Lefamulin Overcomes Acquired Drug Resistance via Regulating Mitochondrial Homeostasis by Targeting ILF3 in Hepatocellular Carcinoma.

Acquired resistance represents a critical clinical challenge to molecular targeted therapies such as tyrosine kinase inhibitors (TKIs) treatment in hepatocellular carcinoma (HCC). Therefore, it is urgent to explore new mechanisms and therapeutics that can overcome or delay resistance. Here, a US Food and Drug Administration (FDA)-approved pleuromutilin antibiotic is identified that overcomes sorafenib resistance in HCC cell lines, cell line-derived xenograft (CDX) and hydrodynamic injection mouse models. It is demonstrated that lefamulin targets interleukin enhancer-binding factor 3 (ILF3) to increase the sorafenib susceptibility of HCC via impairing mitochondrial function. Mechanistically, lefamulin directly binds to the Alanine-99 site of ILF3 protein and interferes with acetyltransferase general control non-depressible 5 (GCN5) and CREB binding protein (CBP) mediated acetylation of Lysine-100 site, which disrupts the ILF3-mediated transcription of mitochondrial ribosomal protein L12 (MRPL12) and subsequent mitochondrial biogenesis. Clinical data further confirm that high ILF3 or MRPL12 expression is associated with poor survival and targeted therapy efficacy in HCC. Conclusively, this findings suggest that ILF3 is a potential therapeutic target for overcoming resistance to TKIs, and lefamulin may be a novel combination therapy strategy for HCC treatment with sorafenib and regorafenib.

In Vivo Immune-Modulatory Activity of Lefamulin in an Influenza Virus A (H1N1) Infection Model in Mice.

Lefamulin is a first-in-class systemic pleuromutilin antimicrobial and potent inhibitor of bacterial translation, and the most recent novel antimicrobial approved for the treatment of community-acquired pneumonia (CAP). It exhibits potent antibacterial activity against the most prevalent bacterial pathogens that cause typical and atypical pneumonia and other infectious diseases. Early studies indicate additional anti-inflammatory activity. In this study, we further investigated the immune-modulatory activity of lefamulin in the influenza A/H1N1 acute respiratory distress syndrome (ARDS) model in BALB/c mice. Comparators included azithromycin, an anti-inflammatory antimicrobial, and the antiviral oseltamivir. Lefamulin significantly decreased the total immune cell infiltration, specifically the neutrophils, inflammatory monocytes, CD4+ and CD8+ T-cells, NK cells, and B-cells into the lung by Day 6 at both doses tested compared to the untreated vehicle control group (placebo), whereas azithromycin and oseltamivir did not significantly affect the total immune cell counts at the tested dosing regimens. Bronchioalveolar lavage fluid concentrations of pro-inflammatory cytokines and chemokines including TNF-α, IL-6, IL-12p70, IL-17A, IFN-γ, and GM-CSF were significantly reduced, and MCP-1 concentrations were lowered (not significantly) by lefamulin at the clinically relevant 'low' dose on Day 3 when the viral load peaked. Similar effects were also observed for oseltamivir and azithromycin. Lefamulin also decreased the viral load (TCID50) by half a log10 by Day 6 and showed positive effects on the gross lung pathology and survival. Oseltamivir and lefamulin were efficacious in the suppression of the development of influenza-induced bronchi-interstitial pneumonia, whereas azithromycin did not show reduced pathology at the tested treatment regimen. The observed anti-inflammatory and immune-modulatory activity of lefamulin at the tested treatment regimens highlights a promising secondary pharmacological property of lefamulin. While these results require confirmation in a clinical trial, they indicate that lefamulin may provide an immune-modulatory activity beyond its proven potent antibacterial activity. This additional activity may benefit CAP patients and potentially prevent acute lung injury (ALI) and ARDS.

Integrative model-based comparison of target site-specific antimicrobial effects: A case study with ceftaroline and lefamulin.

OBJECTIVE: Predictions of antimicrobial effects typically rely on plasma-based pharmacokinetic-pharmacodynamic (PK-PD) targets, ignoring target-site concentrations and potential differences in tissue penetration between antibiotics. In this study, we applied PK-PD modelling to compare target site-specific effects of antibiotics by integrating clinical microdialysis data, in vitro time-kill curves, and antimicrobial susceptibility distributions. As a case study, we compared the effect of lefamulin and ceftaroline against methicillin-resistant Staphylococcus aureus (MRSA) at soft-tissue concentrations. METHODS: A population PK model describing lefamulin concentrations in plasma, subcutaneous adipose and muscle tissue was developed. For ceftaroline, a similar previously reported PK model was adopted. In vitro time-kill experiments were performed with six MRSA isolates and a PD model was developed to describe bacterial growth and antimicrobial effects. The clinical PK and in vitro PD models were linked to compare antimicrobial effects of ceftaroline and lefamulin at the different target sites. RESULTS: Considering minimum inhibitory concentration (MIC) distributions and standard dosages, ceftaroline showed superior anti-MRSA effects compared to lefamulin both at plasma and soft-tissue concentrations. Looking at the individual antibiotics, lefamulin effects were highest at soft-tissue concentrations, while ceftaroline effects were highest at plasma concentrations, emphasising the importance of considering target-site PK-PD in antibiotic treatment optimisation. CONCLUSION: Given standard dosing regimens, ceftaroline appeared more effective than lefamulin against MRSA at soft-tissue concentrations. The PK-PD model-based approach applied in this study could be used to compare or explore the potential of antibiotics for specific indications or in populations with unique target-site PK.

Safety and Pharmacokinetics Following Oral or Intravenous Lefamulin in Adults With Cystic Fibrosis.

PURPOSE: Methicillin-resistant Staphylococcus aureus infections are increasing in prevalence in patients with cystic fibrosis (CF) and are associated with worsening lung function and increased mortality. Lefamulin is a pleuromutilin antimicrobial approved to treat community-acquired bacterial pneumonia based on potent in vitro activity and clinical efficacy. This Phase I, open-label, randomized crossover study assessed the safety and pharmacokinetic profile of oral and intravenous (IV) lefamulin in adults with CF. METHODS: The study comprised 2 dosing periods in which adults with CF (N = 13) received a single dose of lefamulin via a 150-mg IV infusion or 600-mg immediate-release orally administered tablet, separated by a 4- to 7-day washout period. Pharmacokinetic and safety parameters were assessed after lefamulin treatment. FINDINGS: Single doses of lefamulin administered via oral tablet or IV infusion resulted in comparable drug exposure, and sputum analysis suggested rapid penetration of lefamulin into the lung. Comparison of the present results with those obtained from prior single-dose studies of healthy volunteers indicate no meaningful difference in the pharmacokinetic properties of lefamulin in patients with CF. Treatment-emergent adverse events were consistent with previous reports, and the majority were mild in severity. IMPLICATIONS: These results show similar lefamulin pharmacokinetic and safety profiles between patients with CF and healthy volunteers receiving the same oral and IV doses, suggesting no need for lefamulin dose adjustment in patients with CF and indicating the potential of lefamulin as therapy for lung infections in patients with CF. CLINICALTRIALS: gov identifier: NCT05225805.

Pharmacokinetic, Pharmacokinetic/Pharmacodynamic, and Safety Investigations of Lefamulin in Healthy Chinese Subjects.

This study aimed to explore the pharmacokinetics (PK) and safety of oral (PO) and intravenous (IV) lefamulin in healthy Chinese subjects and to evaluate the efficacy of the intravenous administration regimen using pharmacokinetic/pharmacodynamic (PK/PD) analysis. This study was a randomized, open-label, single- and multiple-dose, intravenous and oral administration study. PK parameters were calculated, and the probability of target attainment (PTA) and the cumulative fraction of response (CFR) after IV administration of lefamulin 150 mg 1 h q12 h were analyzed with Monte Carlo simulations. Lefamulin exhibited extensive distribution. The mean steady-state AUC0-24 h of 150 mg lefamulin IV and 600 mg lefamulin PO were 10.03 and 13.96 µg·h/mL, respectively. For Streptococcus pneumoniae and Staphylococcus aureus, based on the free-drug AUC over MIC ratio (fAUC/MIC) target of 1-log10 cfu reduction, the PK/PD breakpoints were 0.25 and 0.125 mg/L, respectively. The CFR was over 90% for both types of strains with 95% protein binding rate, suggesting that the regimen was microbiologically effective. Lefamulin was safe and well-tolerated. The PK of lefamulin in healthy Chinese subjects were consistent with that in foreign countries. Lefamulin demonstrated the microbiological effectiveness against Streptococcus pneumoniae and Staphylococcus aureus.

[New developments in the fight against bacterial infections : Update on antiobiotic research, development and treatment]. / Neue Entwicklungen in der Bekämpfung bakterieller Infektionen : Update Antiobiotikaforschung, - entwicklung und -therapie.

Infections caused by pathogens with antimicrobial resistance (AMR) pose a threat to modern healthcare and have triggered the development of comprehensive national and global action plans against the spread of AMR. These include an increasing global network with the focus on rational antibiotic use, innovative strategies on antibiotic research and development, and new therapeutic approaches in antibacterial drug research. In Europe 671,689 infections associated with AMR pathogens and 33,110 deaths directly related to AMR were counted in just 1 year. Globally, resistant Staphylococcus aureus, Escherichia coli, pneumococci, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa are the most common pathogens in the context of these deaths. Resistance to antibiotics in major drug classes such as beta-lactams and fluoroquinolones is particularly common. Strategies for overcoming the global AMR crisis address research on AMR emergence and spread, promoting campaigns for responsible antibiotic use, and improving infection prevention. The identification of new antibiotics and treatment approaches and the development of new strategies to contain the spread of AMR are essential. Newly approved substances include delafloxacin, lefamulin, and meropenem-vaborbactam. New antibiotics that are well advanced in clinical trials are aztreonam-avibactam, sulbactam-durlobactam, omadacycline, and type II topoisomerase inhibitors. Much interest is also being shown in the development of new therapeutic approaches such as bacteriophage treatment.

Current and emerging drug treatment strategies to tackle invasive community-associated methicillin-resistant Staphylococcus aureus (MRSA) infection: what are the challenges?

INTRODUCTION: Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infections represent a leading cause of purulent skin and soft tissue infections in some geographical regions. Traditionally, 'old antibiotics' such as trimethoprim-sulfamethoxazole, tetracyclines, clindamycin, chloramphenicol,vancomycin, and teicoplanin have been used to treat these infections, but these were often associated with low efficacy and excessive side effects and toxicity, especially nephrotoxicity. Along with the development of new compounds, the last decade has seen substantial improvements in the management of CA-MRSA infections. AREAS COVERED: In this review, the authors discuss the current and emerging drug treatment strategies to tackle invasive CA-MRSA infections. Articles reported in this review were selected from through literature searches using the PubMed database. EXPERT OPINION: The availability of new drugs showing a potent in vitro activity against CA-MRSA represents a unique opportunity to face the threat of resistance while potentially reducing toxicity. All these compounds represent promising options to enhance our antibiotic armamentarium. However, data regarding the use of these new drugs in real-life studies are limited and their best placement in therapy and in terms of optimization of medical resources and balance of cost-effectiveness requires further investigation.

Pharmacodynamic evaluation of lefamulin in the treatment of gonorrhea using a hollow fiber infection model simulating Neisseria gonorrhoeae infections.

The emergence and spread of antimicrobial resistance in Neisseria gonorrhoeae is seriously threatening the treatment and control of gonorrhea globally. Novel treatment options are essential, coupled with appropriate methods to pharmacodynamically examine the efficacy and resistance emergence of these novel drugs. Herein, we used our dynamic in vitro hollow fiber infection model (HFIM) to evaluate protein-unbound lefamulin, a semisynthetic pleuromutilin, against N. gonorrhoeae. Dose-range and dose-fractionation experiments with N. gonorrhoeae reference strains: WHO F (susceptible to all relevant antimicrobials), WHO X (extensively drug-resistant, including ceftriaxone resistance), and WHO V (high-level azithromycin resistant, and highest gonococcal MIC of lefamulin (2 mg/l) reported), were performed to examine lefamulin gonococcal killing and resistance development during treatment. The dose-range experiments, simulating a single oral dose of lefamulin based on human plasma concentrations, indicated that ≥1.2 g, ≥2.8 g, and ≥9.6 g of lefamulin were required to eradicate WHO F, X, and V, respectively. Dose-fractionation experiments, based on human lefamulin plasma concentrations, showed that WHO X was eradicated with ≥2.8 g per day when administered as q12 h (1.4 g twice a day) and with ≥3.6 g per day when administered as q8 h (1.2 g thrice a day), both for 7 days. However, when simulating the treatment with 5-10 times higher concentrations of free lefamulin in relevant gonorrhea tissues (based on urogenital tissues in a rat model), 600 mg every 12 h for 5 days (approved oral treatment for community-acquired bacterial pneumonia) eradicated all strains, and no lefamulin resistance emerged in the successful treatment arms. In many arms failing single or multiple dose treatments for WHO X, lefamulin-resistant mutants (MIC = 2 mg/l), containing an A132V amino acid substitution in ribosomal protein L3, were selected. Nevertheless, these lefamulin-resistant mutants demonstrated an impaired biofitness. In conclusion, a clinical study is warranted to elucidate the clinical potential of lefamulin as a treatment option for uncomplicated gonorrhea (as well as several other bacterial STIs).

Lefamulin: a New Hope in the Field of Community-Acquired Bacterial Pneumonia.

Purpose of Review: Community-acquired bacterial pneumonia (CABP) continues to be a worldwide health concern since it is the major cause of mortality and hospitalisation worldwide. Increased macrolide resistance among Streptococcus pneumoniae and other infections has resulted in a significantly larger illness burden, which has been exacerbated by evolving demography and a higher prevalence of comorbid disorders. Owing to such circumstances, the creation of new antibiotic classes is critical. Recent Findings: Lefamulin, also referred to as BC-3781, is the primary pleuromutilin antibiotic which has been permitted for both intravenous and oral use in humans for the remedy of bacterial infections. It has shown activity against gram-positive bacteria including methicillin-resistant strains as well as atypical organisms which as often implicated in CABP. It has a completely unique mechanism of action that inhibits protein synthesis via way of means of stopping the binding of tRNA for peptide transfer. The C(14) side chain is responsible for its pharmacodynamic and antimicrobial properties, together with supporting in overcoming bacterial ribosomal resistance and mutations improvement amplifying the number of hydrogen bonds to the target site. Summary: This review aims to highlight the pre-existing treatment options and specific purposes to shed some light upon the development of a new drug lefamulin and its specifications and explore this novel drug's superior efficacy to already existing treatment strategies.

Tissue Distribution of [14C]-Lefamulin into the Urogenital Tract in Rats.

Lefamulin, a semisynthetic pleuromutilin antibiotic approved in the United States, Canada, and Europe for intravenous and oral treatment of community-acquired bacterial pneumonia, is highly active in vitro against bacterial pathogens that cause sexually transmitted infections (STIs), including multidrug-resistant strains of Neisseria gonorrhoeae, Chlamydia trachomatis, and Mycoplasma genitalium. This nonclinical study used quantitative whole-body autoradiography (QWBA) and qualitative tape-transfer microautoradiography (MARG) to investigate lefamulin distribution into urogenital tract tissues down to a cellular level in male and female rats. A single intravenous dose (30 mg/kg) of [14C]-lefamulin was administered to 3 male and 3 female Sprague-Dawley rats. At 0.5, 6, and 24 h post dose, rats were euthanized and [14C]-lefamulin distribution was investigated using QWBA and MARG of sagittal planes. [14C]-lefamulin was well distributed throughout the carcasses of male and female rats, with the highest concentrations observed in male bulbourethral gland, urethra, prostate in female clitoral gland, uterus (particularly endometrium), and ovary. In these areas, concentrations were similar to or higher than those observed in the lungs. Concentrations peaked at 0.5 h post dose, remaining detectable in the urogenital tract up to 24 h post dose. [14C]-lefamulin in rats showed rapid, homogeneous distribution into urogenital tissues down to a cellular level, with high tissue:blood ratios in tissues relevant to STI treatment. These results, and the potent in vitro activity of lefamulin against multidrug-resistant bacteria known to cause STIs, will help inform further assessment of lefamulin, including potential clinical evaluation for treatment of STIs.

A plain language summary of how lefamulin alone can be used to treat pneumonia caught outside of the hospital due to common bacterial causes, including drug-resistant bacteria.

WHAT IS THIS SUMMARY ABOUT?: Bacterial pneumonia is an infection of the lung caused by bacteria that is potentially deadly, costly, and affects millions of people worldwide every year. Treatment is becoming more challenging-many current treatments no longer work well because some strains of bacteria that cause pneumonia have become resistant to current antibiotics. Many of the antibiotics that do still work have undesirable side effects. Therefore, new antibiotics that work differently are needed to treat bacterial pneumonia. Lefamulin (brand name, Xenleta®) is an antibiotic that was approved to treat bacterial pneumonia caught outside a hospital (also called community-acquired bacterial pneumonia, or CABP) based on results of two clinical studies. In both studies, participants started treatment with lefamulin before the type of bacteria causing the infection was known. Lefamulin was well tolerated and worked well in 5 to 7 days to kill the bacteria causing the infection and to improve symptoms in almost all participants with CABP. WHAT WERE THE RESULTS?: After the studies were completed, the researchers looked back at what kinds of bacteria were identified from the study participants. Lefamulin worked well to kill bacteria and to improve CABP symptoms for most kinds of infecting bacteria, including bacteria resistant to many current antibiotics. WHAT DO THE RESULTS MEAN?: These results suggest that lefamulin, by itself, provides a much-needed treatment option for CABP that covers most of the key bacteria causing this infection.

Current pharmacotherapy for methicillin-resistant Staphylococcus aureus (MRSA) pneumonia.

INTRODUCTION: Currently, several antibiotics are active against methicillin-resistant Staphylococcus aureus (MRSA) and can be used for the treatment of pneumonia. They show great variability in terms of antibiotic class, indication, pharmacodynamic/pharmacokinetic properties, type of available formulations, spectrum of activity against bacteria other than MRSA, and toxicity profile. AREAS COVERED: In this narrative review, the authors discuss the characteristics of currently available agents for the treatment of MRSA pneumonia. EXPERT OPINION: The availability of different agents with anti-MRSA activity, and approved for the treatment of pneumonia can allow a personalized approach for any given patient based on the severity of the disease, the setting of occurrence, the patient's baseline risk of toxicity and drug interactions, and the possibility of oral therapy whenever early discharge or outpatient treatment are possible. Although some gray areas still remain, like the lack of high certainty evidence on the efficacy of some old agents and on the precise role of companion agents with toxin inhibitory activity in the case of necrotizing pneumonia, the frequent availability of different treatment choices, each with peculiar characteristics, is already allowing an important step toward a precision medicine approach for the treatment of MRSA pneumonia.

Pooled microbiological findings and efficacy outcomes by pathogen in adults with community-acquired bacterial pneumonia from the Lefamulin Evaluation Against Pneumonia (LEAP) 1 and LEAP 2 phase 3 trials of lefamulin versus moxifloxacin.

OBJECTIVES: Lefamulin, a pleuromutilin antibiotic approved for community-acquired bacterial pneumonia (CABP), was evaluated for microbiological efficacy in a prespecified pooled analysis of LEAP 1 and 2 phase 3 clinical trial data in patients with CABP. METHODS: In LEAP 1, adults (PORT risk class III‒V) received intravenous (IV) lefamulin 150 mg every 12 h (q12h) for 5‒7 days or moxifloxacin 400 mg every 24 h (q24h) for 7 days, with optional IV-to-oral switch. In LEAP 2, adults (PORT II‒IV) received oral lefamulin 600 mg q12h for 5 days or moxifloxacin 400 mg q24h for 7 days. Primary outcomes were early clinical response (ECR) at 96 ± 24 h after treatment start and investigator assessment of clinical response (IACR) 5‒10 days after the last dose. Secondary outcomes included ECR and IACR in patients with a baseline CABP pathogen (detected via culture, urinary antigen testing, serology and/or real-time PCR). RESULTS: Baseline CABP pathogens were detected in 709/1289 patients (55.0%; microbiological intention-to-treat population). The most frequently identified pathogens were Streptococcus pneumoniae (61.9% of patients) and Haemophilus influenzae (29.9%); 25.1% had atypical pathogens and 33.1% had polymicrobial infections. Pathogens were identified most frequently by PCR from sputum, followed by culture from respiratory specimens. In patients with baseline CABP pathogens, ECR rates were 89.3% (lefamulin) and 93.0% (moxifloxacin); IACR success rates were 83.2% and 86.7%, respectively. Results were consistent across CABP pathogens, including drug-resistant isolates and polymicrobial infections. CONCLUSION: Lefamulin is a valuable IV and oral monotherapy option for empirical and directed CABP treatment in adults.

Clinical use of lefamulin: A first-in-class semisynthetic pleuromutilin antibiotic.

Lefamulin is a novel antibiotic agent within the pleuromutilin derivative class approved for the treatment of community-acquired bacterial pneumonia (CABP) by the United States Food and Drug Administration and the European Commission in 2019 and 2020, respectively. The objective of this article is to provide a summary of clinically relevant data underlying lefamulin and to provide recommendations for its place in therapy. In vitro data establish lefamulin's activity against a number of Gram-positive, Gram-negative and atypical organisms relevant in the treatment of CABP, including Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Legionella pneumophila, Mycoplasma pneumoniae and Chlamydophila pneumoniae. Two phase-3 studies, the Lefamulin Evaluation Against Pneumonia trials, established non-inferiority of lefamulin against moxifloxacin in the treatment of CABP, including the sequential transition from intravenous to oral therapy and across a broad set of patient demographics and severities. Pooled and post hoc analyses have confirmed these effects for a variety of subgroups and secondary endpoints. Real-world study data post-approval have largely not yet emerged for lefamulin, and there is a need for further investigation into safety/efficacy for off-label indications such as acute bacterial skin and skin structure infections and sexually transmitted infections. Further data regarding tolerability, particularly with long-term use, as well as the emergence of resistance over time, are still undefined.

Lefamulin in Patients with Community-Acquired Bacterial Pneumonia Caused by Atypical Respiratory Pathogens: Pooled Results from Two Phase 3 Trials.

Lefamulin was the first systemic pleuromutilin antibiotic approved for intravenous and oral use in adults with community-acquired bacterial pneumonia based on two phase 3 trials (Lefamulin Evaluation Against Pneumonia [LEAP]-1 and LEAP-2). This pooled analysis evaluated lefamulin efficacy and safety in adults with community-acquired bacterial pneumonia caused by atypical pathogens (Mycoplasma pneumoniae, Legionella pneumophila, and Chlamydia pneumoniae). In LEAP-1, participants received intravenous lefamulin 150 mg every 12 h for 5-7 days or moxifloxacin 400 mg every 24 h for 7 days, with optional intravenous-to-oral switch. In LEAP-2, participants received oral lefamulin 600 mg every 12 h for 5 days or moxifloxacin 400 mg every 24 h for 7 days. Primary outcomes were early clinical response at 96 ± 24 h after first dose and investigator assessment of clinical response at test of cure (5-10 days after last dose). Atypical pathogens were identified in 25.0% (91/364) of lefamulin-treated patients and 25.2% (87/345) of moxifloxacin-treated patients; most were identified by ≥1 standard diagnostic modality (M. pneumoniae 71.2% [52/73]; L. pneumophila 96.9% [63/65]; C. pneumoniae 79.3% [46/58]); the most common standard diagnostic modality was serology. In terms of disease severity, more than 90% of patients had CURB-65 (confusion of new onset, blood urea nitrogen > 19 mg/dL, respiratory rate ≥ 30 breaths/min, blood pressure <90 mm Hg systolic or ≤60 mm Hg diastolic, and age ≥ 65 years) scores of 0-2; approximately 50% of patients had PORT (Pneumonia Outcomes Research Team) risk class of III, and the remaining patients were more likely to have PORT risk class of II or IV versus V. In patients with atypical pathogens, early clinical response (lefamulin 84.4-96.6%; moxifloxacin 90.3-96.8%) and investigator assessment of clinical response at test of cure (lefamulin 74.1-89.7%; moxifloxacin 74.2-97.1%) were high and similar between arms. Treatment-emergent adverse event rates were similar in the lefamulin (34.1% [31/91]) and moxifloxacin (32.2% [28/87]) groups. Limitations to this analysis include its post hoc nature, the small numbers of patients infected with atypical pathogens, the possibility of PCR-based diagnostic methods to identify non-etiologically relevant pathogens, and the possibility that these findings may not be generalizable to all patients. Lefamulin as short-course empiric monotherapy, including 5-day oral therapy, was well tolerated in adults with community-acquired bacterial pneumonia and demonstrated high clinical response rates against atypical pathogens.

The Very First Modification of Pleuromutilin and Lefamulin by Photoinitiated Radical Addition Reactions-Synthesis and Antibacterial Studies.

Pleuromutilin is a fungal diterpene natural product with antimicrobial properties, semisynthetic derivatives of which are used in veterinary and human medicine. The development of bacterial resistance to pleuromutilins is known to be very slow, which makes the tricyclic diterpene skeleton of pleuromutilin a very attractive starting structure for the development of new antibiotic derivatives that are unlikely to induce resistance. Here, we report the very first synthetic modifications of pleuromutilin and lefamulin at alkene position C19-C20, by two different photoinduced addition reactions, the radical thiol-ene coupling reaction, and the atom transfer radical additions (ATRAs) of perfluoroalkyl iodides. Pleuromutilin were modified with the addition of several alkyl- and aryl-thiols, thiol-containing amino acids and nucleoside and carbohydrate thiols, as well as perfluoroalkylated side chains. The antibacterial properties of the novel semisynthetic pleuromutilin derivatives were investigated on a panel of bacterial strains, including susceptible and multiresistant pathogens and normal flora members. We have identified some novel semisynthetic pleuromutilin and lefamulin derivatives with promising antimicrobial properties.

Determination of In Vitro Antimicrobial Susceptibility for Lefamulin (Pleuromutilin) for Ureaplasma Spp. and Mycoplasma hominis.

Lefamulin is the first of the pleuromutilin class of antimicrobials to be available for therapeutic use in humans. Minimum inhibitory concentrations of lefamulin were determined by microbroth dilution for 90 characterised clinical isolates (25 Ureaplasma parvum, 25 Ureaplasma urealyticum, and 40 Mycoplasma hominis). All Mycoplasma hominis isolates possessed lefamulin MICs of ≤0.25 mg/L after 48 h (MIC50/90 of 0.06/0.12 mg/L), despite an inherent resistance to macrolides; while Ureaplasma isolates had MICs of ≤2 mg/L after 24 h (MIC50/90 of 0.25/1 mg/L), despite inherent resistance to clindamycin. Two U. urealyticum isolates with additional A2058G mutations of 23S rRNA, and one U. parvum isolate with a R66Q67 deletion (all of which had a combined resistance to macrolides and clindamycin) only showed a 2-fold increase in lefamulin MIC (1-2 mg/L) relative to macrolide-susceptible strains. Lefamulin could be an effective alternative antimicrobial for treating Ureaplasma spp. and Mycoplasma hominis infections irrespective of intrinsic or acquired resistance to macrolides, lincosamides, and ketolides. Based on this potent in vitro activity and the known good, rapid, and homogenous tissue penetration of female and male urogenital tissues and glands, further exploration of clinical efficacy of lefamulin for the treatment of Mycoplasma and Ureaplasma urogenital infections is warranted.

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.