Pharmacokinetics/pharmacodynamics of lefamulin in a neutropenic murine pneumonia model with Staphylococcus aureus and Streptococcus pneumoniae.

OBJECTIVES: To present results of preclinical studies that supported further development of lefamulin for treating patients with community-acquired bacterial pneumonia (CABP). METHODS: The effect of bovine lung surfactant on the antibacterial activity of lefamulin against Streptococcus pneumoniae and Staphylococcus aureus was determined by broth microdilution assay. In vitro accumulation of lefamulin was evaluated in J774 mouse macrophages. Pharmacokinetics was assessed in female BALB/c (Bagg albino) mice treated with subcutaneous lefamulin (35 or 70 mg/kg). In neutropenic lung infection experiments, BALB/c mice received intraperitoneal cyclophosphamide before challenge with single S. pneumoniae or S. aureus strains; subcutaneous lefamulin (1.25-160 mg/kg) was given twice daily post-infection. Hill models described relationships between AUC/MIC ratios and changes in log10 cfu. RESULTS: Lung surfactant did not significantly increase lefamulin MIC values against test strains. Lefamulin uptake in macrophages was rapid (a plateau was reached in ∼3 h). In mice, distribution of lefamulin [plasma to epithelial lining fluid (ELF)] was rapid, showing an ∼2-fold increase in lefamulin exposure in the ELF during the 5.5 h period. Median plasma AUC/MIC ratios associated with 1 and 2 log10 cfu reductions from baseline were 1.37 and 2.15, respectively, for S. pneumoniae and 2.13 and 6.24 for S. aureus. Corresponding ELF results were 14.0 and 22.0 for S. pneumoniae and 21.7 and 63.9 for S. aureus. CONCLUSIONS: Overall, lefamulin displays desirable pharmacokinetic/pharmacodynamic relationships that are predictive of the clinical effectiveness of lefamulin and other antibacterial agents used to treat CABP.

Development, validation, and application of an LC-MS/MS method for the quantification of the novel antibiotic drug lefamulin (Xenleta®) and its main metabolite 2R-hydroxy lefamulin in human plasma.

Lefamulin (Xenleta®) is a first in-class systemic pleuromutilin antibiotic that inhibits bacterial protein synthesis and selectively binds to a highly conserved region of the peptidyl transferase center of the bacterial 50S ribosomal subunit. A total of twenty-five Phase 1 clinical studies, one Phase 2 study in acute bacterial skin and skin structure infections (ABSSSI), and two pivotal Phase 3 studies in adults with community acquired bacterial pneumonia (CABP) have been completed. Xenleta® (lefamulin) has been approved by the FDA on August 19, 2019, by Health Canada on July 10, 2020, and by the EMA on July 28, 2020 for the oral and IV treatment of CABP in adults. For and during the clinical development, simple, sensitive, precise, and selective LC-MS/MS methods were developed and validated, first for lefamulin alone and later for the simultaneous quantification of lefamulin and its main metabolite 2R-hydroxy lefamulin in human plasma. Chromatographic separation in the current method was achieved on a reverse phase C18 column using gradient elution at a flowrate of 500 µL/min consisting of a mobile phase of water (A) and methanol (B) each containing 0.1 % formic acid (v/v) with a run time of 8.0 min. The detection and quantification of the analytes were performed on AB Sciex Triple Quad 5500 using multiple reaction monitoring operated in positive electrospray ionization mode after a simple plasma protein precipitation cleanup and dilution. The method was linear over the concentration range of 1.00-1 000 ng/mL (r ≥ 0.999) for lefamulin und 1.00-500 ng/mL (r ≥ 0.999) for 2R-hydroxy lefamulin. No significant matrix effects and a good extraction recovery were observed. The within- and between-run precision and accuracy were within the acceptable limits, and both analytes were found to be stable throughout the short term, long term, and freeze thaw stability studies. This current validated method was successfully applied in five Phase 1 and two Phase 3 studies.

Application of capillary electrophoresis-mass spectrometry for the investigation of the binding behavior of oxaliplatin to 5'-GMP in the presence of the sulfur-containing amino acid L-methionine.

Capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) has been used for investigating the influence of the sulfur containing amino acid L-methionine (L-Met) on the binding behavior of oxaliplatin (trans-R,R-diaminocyclohexane-(oxalato)platinum(II)) to 5'-GMP. L-Methionine competes with 5'-GMP for the platinum binding site and forms as well as 5'-GMP adducts with oxaliplatin. The formation of the prognosed complexes [Pt(DACH)(L-Met-S,N)]+ and [Pt(DACH)(5'-GMP)2]2- (DACH = 1,2-diaminocyclohexane) could be proved directly by using CE-ESI-MS. Furthermore, we could now bring forward proofs, that the coordination of 5'-GMP with oxaliplatin is inhibited by L-methionine and could show, that the 5'-GMP ligands of the [Pt(DACH) (5'-GMP)2]2- complex can be replaced slowly by L-methionine whereas methionine can not be replaced by GMP.