INTRODUCTION

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The respiratory pathogens Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis cause a wide spectrum of pediatric and adult infections, including acute otitis media, sinusitis, pneumonia, bacteremia, meningitis, and acute exacerbations of chronic bronchitis. S. pneumoniae is the most common community-acquired respiratory tract pathogen; it causes as many as 20% of all cases of community-acquired pneumonia annually, leading to significant morbidity and mortality rates (2, 5). Recent data indicate that approximately one-third of S. pneumoniae isolates in the United States have some level of resistance to penicillin, and up to 40% of H. influenzae isolates and almost all M. catarrhalis isolates produce -lactamase, which mediates resistance to penicillins and certain cephalosporins (6). Furthermore, the emergence of strains of S. pneumoniae resistant to penicillin as well as extended-spectrum cephalosporins, macrolides, and quinolones has become a considerable concern in many parts of the world (1, 3). LB 10827 (Fig. 1) is a new, nonester-type oral cephalosporin which has an excellent in vitro activity against the major respiratory pathogens and is currently under preclinical trial (S. H. Oh, E. J. Ryu, K. S. Paek, M. Y. Kim, S. H. Lee, and C. S. Lee, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 397, 1999). In this study, we assessed the in vitro and in vivo activities of LB 10827 against these three common bacterial respiratory tract pathogens.

View larger version (9K): [in this window] [in a new window]   FIG. 1.   Chemical structure of LB 10827.

	MATERIALS AND METHODS

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Bacterial strains. The bacterial strains used in this study were obtained from various community hospitals and medical centers in Korea between 1998 and 1999. All strains were stored frozen at 70°C.

Antimicrobial agents. LB 10827 was synthesized at LG Chem., and other antibiotics were obtained from their respective manufacturers and used without further purification. For in vivo experiments, antibiotic powders were freshly diluted with saline before each experiment with animals according to the manufacturers' instructions.

Susceptibility test. MICs were determined by the broth dilution method for S. pneumoniae in cation-adjusted Mueller-Hinton broth (MHB) with 5% lysed defibrinated horse blood, in Haemophilus test medium for H. influenzae isolates, and in MHB supplemented with 3 to 5% sheep blood for M. catarrhalis with inocula of 5 × 10⁵ CFU/ml according to guidelines of the National Committee for Clinical Laboratory Standards (NCCLS) (4). Inocula were prepared from overnight growth suspended in saline to achieve a turbidity equivalent to that of a 0.5 McFarland standard (approximately 10⁸ organisms per ml). The inoculated trays were incubated in ambient air for 20 to 24 h at 35°C. The MICs were read as the lowest concentration of antimicrobial agent that inhibited visible growth. The following control strains were tested on a daily basis: S. pneumoniae ATCC 49619 and H. influenzae ATCC 49247 and H. influenzae ATCC 49766.

Animals. Male Sprague-Dawley rats (80 to 100 g) and female C57BL/6 mice (17 to 19 g) obtained from the Dae-Han Laboratory Animal Research Center, Eumsung, Korea, were used in the pneumonia infection model.

Time-kill study. Initial broth cultures of S. pneumoniae, H. influenzae, and M. catarrhalis were prepared from a 24-h agar plate. Test organisms were diluted to approximately 10⁵ CFU/ml with an appropriate fresh medium: MHB with 5% horse serum for S. pneumoniae, MHB with 5% Fildes enrichment for H. influenzae, and MHB for M. catarrhalis. The diluted cultures were preincubated for 2 h and each drug was added. Aliquots (0.1 ml) of the cultures were removed after 0, 2, 4, 6, 12, and 24 h of incubation, and serial 10-fold dilutions were prepared in saline to minimize antibiotic carryover. The number of viable cells was determined on appropriate drug-free agar plates after 24 h of incubation. The viable count threshold of a 0.1-ml aliquot placed on a plate is theoretically 10 CFU/ml if one colony grows; however, for statistical accuracy, the lower limit was set at 250 CFU/ml, and this threshold was therefore used in all time-kill experiments. Antimicrobials were considered bactericidal at the lowest concentration that reduced the original inoculum by 3 log₁₀ CFU/ml (99.9%) at each of the time points and were considered bacteriostatic when the inoculum was reduced by 0 to 3 log₁₀ CFU/ml.

Experimental pneumonia in neutropenic rats. S. pneumoniae type III was cultured in tryptic soy agar supplemented with 5% sheep blood for 18 h, suspended in the saline, mixed with brain heart infusion broth, and cooled with molten agar. A total of 50 µl of bacterial suspension (1.5 × 10⁵ CFU/rat) was injected by intrabronchial instillation into the neutropenic rats, which were prepared by intraperitoneal injection of cyclophosphamide 4 days before infection (100 mg/kg of body weight), under pentobarbital anesthesia. The drugs were administered orally to groups of six rats each at 18, 26, 42, and 50 h after infection with doses of 2, 10, or 50 mg/kg. At 20 h after the final drug treatment, the lungs were removed aseptically and homogenized in 9 ml of saline, and the viable cells in the lungs of each rat were counted on agar plates containing 5% sheep blood. All animals in a control group developed acute pneumonia and died within 2 days after infection. MICs of comparison drugs used for in vivo tests with S. pneumoniae are as follows: 0.008 µg/ml for LB 10827, 0.063 µg/ml for cefdinir, 0.031 µg/ml for augmentin, 0.031 µg/ml for clarithromycin, and 0.25 µg/ml for trovafloxacin.

Experimental pneumonia in mice. S. pneumoniae type III was incubated on tryptic soy agar supplemented with 5% sheep blood at 35°C, harvested, and suspended in saline. Female C57BL/6 mice were anesthetized by intraperitoneal injection of sodium pentobarbital and then infected by intranasal instillation of bacterial suspension (2.0 × 10⁷ CFU/mouse). Antibacterial agents at various doses were orally administered 6 h after infection and twice daily for the following 3 days, and the survival rate was observed for 2 weeks.

	RESULTS

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The results of agar and broth dilution MIC testing of recent clinical isolates (146 strains) are presented in Table 1. LB 10827 demonstrated activity comparable with those of other -lactams against most of the penicillin-susceptible S. pneumoniae isolates. However, clarithromycin and all tested -lactams showed significantly decreased antibacterial activities against penicillin-resistant strains. Against 19 penicillin-resistant S. pneumoniae isolates, LB 10827 was 4-fold more active than amoxicillin-clavulanate and 32-fold more active than cefdinir. Quinolone resistance was found equally in S. pneumoniae strains that are penicillin sensitive, intermediate, and resistant. Although quinolones retained activity against the penicillin-resistant strains, they were almost inactive against ciprofloxacin-resistant strains. LB 10827 was 32-fold more active than trovafloxacin and 128-fold more active than ciprofloxacin against 18 ciprofloxacin-resistant strains.

LB 10827 also showed potent activity against H. influenzae and M. catarrhalis (MICs at which 90% of isolates tested are inhibited [MIC₉₀s] of 0.5 and 0.13 µg/ml, respectively), and its activity is preserved in the presence of -lactamase. LB 10827 was 2- to 32-fold more active against -lactamase-negative H. influenzae isolates and 2- to 64-fold more active against -lactamase-positive H. influenzae isolates than other comparison -lactams and macrolides. The slight difference in susceptibility between -lactamase-producing and nonproducing strains could come from other mechanisms and not from -lactamases. This assumption is supported by the clear distinction of the susceptibilities to older -lactam antibiotics against -lactamase-positive and -negative H. influenzae isolates (MIC₉₀s of amoxicillin for -lactamase-positive and -negative strains were >32 and 4 µg/ml, respectively). Against 31 -lactamase-positive M. catarrhalis isolates, LB 10827 was 2- to 32-fold more active than other comparison drugs, except quinolones.

The results of time-kill experiments are presented in Fig. 2, 3, and 4 and were analyzed by determining the number of strains which showed viable count decreases of 1, 2, and 3 log₁₀ CFU/ml compared to the counts at 0 h. Only LB 10827 and amoxicillin-clavulanate at the MIC after 12 h was bactericidal against S. pneumoniae. At 3 to 6 h, some degree of killing in the 90 to 99% range was observed for both compounds (Fig. 2). All compounds yielded 99.9% killing at two times and four times the MIC after 12 h, except clarithromycin. LB 10827 also evidenced bactericidal activity against -lactamase-producing H. influenzae and M. catarrhalis isolates (Fig. 3 and 4). H. inflenzae was killed by LB 10827 with a 99% reduction of the original inoculum after 6 h at the MIC. Most of the comparison agents under the same conditions showed less than 90% killing, and regrowth was observed at 12 h. All compounds at two and four times the MIC after 24 h were bactericidal against M. catarrhalis, and only amoxicillin-clavulanate achieved 99.9% killing after 24 h at the MIC.

View larger version (20K): [in this window] [in a new window]   FIG. 2.   Time-kill kinetics of LB 10827 and other oral compounds against S. pneumoniae PN038 at 0, 3, 6, 9, and 12 h. Symbols for LB 10827:  growth control; , 0.13 mg/liter; , 0.25 mg/liter (MIC); *, 0.5 mg/liter; , 1 mg/liter. Symbols for cefprozil: , growth control; , 4 mg/liter; , 8 mg/liter (MIC); *, 16 mg/liter; , 32 mg/liter. Symbols for cefdinir: , growth control; , 4 mg/liter; , 8 mg/liter (MIC); *, 16 mg/liter; , 32 mg/liter. Symbols for amoxicillin-clavulanic acid (Amox/Clav): , growth control; , 1 mg/liter; , 2 mg/liter (MIC); *, 4 mg/liter; , 8 mg/liter. Symbols for cefuroxime: , growth control; , 2 mg/liter; , 4 mg/liter (MIC); *, 8 mg/liter; , 16 mg/liter. Symbols for clarithromycin: , growth control; , 2 mg/liter; , 4 mg/liter (MIC); *, 8 mg/liter; , 16 mg/liter; ......, threshold.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   Time-kill kinetics of LB 10827 and other oral compounds against H. influenzae KY115101 at 0, 3, 6, 9, and 12 h. Symbols for LB 10827: , growth control; , 0.063 mg/liter; , 0.13 mg/liter (MIC ); *, 0.25 mg/liter; , 0.5 mg/liter. Symbols for cefprozil: , growth control; , 2 mg/liter; , 4 mg/liter (MIC); *, 8 mg/liter; , 16 mg/liter. Symbols for cefdinir: , growth control; , 0.25 mg/liter; , 0.5 mg/liter (MIC); *, 1 mg/liter; , 2 mg/liter. Symbols for amoxicillin-clavulanic acid (Amox/Clav): , growth control; , 0.5 mg/liter; , 1 mg/liter (MIC); *, 2 mg/liter; , 4 mg/liter. Symbols for cefuroxime: , growth control; , 0.25 mg/liter; , 0.5 mg/liter (MIC); *, 1 mg/liter; , 2 mg/liter. Symbols for clarithromycin: , growth control; , 4 mg/liter; , 8 mg/liter (MIC); *, 16 mg/liter; , 32 mg/liter; ......, threshold.

View larger version (24K): [in this window] [in a new window]   FIG. 4.   Time-kill kinetics of LB 10827 and other oral compounds against M. catarrhalis MCA027 at 0, 2, 4, 6, and 24 h. Symbols for LB 10827: , growth control; , 0.13 mg/liter; , 0.25 mg/liter (MIC); *, 0.5 mg/liter; , 1 mg/liter. Symbols for cefprozil: , growth control; , 4 mg/liter; , 8 mg/liter (MIC); *, 16 mg/liter; , 32 mg/liter. Symbols for cefdinir: , growth control; , 0.25 mg/liter; , 0.5 mg/liter (MIC); *, 1 mg/liter; , 2 mg/liter. Symbols for amoxicillin-clavulanic acid (Amox/Clav): , growth control; , 0.25 mg/liter; , 0.5 mg/liter (MIC); *, 1 mg/liter; , 2 mg/liter. Symbols for cefuroxime: , growth control; , 1 mg/liter; , 2 mg/liter (MIC); *, 4 mg/liter; , 8 mg/liter. Symbols for clarithromycin: , growth control; , 0.063 mg/liter; , 0.13 mg/liter (MIC); *, 0.25 mg/liter; , 0.5 mg/liter; ......, threshold.

In order to elucidate the correlation between its potent in vitro and in vivo antibacterial activities and the effect of the high protein-binding value (S. W. Lee, personal communication), the in vivo efficacy of LB 10827 for acute pneumonia infections was evaluated and compared with those of amoxicillin-clavulanate, cefdinir, and trovafloxacin in rat and mouse infection models. In the rat pneumonia model, LB 10827 dramatically reduced the numbers of viable cells in the lungs of all three groups (Fig. 5). Futhermore, a 2-mg/kg dose of LB 10827 showed better in vivo activity than 10 mg of amoxicillin-clavulanate per kg and 50 mg of cefdinir or trovafloxacin per kg. Similar efficacy was also observed in a model of pneumonia that was induced by intranasal instillation of the same strain to C57BL/6 mice (Fig. 6). LB 10827 was as effective as amoxicillin-clavulanate and more effective than the other compared antibiotics, and these in vivo activities of LB 10827 were well correlated with its excellent in vitro antibacterial activities in rat and mouse infection models.

View larger version (47K): [in this window] [in a new window]   FIG. 5.   In vivo therapeutic efficacy of LB 10827 against acute pneumonia caused by S. pneumoniae in the neutropenic rat model.

View larger version (36K): [in this window] [in a new window]   FIG. 6.   Survival rates of infected immunocompetent mice treated with LB 10827 (), amoxicillin-clavulanate *, cefdinir (), trovafloxacin (), clarithromycin (), and control () with doses of 1.8 mg/kg (a) and 5.6 mg/kg (b).

	DISCUSSION

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LB 10827 is a new oral cephalosporin with improved activity against S. pneumoniae, H. influenzae, and M. catarrhalis. While LB 10827 has the same side chain as cefdinir at position 7, it contains a new diaminopyrimidine group in the side chain at position 3. This side chain has a chemical structure different from that of cefdinir, suggesting that the new side chain at position 3 enhances the activity of LB 10827 against gram-positive organisms. Thus, LB 10827 represents an advance in the activities of oral expanded-spectrum cephalosporins against respiratory pathogens.

In the rat pneumonia model, LB 10827 was as effective as amoxicillin-clavulanate against a penicillin-susceptible S. pneumoniae isolate and was considerably more efficacious than cefdinir and trovafloxacin. Similar efficacy was observed in a mouse pneumonia model using a penicillin-susceptible pneumococcus. Again, LB 10827 was as effective as amoxicillin-clavulanate and was more effective than the comparison antibiotics. These data are very encouraging, as amoxicillin-clavulanate, the most effective compared compound in the in vivo studies, has proven clinical efficacy against pneumococcal infections and is often the treatment of choice for bacterial pneumonia (7). If the pharmacokinetic data from human studies for LB 10827 are favorable (i.e., around 50% bioavailability and an elimination half-life of some 3 h or more), then the compound should be as effective as amoxicillin-clavulanate in the clinic against sensitive pneumococci and, hopefully, more effective against resistant strains.

The results of the present study indicate that LB 10827 has potent in vitro activity against recent clinical isolates of S. pneumoniae and M. catarrhalis. Although the drug is slightly less active against H. influenzae, the overall activity of LB 10827 against these three respiratory pathogens indicates a potential role for this agent in therapy of infections for which the currently marketed antimicrobials have a limited role because of bacterial resistance.