Discovery of Antibacterial Contezolid Acefosamil: Innovative O-Acyl Phosphoramidate Prodrug for IV and Oral Therapies.

New oral antibiotic contezolid (CZD) is effective against Gram-positive infections but unsuitable for intravenous (IV) administration due to its modest solubility. To address the medical need for an IV form of CZD, its isoxazol-3-yl phosphoramidate derivatives have been explored, and contezolid acefosamil (CZA, 8), the first representative of a novel O-acyl phosphoramidate prodrug class, has been identified. CZA exhibits high aqueous solubility (>200 mg/mL) and good hydrolytic stability at media pH suitable for IV administration. CZA rapidly converts into the active drug CZD in vivo. In a pharmacokinetic (PK) rat model, the exposure of active drug CZD after IV administration of the prodrug CZA was similar to or higher than that from the IV administration of CZD. The prodrug CZA is bioequivalent to or better than CZD in several preclinical infection models. CZA is likewise active upon its oral administration. To date, CZA has been evaluated in Phase 1 and Phase 2 clinical trials in the USA. It is advancing into further clinical studies including step-down therapy with in-hospital intravenous CZA administration followed by outpatient oral CZD treatment.

Clinical Pharmacology and Utility of Contezolid in Chinese Patients with Complicated Skin and Soft-Tissue Infections.

This study aimed to build a population pharmacokinetic (PopPK) model for contezolid tablet (MRX-I) in healthy subjects and adults with complicated skin and soft-tissue infections (cSSTIs) to further evaluate the efficacy and safety of contezolid and recommend the optimal dosing regimen based on pharmacokinetic/pharmacodynamic (PK/PD) analysis. PopPK analysis was performed using a nonlinear mixed-effects model (NONMEM) to examine the effects of age, body weight, sex, liver and renal functions, albumin, food, dosage strength, and subject type on the PK parameters of contezolid. PK/PD analysis was combined with the MIC of contezolid, clinical/microbiological efficacy, and nonclinical study data. Adverse events (AEs) and study drug-related AEs reported were summarized to examine the relationship between contezolid exposure level and safety measures. A two-compartment model was built. An exponential model was used to describe the interindividual variation. A proportional model was used to describe the intraindividual variation of PK parameters. Good clinical and microbiological efficacy are expected for the infections caused by S. aureus when contezolid is administered at 600 mg or 800 mg every 12 h (q12h). The area under the concentration-time curve from 0 to 24 h at steady state and maximum concentration of drug in serum at steady state of contezolid did not show significant association with the incidence of any AE. The dosing regimen of contezolid at 800 mg q12h administered postprandially for 7 to 14 days is expected to achieve satisfactory clinical and microbiological efficacy in cSSTIs, which is slightly better than that of 600 mg contezolid. This administration has been added to the prescribing information of contezolid tablets.

A Phase III multicentre, randomized, double-blind trial to evaluate the efficacy and safety of oral contezolid versus linezolid in adults with complicated skin and soft tissue infections.

OBJECTIVES: Contezolid is a novel oxazolidinone antibacterial agent for managing infections caused by aerobic and anaerobic Gram-positive bacteria including methicillin-resistant strains. A Phase III, multicentre, randomized, double-blind, active-controlled trial evaluated the efficacy and safety of contezolid versus linezolid in adults with complicated skin and soft tissue infections (cSSTIs). METHODS: Adult patients with cSSTI were randomized in a ratio of 1:1 to receive contezolid 800 mg or linezolid 600 mg q12h for 7-14 days. Clinical cure rate and safety were assessed at the test of cure (TOC) visit in the full analysis set (FAS) and clinical evaluable (CE) population. Non-inferiority was defined as a lower limit of the 95% CI around the treatment difference of clinical cure rates greater than -10%. Chinadrugtrials.org.cn registration identifier: CTR20150855. RESULTS: Clinical cure rates at TOC indicated non-inferiority of contezolid 800 mg to linezolid 600 mg q12h for patients in the FAS with clinical evaluation, FAS, and CE populations: 92.8% (271/292) versus 93.4% (284/304) (difference -0.6%, 95% CI: -4.7% to 3.5%), 81.4% (271/333) versus 84.5% (284/336) (difference -3.1%, 95% CI: -8.8% to 2.6%) and 90.5% (267/295) versus 90.1% (282/313) (difference 0.4%, 95% CI: -4.3% to 5.1%). Contezolid and linezolid showed similar efficacy for the cSSTIs caused by methicillin-susceptible or methicillin-resistant Staphylococcus aureus. Contezolid demonstrated significant lower incidence of leucopenia (0.3% versus 3.4%) and thrombocytopenia (0% versus 2.3%) than linezolid. The frequency of treatment-emergent adverse events was comparable between the two groups. CONCLUSIONS: Contezolid 800 mg q12h is as effective as linezolid for treatment of cSSTIs in adults, but safer than linezolid in terms of haematological abnormalities.

Metabolism of MRX-I, a novel antibacterial oxazolidinone, in humans: the oxidative ring opening of 2,3-Dihydropyridin-4-one catalyzed by non-P450 enzymes.

MRX-I is an analog of linezolid containing a 2,3-dihydropyridin-4-one (DHPO) ring rather than a morpholine ring. Our objectives were to characterize the major metabolic pathways of MRX-I in humans and clarify the mechanism underlying the oxidative ring opening of DHPO. After an oral dose of MRX-I (600 mg), nine metabolites were identified in humans. The principal metabolic pathway proposed involved the DHPO ring opening, generating the main metabolites in the plasma and urine: the hydroxyethyl amino propionic acid metabolite MRX445-1 and the carboxymethyl amino propionic acid metabolite MRX459. An in vitro phenotyping study demonstrated that multiple non-cytochrome P450 enzymes are involved in the formation of MRX445-1 and MRX459, including flavin-containing monooxygenase 5, short-chain dehydrogenase/reductase, aldehyde ketone reductase, and aldehyde dehydrogenase (ALDH). H2 (18)O experiments revealed that two (18)O atoms are incorporated into MRX445-1, one in the carboxyethyl group and the other in the hydroxyl group, and three (18)O atoms are incorporated into MRX459, two in the carboxymethyl group and one in the hydroxyl group. Based on these results, the mechanism proposed for the DHPO ring opening involves the metabolism of MRX-I via FMO5-mediated Baeyer-Villiger oxidation to an enol lactone, hydrolysis to an enol, and enol-aldehyde tautomerism to an aldehyde. The aldehyde is reduced by short-chain dehydrogenase/reductase, aldehyde ketone reductase, ALDH to MRX445-1, or oxidized by ALDH to MRX459. Our study suggests that few clinical adverse drug-drug interactions should be anticipated between MRX-I and cytochrome P450 inhibitors or inducers.

Selection and characterisation of Staphylococcus aureus mutants with reduced susceptibility to the investigational oxazolidinone MRX-I.

MRX-I is a new oxazolidinone antimicrobial under development. In this study, the potential for development of resistance to MRX-I in Staphylococcus aureus was investigated and key mutations were characterised. Determination of spontaneous resistance frequency and the mutant selection window (MSW) were performed with meticillin-susceptible S. aureus (MSSA) ATCC 29213, meticillin-resistant S. aureus (MRSA) ATCC 33591 and two clinical MRSA isolates SA 0016 and SA 0017. Selected resistant mutants were sequenced for 23S rRNA as well as genes encoding the ribosomal proteins L3, L4 and L22. Resistance frequencies for the aforementioned strains were <8.25×10(-12), <6.33×10(-12), <2.96×10(-13) and <4.52×10(-13), respectively, and the MSW of MRX-I was 2-4, 1-4, 1-2 and 1-4 mg/L, respectively. After 30 serial passages, MRX-I minimum inhibitory concentrations (MICs) increased up to 8- to 16-fold both against MSSA and MRSA, whilst linezolid MICs increased 128-fold against MSSA and 16- to 32-fold against MRSA. MRX-I resistance mutations were clustered mainly in 23S rRNA and L3 protein regions. The U2504A transversion in 23S rRNA dominated in MRX-I-resistant mutants. No mutations in L4 and L22 proteins were observed. MRX-I exhibits a low potential to develop resistance in S. aureus, with a reduced resistance propensity compared with linezolid.

New potent antibacterial oxazolidinone (MRX-I) with an improved class safety profile.

Oxazolidinones comprise an important class of antibacterial protein synthesis inhibitors. Myelosuppression and monoamine oxidase inhibition (MAOI) are key independent causes for limiting adverse effects in therapy with the sole approved drug of this class, linezolid. This annotation describes a novel oxazolidinone agent, (S)-5-((isoxazol-3-ylamino)methyl)-3-(2,3,5-trifluoro-4-(4-oxo-3,4-dihydropyridin-1(2H)-yl)phenyl)oxazolidin-2-one (MRX-I), distinguished by its high activity against Gram-positive pathogens coupled with markedly reduced potential for myelosuppression and MAOI. The medical need, medicinal chemistry rationale, preclinical data, and phase I clinical trial summary for this new agent are reviewed herein.

Proteomic study of peptide deformylase inhibition in Streptococcus pneumoniae and Staphylococcus aureus.

Peptide deformylase (PDF) is an essential enzyme in both gram-negative and gram-positive bacteria. It hydrolyzes formylated N-terminal peptides to generate free N-terminal peptides during the process of protein maturation. Inhibition of this enzyme results in cessation of bacterial growth. We have examined the effect of a potent PDF inhibitor, LBM-415 (also known as VIC-104959), on the proteomes of Staphylococcus aureus and Streptococcus pneumoniae using two-dimensional electrophoresis. Both S. aureus and S. pneumoniae showed accumulation of many N-terminal formylated peptides/proteins upon PDF inhibition. In S. pneumoniae, formylated peptide/protein accumulation was time dependent. Following inhibition, subsequent removal of the inhibitor resulted in deformylation of formylated peptides/proteins; this recovery process was also time dependent. If instead the inhibited cells were maintained in the presence of sub-MIC levels of the PDF inhibitor, the formylated peptides/proteins remained for a much longer time, which correlated with a prolonged postantibiotic effect in vitro. These observations may have broader implications for the application of this class of antibiotics in vivo.

The evolution of peptide deformylase as a target: contribution of biochemistry, genetics and genomics.

Although peptide deformylase (PDF, EC 3.5.1.27) was first described in 1968, the instability of enzyme preparations prevented it from being seriously considered as a target until this problem was finally solved in 1998. PDFs essentiality was first demonstrated in Escherichia coli in 1994. Genomic analyses have shown this enzyme to be present in all eubacteria. PDF homologs have also been found in eukaryotes including Homo sapiens. The function and relevance of the human chromosomal homolog to the safety of PDF inhibitors as therapeutic agents is not clear at this stage. Although there is considerable sequence variation between the different bacterial PDFs, there are three strongly conserved motifs that together constitute a critical metal binding site. The observation that PDF is a metalloenzyme has led to the design of inhibitors containing metal chelating pharmacophores. The most potent of these synthetic inhibitors are active against a range of clinically relevant respiratory tract pathogens in vitro and in vivo, including those resistant to current antibiotics. Mutants resistant to PDF inhibitors have been obtained in the laboratory; these resulted from mutations in the genes for transformylase (EC 2.1.2.9) or PDF. The mechanism involved and its frequency were pathogen-dependent. The two most advanced PDF inhibitor leads, which are both reverse hydroxamates, have progressed to phase 1 clinical trials and were well tolerated.

Role of the AcrAB-TolC efflux pump in determining susceptibility of Haemophilus influenzae to the novel peptide deformylase inhibitor LBM415.

Haemophilus influenzae isolates vary widely in their susceptibilities to the peptide deformylase inhibitor LBM415 (MIC range, 0.06 to 32 microg/ml); however, on average, they are less susceptible than gram-positive organisms, such as Staphylococcus aureus and Streptococcus pneumoniae. Insertional inactivation of the H. influenzae acrB or tolC gene in strain NB65044 (Rd strain KW20) increased susceptibility to LBM415, confirming a role for the AcrAB-TolC pump in determining resistance. Consistent with this, sequencing of a PCR fragment generated with primers flanking the acrRA region from an LBM415-hypersusceptible H. influenzae clinical isolate revealed a genetic deletion of acrA. Inactivation of acrB or tolC in several clinical isolates with atypically reduced susceptibility to LBM415 (MIC of 16 microg/ml or greater) significantly increased susceptibility, confirming that the pump is also a determinant of decreased susceptibility in these clinical isolates. Examination of acrR, encoding the putative repressor of pump gene expression, from several of these strains revealed mutations introducing frameshifts, stop codons, and amino acid changes relative to the published sequence, suggesting that loss of pump repression leads to decreased susceptibility. Supporting this, NB65044 acrR mutants selected by exposure to LBM415 at 8 microg/ml had susceptibilities to LBM415 and other pump substrates comparable to the least sensitive clinical isolates and showed increased expression of pump genes.

Conformationally restricted analogs of deoxynegamycin.

Deoxynegamycin (1b) is a protein synthesis inhibitor with activity against Gram-negative (GN) bacteria. A series of conformationally restricted analogs were synthesized to probe its bioactive conformation. Indeed, some of the constrained analogs were found to be equal or better than deoxynegamycin in protein synthesis assay (1b, IC(50)=8.2 microM; 44, IC(50)=6.6 microM; 35e(2), IC(50)=1 microM). However, deoxynegamycin had the best in vitro whole cell antibacterial activity (Escherichia coli, MIC=4-16 microg/mL; Klebsiella pneumoniae, MIC=8 microg/mL) suggesting that other factors such as permeation may also be contributing to the overall whole cell activity. A new finding is that deoxynegamycin is efficacious in an E. coli murine septicemia model (ED(50)=4.8 mg/kg), providing further evidence of the favorable in vivo properties of this class of molecules.

New antibacterial tetrahydro-4(2H)-thiopyran and thiomorpholine S-oxide and S,S-dioxide phenyloxazolidinones.

Combinatorial libraries of N-acylated 5-(S)-aminomethyloxazolidinone derivatives of S-oxide and S,S-dioxide tetrahydro-4(2H)-thiopyranyl and thiomorpholine phenyloxazolidinone series have been synthesized on a solid phase and evaluated for antimicrobial activity. Several novel potent leads have been identified, including orally active oxazolidinones with enhanced activity against respiratory tract infection pathogens Haemophilus influenzae and Moraxella catarrhalis.

Targeting metalloenzymes: a strategy that works.

Faced with a wealth of antibacterial drug discovery targets as a result of bacterial genomics, we need to carefully select which ones to work with. Choosing bacterial metalloenzymes is one possible approach that can increase the probability of success. Metalloenzymes can be identified through specific motif searches of bacterial genomes. Current state-of-the-art medicinal chemistry allows for the design of inhibitor libraries targeting metalloenzymes and the efficient optimization of leads identified. This approach has been successfully applied to the discovery of in vivo active antibacterial agents that are inhibitors of bacterial peptide deformylase and UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase. Other bacterial metalloenzymes are open to the same approach.

N- and C-terminal modifications of negamycin.

Negamycin 1 is a bactericidal antibiotic with activity against Gram-negative bacteria, and served as a template in an antibiotic discovery program. An orthogonally protected beta-amino acid derivative 3a was synthesized and used in parallel synthesis of negamycin derivatives on solid support. This advanced intermediate was also used for N- and C-terminal modifications using solution-phase methodologies. The N-terminal modifications have resulted in the identification of active analogues, whereas the C-terminal modifications resulted in complete loss of antibacterial activity. The N-methyl negamycin analogue, 19a, inhibits protein synthesis (IC(50)=2.3 microM), has antibacterial activity (Escherichia coli, MIC=16 microgram/mL), and is efficacious in an E. coli murine septicemia model (ED(50)=16.3mg/kg).

N-alkyl urea hydroxamic acids as a new class of peptide deformylase inhibitors with antibacterial activity.

Peptide deformylase (PDF) is a prokaryotic metalloenzyme that is essential for bacterial growth and is a new target for the development of antibacterial agents. All previously reported PDF inhibitors with sufficient antibacterial activity share the structural feature of a 2-substituted alkanoyl at the P(1)' site. Using a combination of iterative parallel synthesis and traditional medicinal chemistry, we have identified a new class of PDF inhibitors with N-alkyl urea at the P(1)' site. Compounds with MICs of 200 micro M for matrilysin and other mammalian metalloproteases. Structure-activity relationship analysis identified preferred substitutions resulting in improved potency and decreased cytotoxity. One of the compounds (VRC4307) was cocrystallized with PDF, and the enzyme-inhibitor structure was determined at a resolution of 1.7 A. This structural information indicated that the urea compounds adopt a binding position similar to that previously determined for succinate hydroxamates. Two compounds, VRC4232 and VRC4307, displayed in vivo efficacy in a mouse protection assay, with 50% protective doses of 30.8 and 17.9 mg/kg of body weight, respectively. These N-alkyl urea hydroxamic acids provide a starting point for identifying new PDF inhibitors that can serve as antimicrobial agents.

Mining bacterial cell wall biosynthesis with new tools: multitarget screens.

The cytoplasmic steps of peptidoglycan synthesis remain underexplored for the discovery of novel antibiotics. Pathway screens are well suited to screen for novel inhibitors because several targets are tested at once. Whole-cell based assays are easier to set up but they tend to favor identification of compounds that inhibit enzymes that are at the limiting rate in the pathway. Screens that use purified enzymes are optimized to detect with similar probability inhibitors of any of the targets in the pathway. This approach is being used to identify novel promising molecules. Copyright 1999 Harcourt Publishers Ltd.