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Sanfetrinem cilexetil is the oral prodrug of sanfetrinem, the first in a series of novel tricyclic -lactam compounds, the trinems (Fig. 1). Previous reports have demonstrated that sanfetrinem has high stability to many -lactamases and to human renal dehydropeptidase I and has a broad spectrum of activity against gram-positive and gram-negative bacteria including penicillin-resistant Streptococcus pneumoniae (PRSP) (2, 8-10, 13). In this study, we evaluated the in vivo activities of sanfetrinem cilexetil and compared them with those of cefdinir and amoxicillin.

View larger version (13K): [in this window] [in a new window]   FIG. 1.   Structures of sanfetrinem cilexetil and sanfetrinem.

Sanfetrinem cilexetil and sanfetrinem were supplied by Glaxo Wellcome SpA (Verona, Italy). The other antibiotics were provided by the indicated manufacturers, as follows: imipenem, Banyu Pharmaceutical Co. (Tokyo, Japan); faropenem, Suntory Co. (Osaka, Japan); cefdinir and amoxicillin, Fujisawa Pharmaceutical Co. (Osaka, Japan); cefteram, Toyama Chemical Co. (Tokyo, Japan); cefuroxime, Nippon Glaxo Co. (Tokyo, Japan); and levofloxacin, Daiichi Pharmaceutical Co. (Tokyo, Japan).

Most bacterial strains used in the present study were clinical isolates collected at Toho University Hospital in the years 1993 through 1995. These isolates were stored at 80°C until use.

MICs were determined by the broth microdilution method as described in the guidelines of the Japanese Society for Chemotherapy (3, 4). Mueller-Hinton (MH) broth (Difco, Detroit, Mich.) supplemented with 50 mg of Ca²⁺ and 25 mg of Mg²⁺ per liter (cation-adjusted MH broth) was used for all assays except those of Streptococcus spp., which were tested with cation-adjusted MH broth supplemented with 5% lysed horse blood.

The potency of sanfetrinem cilexetil was determined in a mouse model of bacteremia. Male ICR mice (weight, 18 to 22 g; total of 160 mice for each strain) (Sankyo Labo Service, Tokyo, Japan) were injected intraperitoneally with the bacteria, which were suspended in saline containing 5% mucin (Difco). The test antibiotics, suspended in 0.5% metholose, were administered orally at five different doses (10 mice in each group) in a single dose 1 h after infection. The survival of the infected mice was monitored for 7 days, and the 50% effective dose (ED₅₀) was calculated by the Probit method. All untreated mice died within 3 days of infection.

The efficacy of sanfetrinem cilexetil was determined against two models of respiratory infection in mice. A model of respiratory infection caused by penicillin-susceptible S. pneumoniae (PSSP) was used to investigate the therapeutic effect. Another model of respiratory infection caused by PSSP and PRSP was used to investigate the antibiotic's ability to eradicate these organisms (6, 12). In both models, 60 or 80 µl of bacterial suspension was inoculated intranasally into mice under ketamine and xylazine anesthesia.

The ICR mice were used in a respiratory infection model to test the protective dose against PSSP. The test antibiotics, suspended in 0.5% metholose, were administrated orally at five different doses (10 mice in each group) on four occasions separated by 12-h intervals and starting 24 h after infection. The survival of the infected mice was monitored for 7 days, and the ED₅₀ was calculated by the Probit method. All untreated mice died within 6 days of infection.

Male CBA/J mice (weight, 16 to 20 g; total of 50 mice for each experiment) (Charles River Japan, Kanagawa, Japan) were used in a respiratory infection model to determine the effects of two dosage regimens on numbers of organisms in infected lungs (6, 11). The test antibiotics, suspended in 0.5% metholose, were administrated orally at a dose of 10 or 50 mg/kg of body weight (five mice in each group) repeated six times at 12-h intervals and starting 24 h after infection. At 24, 37, 61, and 85 h after infection, lungs were removed aseptically and homogenized with a fourfold excess of saline. The numbers of viable bacteria in the lungs of each mouse were then counted on heart infusion agar (Difco) plates containing 5% horse blood.

For pharmacokinetic studies, each antibiotic was orally administered to male ICR mice (weight, 18 to 22 g; five mice in each group) at a dose of 10 mg/kg. Blood and lung samples were obtained from anesthetized animals at 0.125, 0.25, 0.5, 1, 2, and 4 h after administration. The concentrations of antibiotics were measured by the paper disk method. For sanfetrinem and cefdinir, Bacillus subtilis ATCC 6633 was used as the indicator organism with cephaloridine test medium (0.5% peptone, 0.3% meat extract, 1% sodium citrate, and 1.5% agar [pH 6.5 to 6.6]). Micrococcus luteus ATCC 9341 was used for amoxicillin with Antibiotics Medium No. 2 (Oxoid, Hampshire, England). The assay sensitivities were 0.033 µg/ml for sanfetrinem and amoxicillin and 0.25 µg/ml for cefdinir.

For statistical analysis, Student's t test was used to compare bacterial numbers in the lungs, with P values of 0.05 or less being considered statistically significant. The limit of detection of bacterial numbers was 2.0 log CFU/lung.

Table 1 compares the in vitro antibacterial activity of sanfetrinem against the clinical isolates of five species with those of the reference drugs. These species were used in in vivo studies. Sanfetrinem showed potent activity against Staphylococcus aureus (methicillin-susceptible strains), with a MIC at which 90% of the isolates were inhibited (MIC₉₀) of 0.06 µg/ml, as well as against Streptococcus pyogenes (MIC₉₀, 0.008 µg/ml) and S. pneumoniae (MIC₉₀, 0.125 µg/ml). Against S. aureus and S. pneumoniae, sanfetrinem was more active than any reference drug except imipenem. Against methicillin-resistant S. aureus, the MIC range of sanfetrinem was from 0.125 to 128 (data not shown); sanfetrinem showed activity against a minority of these isolates. Sanfetrinem was also active against Escherichia coli and Klebsiella pneumoniae. The MIC₉₀s of sanfetrinem for these two isolates were 0.25 and 0.5 µg/ml, respectively. Levofloxacin was the only reference drug more active than sanfetrinem against K. pneumoniae.

The protective efficacy of sanfetrinem cilexetil against experimental bacteremic infections caused by S. aureus, S. pyogenes, E. coli, and K. pneumoniae in mice was compared with those of cefdinir and amoxicillin (Table 2). Sanfetrinem cilexetil showed good protective effects against infections caused by all of the tested strains except K. pneumoniae 3K25. Against infections caused by S. aureus Smith, S. aureus 5 (a -lactamase-producing strain), and S. pyogenes, the ED₅₀s of sanfetrinem cilexetil were 0.09, 0.71, and 0.08 mg/kg, respectively. The new trinem was 4.6 to 9.8 times as effective as cefdinir and 1.8 to 112.4 times as effective as amoxicillin in these tests. Sanfetrinem cilexetil was also highly active against E. coli C11 (ED₅₀, 0.28 mg/kg) and E. coli 311 (ED₅₀, 0.66 mg/kg) (a -lactamase producing strain) infections. These ED₅₀s make them 2.3 to 6.2 times as effective as cefdinir. Against K. pneumoniae 3K25, the ED₅₀ of sanfetrinem cilexetil was 25.6 mg/kg, which made it more effective than amoxicillin but less effective than cefdinir.

The therapeutic efficacy of sanfetrinem cilexetil against experimental respiratory tract infection caused by PSSP was compared with those of cefdinir and amoxicillin (Table 2). Sanfetrinem cilexetil had an ED₅₀ of 0.18 mg/kg. This was 1.6 times as effective as amoxicillin, while cefdinir showed little effectiveness in this model.

We compared the efficacy of sanfetrinem cilexetil with that of amoxicillin, usually referred to as the "gold standard" therapy for S. pneumoniae infections (5, 7), in eradicating experimental respiratory tract infections caused by PSSP and PRSP (Fig. 2 through 4). Against infection caused by PSSP, administration of sanfetrinem cilexetil at a dose of 10 mg/kg led to a drastic reduction in the number of viable bacterial cells and to complete elimination of bacteria from the lungs (below the limit of detection) after six doses. Amoxicillin also decreased the viable cell count, but 1.9 × 10² CFU were recovered from the lungs of one mouse (Fig. 2). The viable cell counts after six doses of either compound (safetrinem cilexetil at 10 mg/kg and amoxicillin at 10 mg/kg) were significantly lower than those in control mice (P < 0.01). Figures 3 and 4 show the results for respiratory tract infections caused by PRSP. Sanfetrinem cilexetil at a dose of 50 mg/kg produced a marked decrease in the number of viable bacterial cells. The viable counts at 85 h after challenge were significantly lower than those in control mice and mice treated with amoxicillin at 50 mg/kg (P < 0.01). At a dose of 10 mg/kg, however, viable cell counts in the lungs at 85 h after infection were almost the same as those at 24 h after infection (significant relative to control and amoxicillin-treated mice [P < 0.01]). Amoxicillin did not decrease viable bacterial cell counts at 50 mg/kg (significant relative to control mice [P < 0.01]) and had no effect at 10 mg/kg.

View larger version (16K): [in this window] [in a new window]   FIG. 2.   Effects of sanfetrinem cilexetil and amoxicillin against PSSP in the mouse respiratory tract. Mice were inoculated intranasally with 3.98 log CFU of S. pneumoniae TUH39 per mouse (MICs: sanfetrinem, 0.008 µg/ml; amoxicillin, 0.03 µg/ml). Each antibiotic dose was 10 mg/kg. Each line represents the average of data from five mice. a, value lower than for the control group at 85 h after infection (P < 0.01).

View larger version (19K): [in this window] [in a new window]   FIG. 3.   Effects of sanfetrinem cilexetil and amoxicillin at dosages of 10 mg/kg twice a day against PRSP in the mouse respiratory tract. Mice were inoculated intranasally with 4.00 log CFU of S. pneumoniae TUM741 per mouse (MICs: sanfetrinem, 0.25 µg/ml; amoxicillin, 1 µg/ml). Each line represents the average of data from five mice. a, value lower than for the control group at 85 h after infection (P < 0.01); b, lower than the value for amoxicillin at 85 h after infection (P < 0.01).

View larger version (18K): [in this window] [in a new window]   FIG. 4.   Effects of sanfetrinem cilexetil and amoxicillin at dosages of 50 mg/kg twice a day against PRSP in the mouse respiratory tract. Mice were inoculated intranasally with 4.26 log CFU of S. pneumoniae TUM741 per mouse (MICs: sanfetrinem, 0.25 µg/ml; amoxicillin, 1 µg/ml). Each line represents the average of data from five mice. a, value lower than for the control group at 85 h after infection (P < 0.01); b, lower than the value for amoxicillin at 85 h after infection (P < 0.01).

Concentrations of active drug in mouse plasma and lungs were measured after single oral administrations of sanfetrinem cilexetil, amoxicillin, and cefdinir at doses of 10 mg/kg. The pharmacokinetic parameters are shown in Table 3. The maximum concentration (C_max) of sanfetrinem in plasma was 7.60 µg/ml at 0.25 h after administration, a value higher than those for amoxicillin (3.74 µg/ml) and cefdinir (0.74 µg/ml). In the lungs, the C_maxs of sanfetrinem and amoxicillin were 1.94 and 1.11 µg/ml, respectively. Cefdinir was not detected in the lungs. The half-life of sanfetrinem (0.37 h) in plasma was shorter than those of amoxicillin (1.34 h) and cefdinir (2.32 h). The area under the curve from 0 h to infinity (AUC_0-) of sanfetrinem in plasma was 6.16 µg·h/ml, roughly the same as that of amoxicillin (7.23 µg·h/ml) and higher than that of cefdinir (2.28 µg·h/ml). The AUC_0-s of sanfetrinem and amoxicillin in the lungs were 1.56 and 2.98 µg·h/ml, respectively. Cefdinir was not detected in the lungs.

Recently, the increasing emergence of PRSP has become a critical problem (1). We therefore compared the efficacy of sanfetrinem cilexetil with that of the reference drugs in two mouse models of respiratory tract infections caused by PSSP and PRSP strains. The CBA/J mouse pneumonia model in particular has been reported to resemble human community-acquired S. pneumoniae pneumonia (11, 12). Results of these studies demonstrate not only that sanfetrinem cilexetil is highly effective in protecting mice against infection and in eradicating established infections but that it is more active than the reference drugs. Sanfetrinem cilexetil also shows potent protective efficacy against bacteremic infections in mice caused by S. aureus, S. pyogenes, and E. coli, including -lactamase-producing strains. These results presumably reflect sanfetrinem cilexetil's strong antibacterial activity in vitro and good pharmacokinetic behavior in mice.

In conclusion, these studies indicate that the new oral trinem sanfetrinem cilexetil is suitable for treatment of infections caused by a variety of bacteria.