Antibiotic Telithromycin Research Articles

Discovery of macrozones, new antimicrobial thiosemicarbazone-based azithromycin conjugates: design, synthesis and in vitro biological evaluation

Increasing bacterial resistance to existing antibiotics presents a serious threat to human health, and new antibacterial agents are desperately needed. Unfortunately, the number of newly marketed antibiotics has decreased dramatically in recent years. Withdrawal of the macrolide antibiotic telithromycin and the inability of solithromycin to gain marketing approval have prompted our efforts to search for new anti-infective macrolide compounds. Here we present the design, synthesis and biological evaluation of a novel hybrid class of azithromycin conjugates, the macrozones. Evaluation of prepared compounds against a panel of pathogenic bacteria revealed that these molecules showed excellent activities against susceptible Streptococcus pneumoniae, Streptococcus pyogenes and Enterococcus faecalis strains comparable with or better than azithromycin. Furthermore, prepared macrozones exhibited excellent activity against efflux resistant S. pneumoniae, which was 32 times better than that of azithromycin, and very good activity against an efflux resistant Staphylococcus aureus strain against which azithromycin is inactive. The results described here can serve as a good basis to guide further activities directed toward the discovery of more potent macrolide anti-infectives.

ClpP-independent function of ClpX interferes with telithromycin resistance conferred by Msr(A) in Staphylococcus aureus.

The ABCF family protein Msr(A) confers high resistance to macrolides but only low resistance to ketolides in staphylococci. Mutations in conserved functional regions of ClpX as well as deletion of clpX significantly increased Msr(A)-mediated resistance to the ketolide antibiotic telithromycin. ClpX is the chaperone component of the ClpXP two-component proteolytic system. Nevertheless, no changes in resistance were observed in a clpP knockout strain expressing msr(A), demonstrating that ClpX affects Msr(A) independently of ClpP.

Desmethyl macrolides: synthesis and evaluation of 4-desmethyl telithromycin.

Novel sources of antibiotics are needed to address the serious threat of bacterial resistance. Accordingly, we have launched a structure-based drug design program featuring a desmethylation strategy wherein methyl groups have been replaced with hydrogens. Herein we report the total synthesis, molecular modeling, and biological evaluation of 4-desmethyl telithromycin (6), a novel desmethyl analogue of the third-generation ketolide antibiotic telithromycin (2) and our final analogue in this series. While 4-desmethyl telithromycin (6) was found to be equipotent with telithromycin (2) against wild-type bacteria, it was 4-fold less potent against the A2058G mutant. These findings reveal that strategically replacing the C4-methyl group with hydrogen (i.e., desmethylation) did not address this mechanism of resistance. Throughout the desmethyl series, the sequential addition of methyls to the 14-membered macrolactone resulted in improved bioactivity. Molecular modeling methods indicate that changes in conformational flexibility dominate the increased biological activity; moreover, they reveal 6 adopts a different conformation once bound to the A2058G ribosome, thus impacting noncovalent interactions reflected in a lower MIC value. Finally, fluorescence polarization experiments of 6 with E. coli ribosomes confirmed 6 is indeed binding the ribosome.

Impact of ribosomal modification on the binding of the antibiotic telithromycin using a combined grand canonical monte carlo/molecular dynamics simulation approach.

Resistance to macrolide antibiotics is conferred by mutation of A2058 to G or methylation by Erm methyltransferases of the exocyclic N6 of A2058 (E. coli numbering) that forms the macrolide binding site in the 50S subunit of the ribosome. Ketolides such as telithromycin mitigate A2058G resistance yet remain susceptible to Erm-based resistance. Molecular details associated with macrolide resistance due to the A2058G mutation and methylation at N6 of A2058 by Erm methyltransferases were investigated using empirical force field-based simulations. To address the buried nature of the macrolide binding site, the number of waters within the pocket was allowed to fluctuate via the use of a Grand Canonical Monte Carlo (GCMC) methodology. The GCMC water insertion/deletion steps were alternated with Molecular Dynamics (MD) simulations to allow for relaxation of the entire system. From this GCMC/MD approach information on the interactions between telithromycin and the 50S ribosome was obtained. In the wild-type (WT) ribosome, the 2′-OH to A2058 N1 hydrogen bond samples short distances with a higher probability, while the effectiveness of telithromycin against the A2058G mutation is explained by a rearrangement of the hydrogen bonding pattern of the 2′-OH to 2058 that maintains the overall antibiotic-ribosome interactions. In both the WT and A2058G mutation there is significant flexibility in telithromycin's imidazole-pyridine side chain (ARM), indicating that entropic effects contribute to the binding affinity. Methylated ribosomes show lower sampling of short 2′-OH to 2058 distances and also demonstrate enhanced G2057-A2058 stacking leading to disrupted A752-U2609 Watson-Crick (WC) interactions as well as hydrogen bonding between telithromycin's ARM and U2609. This information will be of utility in the rational design of novel macrolide analogs with improved activity against methylated A2058 ribosomes.

Desmethyl Macrolides: Synthesis and Evaluation of 4,8-Didesmethyl Telithromycin

There is an urgent need for novel sources of antibiotics to address the incessant and inevitable onset of bacterial resistance. To this end, we have initiated a structure-based drug design program that features a desmethylation strategy (i.e., replacing methyl groups with hydrogens). Herein we report the total synthesis, molecular modeling and biological evaluation of 4,8-didesmethyl telithromycin (5), a novel desmethyl analogue of the third-generation ketolide antibiotic telithromycin (2), which is an FDA-approved semisynthetic derivative of erythromycin (1). We found 4,8-didesmethyl telithromycin (5) to be eight times more active than previously prepared 4,8,10-tridesmethyl congener (3) and two times more active than 4,10-didesmethyl regioisomer (4) in MIC assays. While less potent than telithromycin (2) and paralleling the observations made in the previous study of 4,10-didesmethyl analogue (4), the inclusion of a single methyl group improves biological activity thus supporting its role in antibiotic activity.

Understanding the regulatory hurdles for antibacterial drug development in the post‐Ketek world

The antibiotic telithromycin (Ketek, Sanofi-aventis) had two of three treatment indications withdrawn after postmarketing reports of serious and fatal adverse events. The rationale for the withdrawal of specific indications (acute bacterial sinusitis and acute exacerbations of chronic bronchitis), while permitting the drug to remain available for the treatment of community-acquired pneumonia, focused on the lack of demonstrated efficacy from clinical trials that included an active control to which the investigational drug, telethromycin, was determined to be statistically noninferior. This action regarding telithromycin represents a reversal of previous FDA guidelines for the conduct of clinical trials for antibacterial treatment indications and has increased the regulatory risk for future drug development. New clinical trial guidelines have been published that will increase the time and resources required to achieve regulatory marketing approval. This paper reviews recent regulatory actions and discusses the impact these new guidelines will have on future antibacterial clinical trial designs and challenges.

Degradation Kinetics of Telithromycin Determined by HPLC Method

The degradation kinetics of the antibiotic telithromycin using a stability-indicating high-performance liquid chromatography (HPLC) method is demonstrated. The photodegradation is performed by UVC lamp-254 nm (15W), installed in a chamber internally coated with mirrors, where telithromycin solutions prepared from coated tablets are placed in quartz cells. To promote oxidation, the reaction between the telithromycin solution and 3% hydrogen peroxide solution is carried out. The kinetics parameters of order of reaction and the rate constants of the degradation are determined for both conditions. The degradation process of telithromycin can be described by first-order kinetics under both experimental conditions used in this study. The results reveal the photo and oxidation lability of the drug and confirm the reliability of HPLC method for telithromycin in the presence of its degradation products.

Revisiting the structures of several antibiotics bound to the bacterial ribosome

The increasing prevalence of antibiotic-resistant pathogens reinforces the need for structures of antibiotic-ribosome complexes that are accurate enough to enable the rational design of novel ribosome-targeting therapeutics. Structures of many antibiotics in complex with both archaeal and eubacterial ribosomes have been determined, yet discrepancies between several of these models have raised the question of whether these differences arise from species-specific variations or from experimental problems. Our structure of chloramphenicol in complex with the 70S ribosome from Thermus thermophilus suggests a model for chloramphenicol bound to the large subunit of the bacterial ribosome that is radically different from the prevailing model. Further, our structures of the macrolide antibiotics erythromycin and azithromycin in complex with a bacterial ribosome are indistinguishable from those determined of complexes with the 50S subunit of Haloarcula marismortui, but differ significantly from the models that have been published for 50S subunit complexes of the eubacterium Deinococcus radiodurans. Our structure of the antibiotic telithromycin bound to the T. thermophilus ribosome reveals a lactone ring with a conformation similar to that observed in the H. marismortui and D. radiodurans complexes. However, the alkyl-aryl moiety is oriented differently in all three organisms, and the contacts observed with the T. thermophilus ribosome are consistent with biochemical studies performed on the Escherichia coli ribosome. Thus, our results support a mode of macrolide binding that is largely conserved across species, suggesting that the quality and interpretation of electron density, rather than species specificity, may be responsible for many of the discrepancies between the models.

Effects of Telithromycin in In Vitro and In Vivo Models of Lipopolysaccharide-Induced Airway Inflammation*

The ketolide antibiotic telithromycin (TEL) exerts immunomodulatory and antiinflammatory effects in vitro and in a mouse model of septic shock. We studied the antiinflammatory activity of TEL in in vitro and in vivo models of airway inflammation induced by lipopolysaccharide (LPS). We measured the effects of TEL on the response of RAW 264.7 macrophages to LPS and of murine lung epithelial (MLE)-12 cells to supernatants of LPS-stimulated RAW 264.7 macrophages. Macrophage inflammatory protein (MIP)-2 and tumor necrosis factor (TNF)-alpha production, nuclear factor (NF)-kappaB activation, and apoptosis were determined. Acute airway inflammation was induced in untreated and TEL-treated BALB/c mice by nebulization with LPS. Total number of leukocytes, macrophages, and neutrophils, the protein concentration, and nitrite and cytokine levels were determined in the BAL fluid. TEL inhibited in a dose-dependent manner the production of MIP-2 and TNF-alpha by LPS-stimulated RAW 264.7 macrophages, and the production of MIP-2 by MLE-12 epithelial cells to supernatants of LPS-stimulated RAW 264.7 macrophages. NF-kappaB activation was inhibited and apoptosis was increased in both cell lines by TEL. The LPS-induced influx of neutrophils in BAL fluid was decreased by TEL pretreatment. TEL also reduced protein, nitrite, MIP-2, and TNF-alpha levels in the BAL fluid of LPS-nebulized animals. We have provided evidence that TEL exerts potent antiinflammatory effects in LPS-induced airways injury. We propose that TEL acts in the early phase of inflammation by reducing the release of inflammatory mediators through NF-kappaB inhibition, and in the later phase through enhancement of inflammatory cell apoptosis.

Telithromycin and Quinupristin-Dalfopristin Induce Delayed Death in Plasmodium falciparum

Antibacterial agents are used in malaria therapy due to their effect on two prokaryote organelles, the mitochondrion and the apicoplast. We demonstrate here that the ribosome-blocking antibiotics telithromycin and quinupristin-dalfopristin, but not linezolid, inhibit the growth of Plasmodium falciparum. Both drugs induce delayed death in the parasite, suggesting that their effect involves the impairment of apicoplast translation processes.

In Vitro activity of telithromycin and quinupristin/dalfopristin against methicillin-resistant coagulase-negative staphylococci with defined resistance genotypes

We determined the activities of new antibiotics telithromycin (ketolide) and quinupristin/dalfopristin (streptogramins) against 88 macrolide and/or lincosamide resistant coagulase-negative staphylococci (CoNS) isolates with defined resistance gene status. Telithromycin susceptibility was determined only in erythromycin-sensitive isolates (15) indicating the same mechanisms of resistance. In contrast, all erythromycin-resistant isolates (73) were either constitutively resistant to telithromycin (13 isolates with constitutive erm genes) or demonstrated telithromycin D-shaped zone (60 isolates with inducible msr(A) and/or erm). However, the level of inducible resistance conferred by msr(A) (35 isolates) was borderline even after induction by erythromycin. No quinupristin/dalfopristin resistant isolate was observed if tested by disk-diffusion method (DDM) but 18 isolates were intermediate (MIC = 1-3 mg/L) and two isolates resistant (MIC = 8 mg/L) if tested by E-test. All these isolates were resistant to streptogramin A and harbored vga(A) gene (1 isolate) or vga(A)LC gene (19 isolates). MICs for quinupristin/dalfopristin were higher for isolates with combination of streptogramin A resistance and constitutive MLSB resistance (MIC = 3-8 mg/L in 4 isolates) than for streptogramin A-resistant isolates susceptible to streptogramin B (MIC = 0.5-2 mg/L in 16 isolates). In addition to S. haemolyticus, vga(A)LC was newly identified in S. epidermidis and S. warnerii indicating its widespread occurrence in CoNS. Misidentification of low-level resistant isolates by DDM may contribute to dissemination of streptogramin A resistance.

Microbiological Assay for the Determination of Telithromycin in Tablets

This study describes the development and validation of a microbiological assay, applying the cylinder-plate method, for the determination of the antibiotic telithromycin. The microbiological method consisted of a cylinder-plate agar diffusion assay using Micrococcus luteus ATCC 9341 as the test microorganism. The response graphs for standard and sample solutions were linear (r = 0.9987), and no parallelism deviations were detected in the tested concentrations (0.25, 0.5, and 1.0 microg/mL). The interday precision was 2.67%. Recovery values were between 96.75 and 100.91%. A preliminary stability study of telithromycin showed that the microbiological assay is specific for the determination of telithromycin in the presence of its degradation products. The proposed method allows the quantitation of telithromycin in pharmaceutical dosage form and can be used for drug analysis in routine quality control.

Methyltransferase Erm(37) Slips on rRNA to Confer Atypical Resistance in Mycobacterium tuberculosis

Members of the Mycobacterium tuberculosis complex possess a resistance determinant, erm(37) (also termed ermMT), which is a truncated homologue of the erm genes found in a diverse range of drug-producing and pathogenic bacteria. All erm genes examined thus far encode N(6)-monomethyltransferases or N(6),N(6)-dimethyltransferases that show absolute specificity for nucleotide A2058 in 23 S rRNA. Monomethylation at A2058 confers resistance to a subset of the macrolide, lincosamide, and streptogramin B (MLS(B)) group of antibiotics and no resistance to the latest macrolide derivatives, the ketolides. Dimethylation at A2058 confers high resistance to all MLS(B) and ketolide drugs. The erm(37) phenotype fits into neither category. We show here by tandem mass spectrometry that Erm(37) initially adds a single methyl group to its primary target at A2058 but then proceeds to attach additional methyl groups to the neighboring nucleotides A2057 and A2059. Other methyltransferases, Erm(E) and Erm(O), maintain their specificity for A2058 on mycobacterial rRNA. Erm(E) and Erm(O) have a full-length C-terminal domain, which appears to be important for stabilizing the methyltransferases at their rRNA target, and this domain is truncated in Erm(37). The lax interaction of the M. tuberculosis Erm(37) with its rRNA produces a unique methylation pattern and confers resistance to the ketolide telithromycin.

Case report of tolerance to Telithromycin in Erythromycin allergic patients

RATIONALE: Ketolides a new class of antibiotic (telithromycin), contraindicated in those allergic to Macrolides, but no reports of challenge to this drug in Macrolide allergic patient exists. METHODS: Two Macrolide allergic patients 1) 39 y/o WF with reactions to PCN, Cephalosporin, Sulfa, Quinolones but tolerated IV Vancomycin. Prior reaction 10 years ago to Emycin was facial flushing, hives, cough and rash after several doses. Azithromycin (2 years ago) caused immediate anaphylaxis. 2) 44 y/o WM with reactions to PCN, Cephalosporin, and a prior reaction to Erythromycin 5 years ago with pruritic rash. Telithromycin test dose is unknown but prior studies suggest starting dose 10−2 of daily dose. We used 8 mg/ml as our test dose prick then .02cc ID and then oral challenge with 10, 25, 50, 75,100,150 mg of liquid Telithromycin administered every 15 minutes. Total oral dose or 400 mg was tolerated. The patients were observed for an additional hour. They were continued on full doses of 800 mg daily for 3 days. RESULTS: Patients were rechecked by phone in 2 hours, and daily for 2 days after the challenge. Both patients tolerated the testing, challenge then oral full dose therapy without an adverse reaction. CONCLUSION: These two cases are important as they may point the way to further studies on Macrolide-Ketolide cross reactivity. Since this new antibiotic Telithromycin has unique and distinct advantages over Macrolides it would be to our advantage to learn if Macrolide sensitive patients can in fact safely take this new class of antibiotic.

Inhibition of secretion of interleukin-1alpha and tumor necrosis factor alpha by the ketolide antibiotic telithromycin.

The antibiotic telithromycin was examined for its effect on secretion of interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, IL-10, and tumor necrosis factor alpha (TNF-alpha) by lipopolysaccharide (LPS)-stimulated monocytes of eight human donors. Secretion of each cytokine was significantly increased by LPS alone, whereas treatment with telithromycin significantly inhibited secretion of IL-1alpha and TNF-alpha but not secretion of IL-1beta, IL-6, and IL-10. Telithromycin had immunomodulatory effects as a result of alteration of secretion of IL-1alpha and TNF-alpha by monocytes.

Osmolytes stimulate the reconstitution of functional 50S ribosomes from in vitro transcripts of Escherichia coli 23S rRNA.

Functional Escherichia coli 50S ribosomal subunits can be reconstituted from their natural rRNA and protein components. However, when the assembly is performed with in vitro-transcribed 23S rRNA, the reconstitution efficiency is diminished by four orders of magnitude. We tested a variety of chemical chaperones (compounds that are typically used for protein folding), putative RNA chaperones (proteins) and ribosome-targeted antibiotics (small-molecule ligands) that might be reasoned to aid in folding and assembly. Addition of the osmolyte trimethylamine-oxide (TMAO) and the ketolide antibiotic telithromycin (HMR3647) to the reconstitution stimulates its efficiency up to 100-fold yielding a substantially improved system for the in vitro analysis of mutant ribosomes.