 75 μL of cell suspension (375,000 cells) was added to the upper wells of a transwell plate. The lower wells contained 180 μL of 22.22 ng/mL rhIL-8 solution. After 1-hour incubation at 37°C, migrated cells in the lower wells were quantified using a CellTiter Glo reagent to measure ATP levels via luminescence. Cytotoxicity was evaluated similarly by incubating cells with compounds for an additional hour before adding CellTiter Glo. [4]
For chemotaxis assay: Human neutrophils were isolated from buffy coat. Cells (5.55×10⁶ cells/mL) were pre-incubated with lefamulin acetate (BC-3781 acetate) (0.03 to 30 μM) for 30 min at RT. Then, 75 μL of cell suspension (375,000 cells) was added to the upper wells of a transwell plate. The lower wells contained 180 μL of 22.22 ng/mL rhIL-8 solution. After 1-hour incubation at 37°C, migrated cells in the lower wells were quantified using a CellTiter Glo reagent to measure ATP levels via luminescence. Cytotoxicity was evaluated similarly by incubating cells with compounds for an additional hour before adding CellTiter Glo. [5]

Animal Model: LPS-induced lung neutrophilia mouse model[4]
Dosage: 10-140 mg/kg
Administration: Subcutaneous injection (s.c.)
Result: decreased numbers of BALF neutrophil cells. Diminished levels of MMP-9, chemokines (CXCL-1, CXCL-2, and CCL-2) and pro-inflammatory cytokines (TNF-α, IL-6, IL-1β, and GM-CSF) in mouse lung tissue.

---

For pharmacokinetic analysis: Female BALB/c mice (weight ~20 g) received a single subcutaneous (SC) injection of lefamulin acetate (BC-3781 acetate) at 35 mg/kg (dissolved in 0.9% saline). Plasma and bronchoalveolar lavage fluid (BALF) samples were collected at 0.08, 0.25, 0.5, 0.75, 1.5, 3, 6, and 24 hours post-administration. [4]
For the LPS-induced lung neutrophilia model: Six-week-old male BALB/c mice were used. Lefamulin acetate (BC-3781 acetate) was dissolved in 0.9% saline and administered subcutaneously (SC) at doses of 10, 30, 35, 70, 100, or 140 mg/kg (free base equivalent) 30 minutes before intranasal (IN) LPS challenge (5 μg LPS/50 μL saline per mouse). Control mice received IN saline. At 4 hours post-LPS challenge, mice were euthanized, BALF was collected for total and differential neutrophil cell counts, and lungs were harvested for homogenization and analysis of cytokines, chemokines, and MMP-9. [4]
For pharmacokinetic analysis: Female BALB/c mice (weight ~20 g) received a single subcutaneous (SC) injection of lefamulin acetate (BC-3781 acetate) at 35 mg/kg (dissolved in 0.9% saline). Plasma and bronchoalveolar lavage fluid (BALF) samples were collected at 0.08, 0.25, 0.5, 0.75, 1.5, 3, 6, and 24 hours post-administration. [5]
For the LPS-induced lung neutrophilia model: Six-week-old male BALB/c mice were used. Lefamulin acetate (BC-3781 acetate) was dissolved in 0.9% saline and administered subcutaneously (SC) at doses of 10, 30, 35, 70, 100, or 140 mg/kg (free base equivalent) 30 minutes before intranasal (IN) LPS challenge (5 μg LPS/50 μL saline per mouse). Control mice received IN saline. At 4 hours post-LPS challenge, mice were euthanized, BALF was collected for total and differential neutrophil cell counts, and lungs were harvested for homogenization and analysis of cytokines, chemokines, and MMP-9. [5]

In mice, following a single subcutaneous (SC) dose of 35 mg/kg lefamulin acetate (BC-3781 acetate), the mean plasma AUC0-24h was 6.25 ± 0.93 μg·h/mL, and the mean Cmax was 1.33 ± 0.18 μg/mL at a Tmax of 0.50 h. In the epithelial lining fluid (ELF), the mean AUC0-24h was 12.6 ± 1.17 μg·h/mL, mean Cmax was 2.16 ± 0.50 μg/mL at a Tmax of 0.50 h. The ELF-to-plasma AUC ratio was approximately 2-fold. [4]
The plasma AUC0-24h for SC lefamulin in mice was comparable to human exposure following a single 150 mg IV dose (half the daily CABP treatment dose). A daily dose of 70 mg/kg in mice provides equivalent exposure to 300 mg IV lefamulin in humans (recommended daily CABP dose). The anti-inflammatory activity was seen at plasma exposures lower than those achieved at the antimicrobial clinical dose. [4]
In mice, following a single subcutaneous (SC) dose of 35 mg/kg lefamulin acetate (BC-3781 acetate), the mean plasma AUC0-24h was 6.25 ± 0.93 μg·h/mL, and the mean Cmax was 1.33 ± 0.18 μg/mL at a Tmax of 0.50 h. In the epithelial lining fluid (ELF), the mean AUC0-24h was 12.6 ± 1.17 μg·h/mL, mean Cmax was 2.16 ± 0.50 μg/mL at a Tmax of 0.50 h. The ELF-to-plasma AUC ratio was approximately 2-fold. [5]
The plasma AUC0-24h for SC lefamulin in mice was comparable to human exposure following a single 150 mg IV dose (half the daily CABP treatment dose). A daily dose of 70 mg/kg in mice provides equivalent exposure to 300 mg IV lefamulin in humans (recommended daily CABP dose). The anti-inflammatory activity was seen at plasma exposures lower than those achieved at the antimicrobial clinical dose. [5]

In vitro, in J774.2 mouse macrophages, cell viability was reduced at 30 μM (15.2 μg/mL free base) and 100 μM (50.8 μg/mL free base) lefamulin acetate (BC-3781 acetate). [4]
In vitro, in human PBMCs, cell viability was reduced at 100 μM lefamulin acetate (BC-3781 acetate). [4]
In vitro, in human neutrophils, cell viability was reduced to 86% of vehicle control at 100 μM (50.8 μg/mL free base) lefamulin acetate (BC-3781 acetate). No cytotoxicity was observed at 30 μM. [4]
In vitro, in J774.2 mouse macrophages, cell viability was reduced at 30 μM (15.2 μg/mL free base) and 100 μM (50.8 μg/mL free base) lefamulin acetate (BC-3781 acetate). [5]
In vitro, in human PBMCs, cell viability was reduced at 100 μM lefamulin acetate (BC-3781 acetate). [5]
In vitro, in human neutrophils, cell viability was reduced to 86% of vehicle control at 100 μM (50.8 μg/mL free base) lefamulin acetate (BC-3781 acetate). No cytotoxicity was observed at 30 μM. [5]

[1]. Lefamulin: Review of a Promising Novel Pleuromutilin Antibiotic. Pharmacotherapy. 2018 Sep;38(9):935-946. doi: 10.1002/phar.2166. Epub 2018 Aug 20.

[2]. In Vitro Activity of Lefamulin against Sexually Transmitted Bacterial Pathogens. Antimicrob Agents Chemother. 2018 Apr 26;62(5):e02380-17.

[3]. In Vitro Activities of Lefamulin and Other Antimicrobial Agents against Macrolide-Susceptible and Macrolide-Resistant Mycoplasma pneumoniae from the United States, Europe, and China. Antimicrob Agents Chemother. 2017 Jan 24;61(2):e02008-16.

[4]. Anti-inflammatory activity of lefamulin versus azithromycin and dexamethasone in vivo and in vitro in a lipopolysaccharide-induced lung neutrophilia mouse model. PLoS One. 2021 Sep 29;16(9): e0237659.

[5]. Pharmacokinetics/pharmacodynamics of lefamulin in a neutropenic murine pneumonia model with Staphylococcus aureus and Streptococcus pneumonia. J Antimicrob Chemother. 2019 Apr 1;74(Suppl 3):iii11-iii18.

See also: Lefamulin acetate (note moved to).

---

Drug Indications
Xenleta may be used to treat adult CAP when commonly used antimicrobial agents are unsuitable or ineffective for the treatment of community-acquired pneumonia (CAP). Official guidelines for the rational use of antimicrobial agents should be consulted.

---

Lefamulin acetate (BC-3781 acetate) is a new antibiotic belonging to the pleuromutilin class. It selectively inhibits bacterial translation by binding to the peptidyl transferase center (PTC) of the bacterial ribosome, a unique mechanism that minimizes potential for cross-resistance with other antibacterial agents. [2]
The results suggest that lefamulin acetate (BC-3781 acetate) could be a promising first-line antibiotic for the treatment of STIs, particularly in populations with high rates of resistance to standard-of-care antibiotics. [2]
Lefamulin acetate (BC-3781 acetate) might be an effective option for treatment of macrolide-resistant Mycoplasma pneumoniae infections. [3]
The anti-inflammatory activity of lefamulin acetate (BC-3781 acetate) (inhibition of neutrophilic lung infiltration and reduction of pro-inflammatory cytokine/chemokine concentrations) was observed at clinically relevant doses (plasma exposures lower than those achieved at the antimicrobial clinical dose). This activity may be beneficial in patients with acute respiratory distress syndrome (ARDS), cystic fibrosis, or severe inflammation-mediated lung injury. The mechanism does not appear to involve direct interaction with macrophages or neutrophilic chemotaxis based on in vitro results. [4]
The anti-inflammatory activity of lefamulin acetate (BC-3781 acetate) (inhibition of neutrophilic lung infiltration and reduction of pro-inflammatory cytokine/chemokine concentrations) was observed at clinically relevant doses (plasma exposures lower than those achieved at the antimicrobial clinical dose). This activity may be beneficial in patients with acute respiratory distress syndrome (ARDS), cystic fibrosis, or severe inflammation-mediated lung injury. The mechanism does not appear to involve direct interaction with macrophages or neutrophilic chemotaxis based on in vitro results. [5]

C30H49NO7S

567.77756857872

567.322

C, 63.46; H, 8.70; N, 2.47; O, 19.72; S, 5.65

1350636-82-6

Lefamulin;1061337-51-6

86346053

Solid powder

4

9

6

39

882

11

S([C@@H]1CC[C@H](C[C@H]1O)N)CC(=O)O[C@@H]1C[C@](C=C)(C)[C@H]([C@H](C)[C@]23CCC([C@H]2[C@@]1(C)C(C)CC3)=O)O.OC(C)=O

WSMXIQXWHPSVDE-ZPJPNJFZSA-N

InChI=1S/C28H45NO5S.C2H4O2/c1-6-26(4)14-22(34-23(32)15-35-21-8-7-18(29)13-20(21)31)27(5)16(2)9-11-28(17(3)25(26)33)12-10-19(30)24(27)281-2(3)4/h6,16-18,20-22,24-25,31,33H,1,7-15,29H2,2-5H31H3,(H,3,4)/t16-,17+,18-,20-,21-,22-,24+,25+,26-,27+,28+/m1./s1

(3aR,4R,5R,7S,8S,9R,9aS,12R)-8-hydroxy-4,7,9,12-tetramethyl-3-oxo-7-vinyldecahydro-4,9a-propanocyclopenta[8]annulen-5-yl 2-(((1R,2R,4R)-4-amino-2-hydroxycyclohexyl)thio)acetate acetic acid (1:1)

BC3781.Ac; BC-3781; BC 3781; BC3781; Xenleta;BC-3781.Ac; BC 3781.Ac;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, 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)

DMSO : ≥ 100 mg/mL (176.12 mM )
Ethanol : ~100 mg/mL
H2O : 1~100 mg/mL (~1.76 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.40 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 (4.40 mM) 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; 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 (4.40 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..

---

Solubility in Formulation 4: 10% DMSO+90% (20% SBE-β-CD in Saline): 2.5 mg/mL (4.40 mM)

---

Solubility in Formulation 5: 100 mg/mL (176.12 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

---

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