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Antibiotic macrolides are experiencing renewed interest in anti-infective therapy since the advent of ketolides. This therapeutic class was introduced in order to broaden the narrow antibacterial spectrum of macrolides and to cope with the emergence of germs resistant to Erythromycin A and its hemisynthetic derivatives or neomacrolides (Clarithromycin, Roxithromycin, Azithromycin, Dirithromycin). From a pharmacochemical point of view, ketolides were first of all obtained by operating chemical modulations on Erythromycin A to obtain the neomacrolides, then, by replacing the neutral sugar (L-cladinose) in C3 by a ketone function coupled with the creation of an oxazolidinone like heterocycle in C11 and C12 in place of the hydroxyls present in these positions (Telithromycin, Cethromycin, Solithromycin). These modulations have enabled the improvement of the chemical stability of ketolides in gastric acid medium and increase their affinity for the ribosomal target, hence the broadening of their spectrum of action towards Gram positive germs including strains resistant to other macrolides and to neomacrolides. Therefore, the objective of this systematic review is to report the various pharmacochemical aspects undertaken since 1952 in the macrolide series based on the structure of Erythromycin A. These aspects will focus on the pharmacomodulations that have led, year after year, to the optimization of stability, the improvement of the pharmacodynamic and pharmacokinetic profile and that have allowed the development of neomacrolides, ketolides and neoketolides, which are today essential in the management of severe bronchopulmonary infections.

Macrolide antibiotics bind to the exit tunnel of the ribosome and inhibit protein synthesis blocking its translocation. Thus, antibiotics including the known macrolide Erythromycin (ERY) are active against bacteria. However, at present, some bacteria show resistance to drugs, which requires the development of new powerful antibacterial agents. One possible way is to use the ERY structure, but change its side chains, while the size of the lactone ring can remain unchanged or change. In this work we consider Cethromycin (CET) and Solithromycin (SOL), which are ketolides with quinolylallyl group at C6 and aminophenyl at C11, respectively (both of them have the same lactone ring as ERY). Experiments have shown that these ketolides have improved efficacy against pathogens, but their binding affinity to the E. coli's ribosome is almost identical. To clarify this issue, we have studied in detail the binding mechanisms of ERY, CET and SOL using the docking and molecular dynamic simulations. In agreement with the experiments, we showed that these compounds have similar binding affinities. Desosamine and lactone ring groups play a critical role in the binding of ERY to the ribosome. In CET and SOL, the contribution of keto and alkylaryl groups is balanced by cyclic carbamate. We have demonstrated that increased fluctuations in the ribosomal residues at the binding site led to an increase in the entropic term in the free binding energy of ERY compared to SOL and CET. The alkyl-aryl arm of both ketolides strongly interacts with A752 and U2609. In addition, the presence of macrolides in the exit tunnel can alter the conformation of U2585, which is located in the peptidyl transferase center, through non-bonded interaction. Therefore, the side chain of ketolides affects not only the binding site but also other residues possibly leading to a strong effect on the protein synthesis process. We predict that to combat bacterial mutations, it is necessary either to design a bulk and charged group as a cladinose, or to use several groups with different signs of charges. This prediction can be used for the development of new efficient antibiotics.

Ketolides, the third generation of expanded-spectrum macrolides, have in the last years become a successful weapon in the endless war against macrolide-resistant pathogens. Ketolides are semisynthetic derivatives of the naturally produced macrolide erythromycin, displaying not only improved activity against some erythromycin-resistant strains but also increased bactericidal activity as well as inhibitory effects at lower drug concentrations. In this study, we present a series of novel ketolides carrying alkyl-aryl side chains at the C-6 position of the lactone ring and, additionally, one or two fluorine atoms attached either directly to the lactone ring at the C-2 position or indirectly via the C-13 position. According to our genetic and biochemical studies, these novel ketolides occupy the known macrolide binding site at the entrance of the ribosomal tunnel and exhibit lower MIC values against wild-type or mutant strains than erythromycin. In most cases, the ketolides display activities comparable to or better than the clinically used ketolide telithromycin. Chemical protection experiments using Escherichia coli ribosomes bearing U2609C or U754A mutations in 23S rRNA suggest that the alkyl-aryl side chain establishes an interaction with the U2609-A752 base pair, analogous to that observed with telithromycin but unlike the interactions formed by cethromycin. These findings reemphasize the versatility of the alkyl-aryl side chains with respect to species specificity, which will be important for future design of improved antimicrobial agents.

A series of novel 6-O-substituted and 6,12-di-O-substituted 8a-aza-8a-homoerythromycin A and 9a-aza-9a-homoerythromycin A ketolides were synthesized and evaluated for in vitro antibacterial activity against a panel of representative erythromycin-susceptible and erythromycin-resistant test strains. Another series of ketolides based on 14-membered erythromycin oxime scaffold was also synthesized and their antibacterial activity compared to those of 15-membered azahomoerythromycin analogues. In general, structure–activity studies have shown that 14-membered ketolides displayed favorable antibacterial activity in comparison to their corresponding 15-membered analogues within 9a-azahomoerythromycin series. However, within 8a-azahomoerythromycin series, some compounds incorporating a ketolide combined with either quinoline or quinolone pharmacophore substructures showed significantly potent activity against a variety of erythromycin-susceptible and macrolide-lincosamide-streptogramin B (MLSB)-resistant Gram-positive pathogens as well as fastidious Gram-negative pathogens. The best compounds in this series overcome all types of resistance in relevant clinical Gram-positive pathogens and display hitherto unprecedented in vitro activity against the constitutively MLSB-resistant strain of Staphylococcus aureus. In addition, they also represent an improvement over telithromycin (2) and cethromycin (3) against fastidious Gram-negative pathogens Haemophilus influenzae and Moraxella catarrhalis.

A practical and efficient synthesis of ketolide antibiotic cethromycin (ABT-773) (1) is described. An effective protection strategy allows high yielding, regioselective C6-O-alkylation and subsequent stereoselective modification of the erythromycin nucleus. ABT-773 was prepared in 10 steps from commercially available erythromycin A oxime.

Mycoplasma pneumoniae is a major etiologic agent of acute lower respiratory infections. We evaluated the antimicrobial and immunologic effects of cethromycin (ABT-773), a ketolide antibiotic, for the treatment of M. pneumoniae pneumonia in a mouse model. Eight-week-old BALB/c mice were inoculated intranasally once with 10 6 CFU of M. pneumoniae on day 0. Treatment was started 24 h after inoculation. Groups of mice were treated subcutaneously with cethromycin at 25 mg/kg of body weight or with placebo daily until sacrifice. Five to ten mice per group were evaluated at days 1, 4, 7, and 10 after inoculation. Outcome variables included bronchoalveolar lavage (BAL) for M. pneumoniae quantitative culture and cytokine and chemokine concentration determinations by enzyme-linked immunosorbent assay (tumor necrosis factor alpha [TNF-α], gamma interferon [IFN-γ], interleukin-1β [IL-1β], IL-2, IL-4, IL-12, granulocyte-macrophage colony-stimulating factor, IL-8, monocyte chemoattractant protein 1 [MCP-1], and macrophage inflammatory protein 1α [MIP-1α]), histopathologic score of the lungs (HPS), and pulmonary function tests (PFT) using whole-body, unrestrained plethysmography at the baseline and post-methacholine exposure as indicators of airway obstruction (AO) and airway hyperresponsiveness (AHR), respectively. The cethromycin-treated mice had a greater reduction in M. pneumoniae culture titers than placebo-treated mice, reaching statistical significance on days 7 and 10 ( P < 0.05). HPS was significantly reduced in cethromycin-treated mice compared with placebo-treated mice on days 4, 7, and 10 ( P < 0.05). Cytokine concentrations in BAL samples were reduced in mice that received cethromycin, and the differences were statistically significant for 7 of the 10 cytokines measured (TNF-α, IFN-γ, IL-1β, IL-8, IL-12, MCP-1, and MIP-1α) on day 4 ( P < 0.05). PFT values were improved in the cethromycin-treated mice, with AO and AHR significantly reduced on day 4 ( P < 0.05). In this mouse model, treatment with cethromycin significantly reduced M. pneumoniae culture titers in BAL samples, cytokine and chemokine concentrations in BAL samples, histologic inflammation in the lungs, and disease severity as defined by AO and AHR.

The efficacy of cethromycin was assessed against isolates of Streptococcus pneumoniae in the presence of neutrophils. Comparison with data from our previous neutropenic model revealed that the presence of neutrophils enhanced the bacteriostatic and bactericidal effect of cethromycin by an average of two- to four-times, respectively.

A method is developed for the specific and sensitive determination of cethromycin concentrations in plasma, bronchoalveolar lavage (BAL), and alveolar cells (AC), using a high-performance liquid chromatographic-tandem mass spectrometry (MS) method. The mobile phase consists of 50% acetonitrile-0.05% acetic acid-5mM ammonium acetate; the column used is a C(8) reversed-phase stationary phase. The preparation of samples requires a solvent extraction step. The retention times for cethromycin and the internal standard are approximately 2.0 and 2.7 min, respectively, with a total run time of 3.5 min. Detection is carried out using electrospray MS in a multiple reaction monitor mode. The detection limits for cethromycin are 1 ng/mL for plasma and 0.2 ng/mL for BAL supernatants and AC suspensions. The assay has excellent performance characteristics and has been used to support a study of the intrapulmonary pharmacokinetics of cethromycin in human subjects.

The in vitro activities of telithromycin and cethromycin (ABT-773) against 412 Streptococcus pyogenes isolates, consecutively collected in 17 Spanish hospitals from different geographical areas, were evaluated and compared with those of erythromycin A, penicillin G, clindamycin and quinupristin-dalfopristin. With a susceptibility testing breakpoint ofor = 1 mg/L for both compounds, 96.1% of isolates were susceptible to telithromycin and 99.8% to cethromycin. Erythromycin non-susceptible isolates (intermediate plus resistant, MICor = 0.5 mg/L) comprised 23% of those tested, and were analysed for the genetic basis of their resistance by PCR. Among these isolates (n = 95), 72.6% harboured mef(A), 8.4%, erm(B), and 3.2%, erm(A), as sole macrolide resistance gene, whereas the presence of mef(A) plus erm(A) (11.6%) or mef(A) plus erm(B) (4.2%) was also observed. Both ketolides displayed a significant in vitro activity against S. pyogenes regardless of the macrolide resistance mechanisms. Nevertheless, in the case of telithromycin, 11 out of 19 of the erm(B)-positive isolates (2.7% of total population) exhibited an MIC range of 4-32 mg/L. According to the present results, telithromycin and cethromycin offer a wide coverage against S. pyogenes isolates in a geographic area with a high incidence of resistance to currently used macrolides.

Clarithromycin and azithromycin, which are more acid-stable than erythromycin A (EM), have been widely prescribed for the treatment of respiratory tract infections because of their high efficacy and safety. However, these macrolide antibiotics are only weakly active against pathogens with an efflux gene (mef) and are inactive against pathogens with a methyltransferase-inducible gene (erm) and constitutively resistant organisms. To address the drug resistance issue, tremendous efforts have been devoted to the modification of the macrolide structure. As a consequence, several types of decladinosyl derivatives, such as ketolide and acylides, have been recognized to be effective against mef-type resistant streptococci and methylase-inducible staphylococci. It has also been recognized that derivatives containing certain 11-, 6- or 4 -tethered aryl substituents, such as telithromycin (HMR 3647), cethromycin (ABT-773) and CP-544372, are effective against erm(B)-type resistant streptococci. Telithromycin was recently approved in several European countries for the treatment of respiratory tract infections and cethromycin is now in the final stage of clinical study. Macrolide antibiotics have been modified to address the issues of acid-instability and inactivity against resistant strains. In this review, we will summarize the progress in the macrolide research area and discuss the desirable features of the next generation macrolide antibiotics.

Pneumococcal resistance to antimicrobials presents problems to physicians for empirical treatment of acute otitis media (AOM). Three hundred thirty-three isolates of Streptococcus pneumoniae selected for nonsusceptibility to penicillin (MIC >0.1 μg/ml) from the middle ear ( n = 325) or mastoid ( n = 8) of children seen between 1994 and 2000 at four children's hospitals in the United States were tested by broth microdilution for susceptibility to nine antibiotics. Using NCCLS 2002 breakpoints, resistance to the following drugs was as indicated: amoxicillin, 1%; azithromycin, 71%; cefprozil, 71%; ceftriaxone, 2%; cefdinir, 98%; erythromycin, 70%; levofloxacin, 0%; and trimethoprim-sulfamethoxazole, 93%. Of the penicillin- and erythromycin-nonsusceptible isolates, 97% were inhibited by cethromycin (ABT-773) and 83% were inhibited by telithromycin at a concentration of ≤0.125 μg/ml. Macrolide resistance among penicillin-nonsusceptible pneumococci increased from 44 to 80% in the 6 years of the study from which the isolates were selected; however, the proportion of isolates with M or MLS B phenotypes remained constant over the time period (53 and 18%, respectively). Prior treatment with a macrolide or clindamycin alone or in combination with a β-lactam resulted in 94 or 85% of isolates causing infections being macrolide and or clindamycin resistant. No prior individual macrolide (azithromycin, erythromycin, or clarithromycin) resulted in more macrolide resistance or in a more prevalent resistance phenotype. The ketolides appear to be active antimicrobials against penicillin- and macrolide-resistant pneumococci.

Objectives: (i) To determine the inhibitory and bactericidal activities of ABT-773, a novel ketolide, against sensitive and erythromycin-resistant pneumococci; (ii) to subdivide erythromycin-resistant pneumococci into resistance phenotypes, more extensive than the conventional M and MLS B groups, by assessing susceptibilities to, and interactions between, erythromycin (14-membered macrolide), clindamycin (lincosamide), rokitamycin (16-membered macrolide), ABT-773 (ketolide), quinupristin (streptogramin B) and dalfopristin (streptogramin A). Methods: MICs and MBCs of ABT-773 were determined for 165 strains of pneumococci (113 resistant to erythromycin). Extended phenotypes for the erythromycin-resistant strains were described in terms of intrinsic susceptibility to, and induction of resistance by, the antibiotics listed above. Results: Erythromycin-resistant strains could be divided into 10 extended phenotypes (designated II-XI), two of which (II and IX) predominated. ABT-773 at 0.12 mg/L inhibited 109 strains (median 0.03 mg/L). MICs for the other four strains (of phenotypes X and XI) were 0.25-1 mg/L. MICs were only slighter higher when measured on agar in CO 2 than by the NCCLS method (in broth in air). MBCs were usually ≤2 x MIC, but for 10 strains (eight of phenotype X, one each of types IX and XI) MBCs were >1 mg/L, and three of the latter (all type X) were tolerant. Clones of reduced susceptibility (MICs 1-8 mg/L, increased by up to 32-fold) could be isolated from some strains of phenotypes VII, IX and X, but not from those of type II (efflux mechanism) or from erythromycin-sensitive strains. Conclusions: ABT-773 was active against all 113 erythromycin-resistant pneumococci tested, which belonged to 10 phenotypes. Extended phenotyping of pneumococci revealed interesting and potentially useful subdivisions of the classical phenotypes.

Cethromycin (ABT-773), a new ketolide, possesses potent in vitro activity against Streptococcus pneumoniae . The objective of this study was to investigate the in vivo bactericidal activity of cethromycin against macrolide-susceptible and -resistant S. pneumoniae in a murine pneumonia model and to describe the pharmacodynamic (PD) profile of cethromycin. Eight (two macrolide susceptible, six macrolide resistant) clinical isolates of S. pneumoniae were investigated. Cyclophosphamide administration rendered ICR mice transiently neutropenic prior to intratracheal inoculation with 0.05 ml of an S. pneumoniae suspension containing 10 7 to 10 8 CFU/ml. Oral cethromycin was initiated 12 to 14 h postinoculation over a dosage range of 0.1 to 800 mg/kg of body weight/day. Lungs from seven to eight mice per treatment and control groups were collected at 0 and 24 h posttherapy to assess bacterial density. The cumulative mortality ( n = 12 to 13) was assessed at 120 h (end of therapy) and at 192 h (3 days posttherapy). Recovery of pneumococci from the lungs of infected animals prior to the initiation of therapy ranged from 4.6 to 7.2 log 10 CFU. Growth in untreated control animals over a 24-h study period increased 0.3 to 2.7 log 10 CFU. Cethromycin demonstrated a substantial bactericidal effect, regardless of macrolide susceptibility. Correlation between changes in bacterial density (24 h) and survival over both 120 and 192 h were statistically significant. All three PD parameters demonstrated a significant correlation with changes in log 10 CFU/lung (Spearman's correlation coefficient, P < 0.001); however, the goodness of fit as assessed with the maximum effect ( E max ) model revealed that the maximum concentration of free drug in serum ( C max free )/MIC and the area under the free drug concentration-time curve (AUC free )/MIC best explained the relationship between drug exposure and reductions in viable bacterial counts. These data reveal that an approximate cethromycin AUC free /MIC of 50 or C max free /MIC of 1 results in bacteriostatic effects, while higher values (twofold) maximize survival.

Infections caused by antibiotic-resistant bacteria pose an increasing risk for clinical treatment. Macrolide-lincosamide-streptogramin B is becoming increasingly ineffective due to the methylation at the binding site of bacteria. Despite great efforts on the natural product, erythromycin, only one derivative, that is, telithromycin, capable of fighting against resistant bacteria has so far been marketed. However, the 3'-dimethylamino group is readily metabolized to a nitroso group, which would inhibit CYP3A4, a very important metabolic enzyme responsible for nearly half of all marketed drugs.Modifications at C-10 of erythromycin were seldom reported. This invention disclosed novel ketolides that had a side chain comprising additional nitrogen atoms in place of the original 10-methyl group. Surprisingly, introduction of the side chain at C-10 led to reduced cytochrome inhibition and increased metabolic stability. As a result, the limited ability to inhibit CYP3A4 would relieve the drug-drug interaction and improve the safety of drug co-administration.This invention opens a new avenue for future modifications to the erythromycin family. It remains unclear how the side chain effected on reduction of CYP inhibition. To fully identify structure-activity relationships, the MIC data of the derivatives on gram-negative bacteria is desirable.

In the search for new antibiotics active against macrolide-resistant pneumococci and Haemophilus influenzae, we synthesized a new class of 3-oxo-6-O-methylerythromycin derivatives, so-called "ketolides". A keto function was introduced in position 3 after removal of L-cladinose, a sugar which has long been thought essential. Further modifications of the macrolactone backbone allowed us to obtain three different series of 9-oxime, 11,12-carbamate, and 11, 12-hydrazonocarbamate ketolides. These compounds were found to be very active against penicillin/erythromycin-resistant pneumococci and noninducers of MLSB resistance. The 11,12-substituted ketolide 61 (HMR 3004) demonstrated a potent activity against multiresistant pneumococci associated with a well-balanced activity against all bacteria involved in respiratory infections including H. influenzae, Mycoplasma catarrhalis, group A streptococci, and atypical bacteria. In addition HMR 3004 displayed high therapeutic activity in animals infected by all major strains, irrespective of their resistance phenotype.

The in vitro activities of two investigational ketolides, cethromycin (formerly ABT-773) and telithromycin, were determined for a selected group of 312 Streptococcus pneumoniae isolates from a national surveillance program. The MIC of cethromycin at which 50% of the isolates were inhibited was 0.008 μg/ml, and the MIC at which 90% of the isolates were inhibited was 0.06 μg/ml; the corresponding values for telithromycin were ≤0.015 and 0.25 μg/ml, respectively. For six quinupristin-dalfopristin-resistant strains, the cethromycin MICs were 0.25 to 16 μg/ml and the telithromycin MICs were 1 to 4 μg/ml. However, there was only 0.3% resistance to telithromycin.

Objectives To investigate the in vitro activity of cethromycin, a new ketolide, against Chlamydia pneumoniae. Methods The in vitro activity of cethromycin against 20 isolates of C. pneumoniae was compared with the activities of telithromycin, erythromycin A, azithromycin and clarithromycin against those isolates. Results The MIC at which 90% of the isolates were inhibited and the minimal chlamydiacidal concentration at which 90% of the isolates were killed by cethromycin were both 0.016 mg/L (range 0.016-0.031 mg/L). Cethromycin was the most active antibiotic tested in this study. Conclusions Our results appear to indicate that cethromycin is an effective antibiotic that should play some role in the treatment of respiratory tract infections caused by C. pneumoniae.

Novel sources of antibiotics are required to keep pace with the inevitable onset of bacterial resistance. Continuing with our macrolide desmethylation strategy as a source of new antibiotics, we report the total synthesis, molecular modeling and biological evaluation of 4,10-didesmethyl telithromycin (4), a novel desmethyl analogue of the 3rd-generation drug telithromycin (2). Telithromycin is an FDA-approved ketolide antibiotic derived from erythromycin (1). We found 4,10-didesmethyl telithromycin (4) to be four times more active than previously prepared 4,8,10-tridesmethyl congener (3) in MIC assays. While less potent than telithromycin (2), the inclusion of the C-8 methyl group has improved biological activity suggesting it plays an important role in antibiotic function.

The macrolides and ketolides represent a class of antibacterial agents with activities against most Gram-positive and selected Gram-negative as well as atypical pathogens including penicillin-resistant microorganisms. Erythromycin, clarithromycin, and azithromycin, the most extensively used macrolides in North America, and the only approved ketolide telithromycin have demonstrated efficacy and safety in a variety of clinical conditions and have been approved for use in both upper and lower-respiratory tract infections as well as skin and skin structure and genital infections. The introduction of clarithromycin and azithromycin provided the medical community with macrolide agents with not only increased pathogen coverage but also a significant improvement in adverse event profile when compared with the first macrolide agent, erythromycin. However, the global increase in macrolide-resistant Streptococcus pneumoniae is limiting their use. The ketolide telithromycin has great promise with efficacy to treat multidrug-resistant S. pneumoniae but its use has been significantly limited due to a number of serious safety issues which have surfaced since approval. Cethromycin is the only ketolide in late stage development. This chapter will provide a comprehensive review of these agents regarding the following aspects: in vitro antibacterial activity, mechanism of action, mechanism of resistance, pharmacokinetic and pharmacodynamic profile, clinical use, and safety issues. Future directions and challenges for the discovery and development of new macrolides and ketolides are also discussed.

A series of novel 6-O-substituted 8a-aza-8a-homoerythromycin A ketolides was synthesized and evaluated for in vitro antibacterial activity. Key strategic elements of the synthesis include the base-induced E-Z isomerization of 3-O-descladinosyl-6-O-allylerythromycin A 9(E)-oxime followed by ring-expanding reaction of the resulting 9(Z)-oxime via Beckmann rearrangement. The ketolides showed potent activity against a variety of erythromycin-susceptible and macrolide-lincosamide-streptogramin B (MLS(B)) resistant Gram-positive and fastidious Gram-negative pathogens. The best compounds in this series overcome all types of resistance in relevant clinical Gram-positive pathogens and display in vitro activity comparable to telithromycin and cethromycin.

Ketolides differ from macrolides by removal of the 3-O-cladinose (replaced by a keto group), a 11,12- or 6,11-cyclic moiety and a heteroaryl-alkyl side chain attached to the macrocyclic ring through a suitable linker. These modifications allow for anchoring at two distinct binding sites in the 23S rRNA (increasing activity against erythromycin-susceptible strains and maintaining activity towards Streptococcus pneumoniae resistant to erythromycin A by ribosomal methylation), and make ketolides less prone to induce methylase expression and less susceptible to efflux in S. pneumoniae. Combined with an advantageous pharmacokinetic profile (good oral bioavailability and penetration in the respiratory tract tissues and fluids; prolonged half-life allowing for once-a-day administration), these antimicrobial properties make ketolides an attractive alternative for the treatment of severe respiratory tract infections such as pneumonia in areas with significant resistance to conventional macrolides. For telithromycin (the only registered ketolide so far), pharmacodynamic considerations suggest optimal efficacy for isolates with minimum inhibitory concentration values < or = 0.25 mg/l (pharmacodynamic/pharmacokinetic breakpoint), calling for continuous and careful surveys of bacterial susceptibility. Postmarketing surveillance studies have evidenced rare, but severe, side effects (hepatotoxicity, respiratory failure in patients with myasthenia gravis, visual disturbance and QTc prolongation in combination with other drugs). On these bases, telithromycin indications have been recently restricted by the US FDA to community-acquired pneumonia, and caution in patients at risk has been advocated by the European authorities. Should these side effects be class related, they may hinder the development of other ketolides such as cethromycin (in Phase III, but on hold in the US) or EDP-420 (Phase II).

Cethromycin is a ketolide with in vitro activity against macrolide-sensitive and -resistant strains of Streptococcus pneumoniae . We compared its in vivo efficacy to erythromycin in a mouse model of acute pneumonia induced by two virulent clinical strains: a serotype 3 susceptible strain (P-4241) (MICs: erythromycin, 0.03 μg/ml; cethromycin, 0.015 μg/ml) and a serotype 1 strain resistant to erythromycin (P-6254; phenotypically MLSB constitutive) (MICs: erythromycin, 1,024 μg/ml; cethromycin, 0.03 μg/ml). Immunocompetent mice were infected with 10 5 CFU of each strain. Six treatments given either subcutaneously (s.c.) or per os (p.o.) at 12-h intervals were initiated at 6 or 12 h after infection. Against P-4241, cethromycin given s.c. at 25 or 12.5 mg/kg protected 100% of the animals, with lungs and blood completely cleared of bacteria. Given p.o., cethromycin maintained its efficacy with 100 and 86% survival at 25 and 12.5 mg/kg, respectively. Erythromycin, given s.c. at 50 or 37.5 mg/kg, provided 50 and 38% survival rates, respectively. Against P-6254, cethromycin was effective at 25 mg/kg (100% survival) regardless of the administration route, whereas only 25 and 8% of animals survived after a 75-mg/kg erythromycin treatment given s.c. and p.o., respectively. The serum protein binding levels of cethromycin were 94.8 and 88.5% after doses of 12.5 and 25 mg/kg, respectively. The higher in vivo activity of cethromycin compared to erythromycin could be explained by favorable pharmacokinetic/pharmacodynamic indexes against P-6254 but not against P-4241.

Macrolide antibiotics have an outstanding ability to concentrate within host cells, particularly phagocytes. In the study described in this paper five different macrolide antibiotics were compared regarding the uptake and release kinetics in human peripheral blood polymorphonuclear neutrophils (PMNs) and three different cell lines, two phagocytic cell lines (RAW 264.7 and THP-1) and an epithelial cell line (MDCK). Based on the results obtained, the substances tested could be clustered into different groups. Azithromycin constituted the first group, characterized by rapid and nonsaturable uptake into phagocytic cells and a high degree of retention in the preloaded cells. The second group included erythromycin and clarithromycin. These two substances do not exhibit cell specificity; consequently, they are taken up to a similar extent and are released by all cell types studied. Ketolides constituted the last group. Their uptake was saturable in cells of monocytic lineage as well as in nondifferentiated cells of myeloid lineage, and they were rapidly released from all the cell lines studied. However, in PMNs, ketolide uptake was not saturable; and unlike telithromycin, cethromycin rapidly egressed from the loaded cells.

The objective of this study was to determine the steady-state plasma and intrapulmonary pharmacokinetic parameters of orally administered cethromycin in healthy volunteers. The study design included administering 150 or 300 mg of cethromycin once daily to 25 or 35 healthy adult subjects, respectively, for a total of five doses. Standardized and timed bronchoalveolar lavage (BAL) was performed after the last dose. Blood was obtained for drug assay prior to the first and last dose, at multiple time points following the last dose, and at the time of BAL. Cethromycin was measured in plasma, BAL, and alveolar cell (AC) by using a combined high-performance liquid chromatography-mass spectrometric technique. Plasma, epithelial lining fluid (ELF), and AC pharmacokinetics were derived by noncompartmental methods.Cmax/90% minimum inhibitory concentration (MIC90) ratios, area under the concentration-time curve (AUC)/MIC90ratios, intrapulmonary drug exposure ratios, and percent time above MIC90during the dosing interval (%T> MIC90) were calculated for recently reported respiratory pathogens. The kinetics were nonlinear, i.e., not proportional to dose. In the 150-mg-dose group, theCmax(mean ± standard deviations), AUC0-24, and half-life for plasma were 0.181 ± 0.084 μg/ml, 0.902 ± 0.469 μg · h/ml, and 4.85 ± 1.10 h, respectively; for ELF the values were 0.9 ± 0.2 μg/ml, 11.4 μg · h/ml, and 6.43 h, respectively; for AC the values were 12.7 ± 6.4 μg/ml, 160.8 μg · h/ml, and 10.0 h, respectively. In the 300-mg-dose group, theCmax(mean ± standard deviations), AUC0-24, and half-life for plasma were 0.500 ± 0.168 μg/ml, 3.067 ± 1.205 μg · h/ml, and 4.94 ± 0.66 h, respectively; for ELF the values were 2.7 ± 2.0 μg/ml, 24.15 μg · h/ml, and 5.26 h, respectively; for AC the values were 55.4 ± 38.7 μg/ml, 636.2 μg · h/ml, and 11.6 h, respectively. We concluded that theCmax/MIC90ratios, AUC/MIC90ratios, %T> MIC90values, and extended plasma and intrapulmonary half-lives provide a pharmacokinetic rationale for once-daily administration and are favorable for the treatment of cethromycin-susceptible pulmonary infections.

Ketolides are a new class of semi-synthetic agents derived from erythromycin A designed to overcome erythromycin A resistance in Streptococcus pneumoniae. Telithromycin (HMR 3647) is the first member of this new class to be approved for clinical use. Cethromycin (ABT-773) has been developed up to Phase III, but its further development seems questionable at the moment. Other ketolides are only in the first stages of preclinical development and may not be available within the foreseeable future. Ketolide compounds inhibit bacterial protein synthesis by interacting with the peptidyl transferase site of the 50S ribosomal subunit, and interact closely with domains II at A752 and V at A2058 and A2059 of the 23S rRNA. These compounds also inhibit the formation of the 50S subunit of the ribosome. Ketolides show good activity against the Gram- positive bacteria responsible for respiratory tract infections including penicillin G- and erythromycin A-resistant S. pneumoniae. The 15 clinical trials with telithromycin published to date include four randomized, double-blind comparative trials and three open-label studies in community-acquired pneumonia, three randomized double-blind trials in acute exacerbation of chronic bronchitis, two randomized double-blind trials in pharyngitis, and two double-blind comparative trials and one open-label trial in acute maxillary sinusitis. Clinical response rates were favourable in all clinical trials, with eradication rates in patients with pneumococcal bacteraemia and penicillin G- and erythromycin A-resistant pneumococcal infections at least as high as those of comparators. As resistance to macrolides continues to emerge, the availability of other ketolides besides telithromycin and a development programme for the application of ketolides in children would appear to be warranted to obtain a new class of antibiotics that may one day replace macro- lides.

A classical velocity centrifugation technique was used to study the in vitro uptake of the new ketolide ABT-773 by human polymorphonuclear neutrophils (PMNs) and a myelomonoblastic cell line, PLB-985, which can be differentiated into PMNs under certain culture conditions, compared to that of HMR 3004. ABT-773 was rapidly taken up by PMNs (cellular concentration to extracellular concentration ratio [C/E], about 34 at 30 s and up to 207 at 5 min), and uptake plateaued from 30 to 180 min (C/E, about 300). ABT-773 was accumulated significantly better than HMR 3004 from 5 to 180 min. Nondifferentiated PLB-985 cells (ND-PLB) accumulated significantly less ABT-773 and HMR 3004 than PMNs and PLB-985 cells differentiated into PMNs (D-PLB). Whatever the cell type and in contrast to the results obtained with HMR 3004, ABT-773 was mainly located in the cytosol (about 75%) and was rapidly released from loaded cells (about 40% at 5 min), followed by a plateau, likely owing to avid reuptake. Verapamil and H89, an inhibitor of protein kinase A, increased drug efflux. Uptake was sensitive to external pH, and the activation energy was moderate (about 50 kJ/mol). The existence of an active transport system on the PMN membrane was suggested by the following findings: concentration-dependent and saturable uptake (Vmax, about 10 000 ng/2.5 × 106PMNs/5 min;Km, about 60 μg/ml) the inhibitory effects of PMN activators or inhibitors (phorbol myristate acetate, verapamil, Ni2+) and the significantly decreased levels of accumulation by killed cells and cells treated at low temperatures. In addition, various macrolides impaired ABT-773 uptake, contrary to the findings for the quinolone levofloxacin. ND- and D-PLB also presented saturation kinetics that defined an active transport system (VmaxandKmvalues were similar to those obtained with PMNs), but the activation pathway of the carrier system did not seem to be fully functional in ND-PLB. As has been observed with other erythromycin A derivatives, ABT-773 impaired oxidant production by phagocytes in a time- and concentration-dependent manner. These data extend our previous results on the existence of an active transport system common to all macrolides and ketolides, at least in PMNs.

Two ketolides, three macrolides, and one azalide were tested in vitro against 17 isolates of the B. burgdorferi s.l. complex. As measured in micrograms per milliliter, activity was highest for cethromycin (MIC at which 90% of the tested isolates were inhibited [MIC 90 ], 0.0019 μg/ml) and telithromycin (MIC 90 , 0.0078 μg/ml). Electron-microscope analysis and time-kill studies also supported enhanced effectiveness of both ketolides.

Sir, Ketolides are a new class of semisynthetic macrolide derivatives that show excellent activity against Streptococcus pneumoniae, even against erythromycin-resistant isolates. Previous investigators have not found any telithromycin-resistant isolates among S. pneumoniae with the constitutive macrolide–lincosamide–streptogramin B (cMLSB) phenotype, even though staphylococci or Streptococcus pyogenes with the cMLSB phenotype can develop resistance to this drug.1–3 The lack of induction of methylase production by this drug is one reason for such a difference.4 Recently, Hamilton-Miller & Shah5 reported that resistance to the ketolide cethromycin (formerly ABT-773) could be induced in S. pneumoniae with the cMLSB phenotype by erythromycin or other related antibiotics. In this study, we examined whether 55 isolates of erythromycin-resistant S. pneumoniae (MIC ≥ 1 mg/L) carrying the mef(A) and/or erm(B) genes could develop resistance to telithromycin, a ketolide with different substitutions from cethromycin (ABT-773), when exposed to erythromycin. Fifty-five clinical isolates of erythromycin-resistant S. pneumoniae (obtained from the sputum of patients with lower respiratory tract infections between 1998 and 2000) were used. These isolates were identified by their sensitivity to optochin and the bile solubility test, and by PCR amplification of the lytA gene. S. pneumoniae ATCC 6305 was used as the quality control strain. Reference samples of the following antimicrobial agents of known potency were kindly supplied in powder form by the indicated manufacturers: erythromycin (Shionogi Pharmaceutical Co., Osaka, Japan), clarithromycin (Taisho Pharmaceutical Co., Tokyo, Japan), azithromycin (Pfizer Laboratories, Groton, CT, USA), rokitamycin (Asahi Kasei, Tokyo, Japan), telithromycin (Nippon HoechstMarion-Roussel, Tokyo, Japan) and clindamycin (Upjohn, Tokyo, Japan). All isolates were tested for susceptibility to the six antibiotics listed above at concentrations between 0.015 and 128 mg/L. MICs were determined by the two-fold agar dilution method using susceptibility test agar (Mueller–Hinton agar medium; Eiken Chemicals, Tokyo, Japan) with 8% Strepto Haemo supplement (SHS; Eiken Chemicals). DNA was obtained as previously reported, and the presence of macrolide resistance genes was investigated by PCR using primers and amplification conditions that have been described previously.6 Induction of telithromycin resistance was examined by the following two methods. (i) Agar dilution method: induction of telithromycin resistance was evaluated by comparing the MICs for telithromycin in the presence and absence of a subinhibitory concentration of erythromycin (0.1 mg/L). Two susceptibility agar plates were prepared for each isolate: one contained only telithromycin and the other contained telithromycin plus erythromycin. The MIC was determined in accordance with the susceptibility test. (ii) Disc diffusion method: A bacterial suspension (2 mL of ∼108 cfu/mL) was inoculated onto 10 mL of susceptibility test agar containing 8% SHS and spread over the surface. After excess suspension was removed, paper discs (8 mm diameter high discs; Tokyo Roshi Kaisha, Tokyo, Japan) containing erythromycin or rokitamycin at 20 μg/disc or telithromycin at 5 μg/disc were placed on the surface of the agar plate. Then the plates were incubated overnight at 35°C and induction of telithromycin resistance was assessed from the shape of the zone of inhibition around the telithromycin disc nearest either the erythromycin or the rokitamycin disc. Fifteen isolates carrying only the mef(A) gene had the M phenotype. Among the other 40 isolates, 25 carried the erm(B) gene, and 15 had both the mef(A) and erm(B) genes. All 40 isolates showed a high

Using six Enterococcus faecalis and five Enterococcus faecium strains, the ketolide ABT-773 (ABT), now known as cethromycin, was found to have in vivo efficacy against both erythromycin (ERY)-susceptible (Ery s ) and -intermediate (Ery i ) enterococci (ABT 50% protective doses [PD 50 s], 0.5 to 4.1 and 10.3 to 16.2 mg/kg of body weight, respectively). Against four highly Ery-resistant (Ery r ) strains for which ABT MICs were low, ABT showed much greater activity (PD 50 , 6.3 to 32.5 mg/kg) than ERY (PD 50 , >200 mg/kg) but was not protective for strains for which ABT MICs were high. In conclusion, ABT-773 showed in vivo efficacy and considerably greater activity than ERY in a mouse peritonitis model.

Nasopharyngeal specimens for culture of Chlamydia pneumoniae were obtained from patients with community-acquired pneumonia enrolled in a randomized study comparing the novel ketolide antibiotic ABT-773 at a dose of 150 mg once a day to 150 mg twice a day, by mouth for 10 days. C. pneumoniae was eradicated from the nasopharynx of 10 of 10 (100%) microbiologically evaluable patients. MICs and MBCs for 13 isolates of C. pneumoniae from 12 patients obtained before and after therapy were performed against ABT-773. The MICA and MBC 90 of ABT-773 were 0.015 mg/L.

The prevalence of antibiotic resistance in bacterial pathogens associated with community-acquired respiratory tract infections is increasing. Ketolides, semi-synthetic derivatives of erythromycin, overcome the macrolide resistance mechanisms found in Streptococcus pneumoniae and Streptococcus pyogenes, two key pathogens. They also have improved potency and longer post-antibiotic effects, while maintaining the antibacterial spectrum of the macrolide class. The new ketolides cethromycin (ABT-773) and telithromycin have overall antibacterial properties that suggest they will be clinically useful new antibiotics and are undergoing clinical development and regulatory review.

The activity of a new ketolide, ABT-773, was compared to the activity of the ketolide telithromycin (HMR-3647) against over 600 gram-positive clinical isolates, including 356 Streptococcus pneumoniae , 167 Staphylococcus aureus , and 136 Streptococcus pyogenes isolates. Macrolide-susceptible isolates as well as macrolide-resistant isolates with ribosomal methylase (Erm), macrolide efflux (Mef), and ribosomal mutations were tested using the NCCLS reference broth microdilution method. Both compounds were extremely active against macrolide-susceptible isolates, with the minimum inhibitory concentrations at which 90% of the isolates tested were inhibited (MIC 90 s) for susceptible streptococci and staphylococci ranging from 0.002 to 0.03 μg/ml for ABT-773 and 0.008 to 0.06 μg/ml for telithromycin. ABT-773 had increased activities against macrolide-resistant S. pneumoniae (Erm MIC 90 , 0.015 μg/ml; Mef MIC 90 , 0.12 μg/ml) compared to those of telithromycin (Erm MIC 90 , 0.12 μg/ml; Mef MIC 90 , 1 μg/ml). Both compounds were active against strains with rRNA or ribosomal protein mutations (MIC 90 , 0.12 μg/ml). ABT-773 was also more active against macrolide-resistant S. pyogenes (ABT-773 Erm MIC 90 , 0.5 μg/ml; ABT-773 Mef MIC 90 , 0.12 μg/ml; telithromycin Erm MIC 90 , >8 μg/ml; telithromycin Mef MIC 90 , 1.0 μg/ml). Both compounds lacked activity against constitutive macrolide-resistant Staphylococcus aureus but had good activities against inducibly resistant Staphylococcus aureus (ABT-773 MIC 90 , 0.06 μg/ml; telithromycin MIC 90 , 0.5 μg/ml). ABT-773 has superior activity against macrolide-resistant streptococci compared to that of telithromycin.

MICs, time-kills, and postantibiotic effects (PAEs) of ABT-773 (a new ketolide) and 10 other agents were determined against 226 pneumococci. Against 78 ermB - and 44 mefE -containing strains, ABT-773 MICs at which 50% of the isolates tested were inhibited (MIC 50 s) and MIC 90 s were 0.016 to 0.03 and 0.125 μg/ml, respectively. Clindamycin was active only against macrolide-resistant strains containing mefE (MIC 50 , 0.06 μg/ml; MIC 90 , 0.125 μg/ml). Activities of pristinamycin (MIC 90 , 0.5 μg/ml) and vancomycin (MIC 90 , 0.25 μg/ml) were unaffected by macrolide or penicillin resistance, while β-lactam MICs rose with those of penicillin G. Against 19 strains with L4 ribosomal protein mutations and two strains with mutations in domain V of 23S rRNA, ABT-773 MICs were 0.03 to 0.25 μg/ml, while macrolide and azalide MICs were all ≥16.0 μg/ml. ABT-773 was bactericidal at twice the MIC after 24 h for 8 of 12 strains (including three strains with erythromycin MICs greater than or equal to 64.0 μg/ml). Kill kinetics of erythromycin, azithromycin, clarithromycin, and roxithromycin against macrolide-susceptible strains were slower than those of ABT-773. ABT-773 had longer PAEs than macrolides, azithromycin, clindamycin, or β-lactams, including against ermB -containing strains. ABT-773, therefore, shows promising in vitro activity against macrolide-susceptible as well as -resistant pneumococci.

The in vitro activity of ABT-773 was evaluated against Streptococcus pneumoniae , Haemophilus influenzae , and Moraxella catarrhalis isolates. ABT-773 was the most active antimicrobial tested against S. pneumoniae . ABT-773 and azithromycin were equivalent in activity against H. influenzae and M. catarrhalis and more active than either clarithromycin or erythromycin.

Sir, Historically, methicillin-resistant Staphylococcus aureus (MRSA) has been regarded as a nosocomial pathogen responsible for severe toxin-mediated disease or invasive pyogenic infections. In recent years, however, community-associated MRSA (CA-MRSA) has been reported from around the world. Although the first CA-MRSA strains were susceptible to most antimicrobials, antimicrobial resistance has increased. One CA-MRSA clone, identified by the CDC as pulsotype USA300, has been implicated in outbreaks within the USA and is resistant to many currently marketed antimicrobial agents. This clone carries both the SCCmec IV element and the Panton–Valentine leukocidin (PVL) locus. The aim of this study was to compare the activity of a novel ketolide, cethromycin (previously ABT-773), with potentially improved pharmacodynamic and therapeutic benefits, three other macrolides (azithromycin, clarithromycin and erythromycin) and one lincosamide (clindamycin) against the 170 MRSA USA300 isolates that are in the Center for Biological Defense (CBD) collection. Seventeen MRSA isolates were procured from two hospital laboratories in Seattle, WA, USA. These were isolated from cultures of blood, skin lesions and tissue abscesses of patients seen in their clinics or emergency department in 2003 and 2004. Of the remaining 153 MRSA, 121 isolates were obtained from cultures of lesions, abscesses and nasopharyngeal specimens from outpatients seen at west central Florida clinical laboratories and doctor’s offices, whereas 32 MRSA were isolated from lesions, abscesses, nasopharyngeal and blood specimens acquired from patients seen at hospitals in the Tampa Bay area from 2004 to 2006. These isolates were previously characterized as USA300 by PFGE using Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) standards for comparison. Cethromycin (obtained from Advanced Life Sciences, Woodridge, IL, USA) was prepared and diluted with calciumadjusted Mueller–Hinton broth (Remel, Lenexa, KS, USA) using 96-well microtitre plates following standard protocols. The MIC and MBC were determined using orthodox procedures. Clarithromycin, erythromycin and clindamycin were tested with Sensititre by TREK Diagnostic Systems (Cleveland, OH, USA), whereas azithromycin, erythromycin and clindamycin were tested using Etest (AB Biodisk, North America Inc., Piscataway, NJ, USA), according to manufacturer’s instructions. All MIC interpretations followed CLSI guidelines. Clindamycin resistance was examined further with the D-test. Controls were S. aureus ATCC 29213, S. aureus ATCC BAA977 and S. aureus ATCC BAA976. Cethromycin was effective against all 170 isolates (Table 1). The cethromycin MIC range was 0.002–0.125 mg/L, whereas the MIC50 and MIC90 were both 0.002 mg/L. The cethromycin MBC range was 0.002–1.0 mg/L, with the MBC50 of 0.002 mg/L and the MBC90 of 0.008 mg/L. Of the 170 CA-MRSA, 169 (99.4%) were intermediate or resistant to at least one macrolide. In contrast, 164 (96.5%) isolates were susceptible to clindamycin. Only six (3.5%) isolates demonstrated either constitutive resistance [4 (2.4%)] or inducible resistance [2 (1.2%)] to clindamycin. This is unlike the USA300 isolates in Boston where 57% of the isolates were clindamycin-resistant. Against 10 erythromycin-susceptible isolates, the cethromycin MIC and MBC ranges were 0.002–0.008 mg/L, compared with the MIC range of 0.002–0.25 mg/L and MBC range of 0.002–1.0 mg/L obtained with the erythromycin-resistant isolates. However, MIC50 and MBC50 values, as well as MIC90 and MBC90 values, did not change regardless of erythromycin susceptibility. There were six isolates resistant to both erythromycin and clindamycin, against which cethromycin MIC and MBC ranges were 0.002–0.06 mg/L. This population’s susceptibility to cethromycin is especially important in the light of the fact that most isolates were resistant to one or more macrolides. This collection of MRSA USA300 isolates was overwhelmingly macrolide-resistant and clindamycinsusceptible. The study of Chavez-Bueno et al. demonstrated a similar pattern in CA-MRSA isolates among children in Texas where the inducible resistance to clindamycin was slowly replaced by clindamycin susceptibility (non-inducible resistance). Most of the CA-MRSA isolates in the Texas study carried the erm(C) gene, with others carried erm(A) or msr(A) with or without erm(C). This phenotypic change suggests an alteration in the proportion of the macrolide resistance determinants in the population. Tenover et al. reported the msr(A) gene in isolates comprising a specific clone (USA300-0114). Accordingly, we hypothesized that most of the CBD collection of MRSA USA300 isolates (only one being USA300-0114) also carried a different determinant, putatively the msr(A)/msr(B) gene encoding an efflux protein that normally renders a bacterial cell resistant only to macrolides and streptogramin B compounds (MS phenotype) instead of the MLSB resistance genes [i.e. erm(A) and erm(C)]. Using the oligonucleotides described by Sutcliffe et al. for PCR primers and a well-characterized MRSA strain NARSA 384 (USA300-0114) as positive control, we performed a PCR assay for the msr(A) gene on DNA extracted from all 170 isolates. After the amplicons were electrophoresed on a 1% agarose gel, a positive PCR reaction yielded a DNA band of 399 bp. All negative and questionable results were performed three times. The assay produced a positive reaction in 160 (94.1%) isolates, whereas only 10 (5.9%) of the 170 MRSA yielded negative PCR reactions. These Research letters

Ketolides belong to a new class of semi-synthetic 14-membered-ring macrolides, which differ from erythromycin A by having a 3-keto group instead of the neutral sugar L-cladinose. The ability of these molecules and their L-cladinose counterparts to induce MLS B resistance in staphylococci (one strain) and streptococci (two strains) was investigated using a disc agar susceptibility method as well as measuring induction kinetics. All 14- and 15-membered ring macrolides tested showed inducing activity. In contrast, ketolides were clearly unable to induce MLS B resistance, a result consistent with the high in-vitro activity of this new class of antibiotics against erythromycin A-inducible resistant bacteria.

The in vitro activities of ABT 773, telithromycin (HMR 3647), azithromycin, clarithromycin, erythromycin, and levofloxacin were tested against 20 strains of Chlamydia pneumoniae . The MIC at which 90% of the isolates were inhibited and the minimal bactericidal concentration at which 90% of the isolates were killed by ABT 773 were 0.015 μg/ml (range, 0.008 to 0.015 μg/ml). ABT 773 was the most active antibiotic tested in this study.

OBJECTIVES To evaluate the penetration of ketolide cethromycin (ABT-773) into human polymorphonuclear leucocytes (PMNs) and its intracellular activity. METHODS The uptake of radiolabelled cethromycin by PMNs was determined by a velocity gradient centrifugation technique. The activity of cethromycin against intracellular Staphylococcus aureus ATCC 25923 in PMNs was also evaluated. RESULTS The cellular to extracellular concentration (C/E) ratio for cethromycin was >200 at an extracellular concentration of 2 mg/L. The uptake of cethromycin into PMNs was rapid and saturable. Cethromycin was slowly released from the loaded PMNs (cell associated drug>50% after 2 h of incubation). Intracellular penetration was significantly affected by the environmental temperature (C/E ratio at 4 degrees C and 37 degrees C: 13 +/- 6 and 226 +/- 31, respectively; P < 0.05), by cell viability (C/E ratio for dead and viable cells: 100 +/- 38 and 226 +/- 31, respectively; P < 0.05), by pH (C/E ratio was significantly increased at basic pH) and by the metabolic inhibitors 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone. The intracellular accumulation of cethromycin also decreased significantly when cells were activated with phorbol myristate acetate or opsonized zymosan. These data indicate that a potentially active mechanism could be involved in the uptake of cethromycin by PMNs. At high extracellular concentrations of 5 and 10 mg/L, cethromycin showed significant intracellular activity against S. aureus. CONCLUSIONS Cethromycin achieves high intracellular concentrations within human PMNs, remaining active intracellularly.