Assessment of the in vitro cytochrome P450 (CYP) inhibition potential of nafithromycin, a next generation lactone ketolide antibiotic.

Nafithromycin is a next generation lactone ketolide antibiotic slated to enter phase III clinical development in India for the treatment of CABP as a shorter 800 mg-OD X3 day therapeutic regimen. Nafithromycin exhibits potent activity against MDR Streptococcus pneumoniae including erythromycin and telithromycin-resistant resistant strains. Older macrolides/ketolides are reported to be potent inhibitors of CYP3A4/5. To facilitate comparative assessment of drug-drug interaction potential, CYP inhibitory activities of nafithromycin was evaluated in comparison with clarithromycin, telithromycin, cethromycin and solithromycin. CYP inhibitory activities were assessed against key CYP isoforms (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and CYP3A4/5) using human liver microsomes. Additionally, time-dependent inhibition (TDI), metabolism-based inhibition (MBI) and k inact /K I activities were also investigated for CYP3A4/5. Nafithromycin did not inhibit key CYP enzymes and was found to be a weak inhibitor of CYP3A4/5. Comparator antibiotics were found to be potent inhibitors with 2- to 50-fold leftward shifts in CYP3A4/5 IC50 values, while such shift was not noted for nafithromycin. k inact /K I ratio of nafithromycin was 3- to 153-fold lower than comparator drugs, further substantiating its lower affinity for CYP3A4/5. In sum, weaker inhibition and lower k inact /K I ratio for CYP3A4/5, points towards nafithromycin's lower propensities towards clinical drug-drug interactions as compared to other macrolides/ketolides antibiotics.

Preclinical safety evaluation of nafithromycin (WCK 4873) with emphasis on liver safety in rat and dog.

Ketolide antibiotics are known to cause hepatic injury. Nafithromycin, a novel lactone ketolide was therefore assessed for hepatic safety through range of preclinical in vitro (metabolic stability, CYP inhibition/induction assays) and in vivo (mass balance and repeat dose toxicity) studies. Repeat-dose toxicity studies in rat and dog revealed that nafithromycin did not cause adverse hematological, biochemical and histopathological changes suggestive of systemic or hepatobiliary safety concern at exposures 3-8 fold higher than targeted therapeutic exposures. The only histological finding noticed was reversible phospholipidosis, mainly in lung and lymphoid organs but not in liver, indicating lower nafithromycin accumulation in liver. This observation was corroborated with lack of biologically relevant elevation of hepatic enzymes linked to hepatic injury. In vitro studies showed that nafithromycin undergoes moderate CYP3A mediated metabolism. Unlike other ketolides, nafithromycin and its metabolites showed weak inhibition of CYP3A isoform and lacked CYP2D6 inhibition. Due to hydrophilic nature, nafithromycin in addition to hepatic clearance is also eliminated unchanged by kidneys in significant amount, thereby minimizing hepatic burden. Based on the scientifically integrated evidences such as moderate metabolism, weak CYP inhibition, lack of CYP induction, minimal accumulation in liver, nafithromycin showed promising hepatic safety profile suitable for its intended community-based usage.

Pretreatment of nafithromycin attenuates inflammatory response in murine lipopolysaccharide induced acute lung injury.

Acute respiratory distress syndrome following an acute lung injury (ALI) is a life threatening inflammatory condition predominantly characterized by vascular protein leakage, neutrophil recruitment and overexpression of proinflammatory cytokines. Pulmonary and systemic bacterial infections are the major cause of ALI wherein the bacterial cell components play a crucial role. Macrolide/ketolide antibiotics are reported to possess immunomodulatory activity; as a result improved survival has been noted in pneumonia patients. Hence immunomodulatory activity of nafithromycin, a novel lactone ketolide antibacterial agent was assessed in the murine LPS induced ALI model. Vehicle, nafithromycin (100 mg/kg), azithromycin (600 mg/kg) and dexamethasone (20 mg/kg) were administered orally, 1 h prior to LPS challenge and bronchoalveolar lavage (BAL) fluid was collected thereafter at 18, 24 and 48 h to determine the total cell count, total protein, myeloperoxidase (MPO), tumor necrosis factor (TNF)-α and interleukin (IL)-6. Results from the current study showed that pretreatment with nafithromycin significantly reduced the total cell count, total protein, MPO, TNF-α and IL-6 levels in BAL fluid compared to LPS control group. Histopathological evaluations also suggest significant reduction in neutrophil infiltration by nafithromycin. Dexamethasone, a positive reference standard as expected exhibited potent anti-inflammatory activity. The immunomodulatory effect of nafithromycin at dose of 100 mg/kg was comparable to azithromycin dosed at 600 mg/kg. As a result of immunomodulatory activity, nafithromycin is expected to provide additional clinical benefits by resolving the secondary complications associated with severe pneumonia and thereby improving survival in such patients.

Comparison of Plasma and Intrapulmonary Concentrations of Nafithromycin (WCK 4873) in Healthy Adult Subjects.

The nafithromycin concentrations in the plasma, epithelial lining fluid (ELF), and alveolar macrophages (AM) of 37 healthy adult subjects were measured following repeated dosing of oral nafithromycin at 800 mg once daily for 3 days. The values of noncompartmental pharmacokinetic (PK) parameters were determined from serial plasma samples collected over a 24-h interval following the first and third oral doses. Each subject underwent one standardized bronchoscopy with bronchoalveolar lavage (BAL) at 3, 6, 9, 12, 24, or 48 h after the third dose of nafithromycin. The mean ± standard deviation values of the plasma PK parameters after the first and third doses included maximum plasma concentrations (Cmax) of 1.02 ± 0.31 µg/ml and 1.39 ± 0.36 µg/ml, respectively; times to Cmax of 3.97 ± 1.30 h and 3.69 ± 1.28 h, respectively; clearances of 67.3 ± 21.3 liters/h and 52.4 ± 18.5 liters/h, respectively, and elimination half-lives of 7.7 ± 1.1 h and 9.1 ± 1.7 h, respectively. The values of the area under the plasma concentration-time curve (AUC) from time zero to 24 h postdosing (AUC0-24) for nafithromycin based on the mean or median total plasma concentrations at BAL fluid sampling times were 16.2 µg · h/ml. For ELF, the respective AUC0-24 values based on the mean and median concentrations were 224.1 and 176.3 µg · h/ml, whereas for AM, the respective AUC0-24 values were 8,538 and 5,894 µg · h/ml. Penetration ratios based on ELF and total plasma AUC0-24 values based on the mean and median concentrations were 13.8 and 10.9, respectively, whereas the ratios of the AM to total plasma concentrations based on the mean and median concentrations were 527 and 364, respectively. The sustained ELF and AM concentrations for 48 h after the third dose suggest that nafithromycin has the potential to be a useful agent for the treatment of lower respiratory tract infections. (This study has been registered at ClinicalTrials.gov under registration no. NCT02453529.).

Determination of Disk Diffusion and MIC Quality Control Ranges for Nafithromycin (WCK 4873), a New Lactone-Ketolide.

Disk diffusion and MIC quality control (QC) ranges were determined for nafithromycin, a new lactone-ketolide, following the completion of a nine-laboratory, Clinical and Laboratory Standards Institute (CLSI) document M23-defined tier 2 study. Five QC strains consistent with the spectrum of activity of nafithromycin were tested: Staphylococcus aureus ATCC 25923 (disk only), S. aureus ATCC 29213 (broth only), Enterococcus faecalis ATCC 29212 (broth only), Streptococcus pneumoniae ATCC 49619 (disk and broth), and Haemophilus influenzae ATCC 49247 (disk and broth). Nafithromycin disk diffusion QC ranges were determined to be 25 to 31 mm for S. aureus ATCC 25923, 25 to 31 mm for S. pneumoniae ATCC 49619, and 16 to 20 mm for H. influenzae ATCC 49247. Nafithromycin MIC QC ranges were determined to be 0.06 to 0.25 µg/ml for S. aureus ATCC 29213, 0.016 to 0.12 µg/ml for E. faecalis ATCC 29212, 0.008 to 0.03 µg/ml for S. pneumoniae ATCC 49619, and 2 to 8 µg/ml for H. influenzae ATCC 49247. All disk diffusion and MIC QC ranges established in this study were approved by the CLSI Subcommittee on Antimicrobial Susceptibility Testing at their June 2015 meeting and were initially reported in the 2017 M100S document. The QC ranges established in this study should be used for determining the in vitro activity of nafithromycin in phase 2 and phase 3 human clinical trials and subsequently for testing patient isolates and isolates in phase 4 surveillance studies.

Simultaneous determination of novel ketolide antibiotic nafithromycin and its major metabolite in human plasma using liquid chromatography tandem mass spectrometry.

Aim: A sensitive method to quantify nafithromycin and its N-desmethyl metabolite in human plasma was necessary for Phase I pharmacokinetic studies. Methodology: A precise and accurate LC-MS/MS bioanalytical method has been developed and validated for the simultaneous quantification of nafithromycin (NFT, WCK 4873) and N-desmethyl metabolite (M1, WCK 4978) in human plasma. Clarithromycin was used as an internal standard. Protein precipitation technique was used as sample preparation approach. The calibration curve was linear (r ≥ 0.99) over the concentration range of 10-5000 ng/ml for NFT and M1. Method was validated as per US FDA guideline. Conclusion: The proposed method was successfully applied for determination of plasma levels of the NFT and M1 during Phase I clinical studies.