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Antimicrobial Agents and Chemotherapy, October 2005, p. 4185-4196, Vol. 49, No. 10
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.10.4185-4196.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

In Vitro and In Vivo Antibacterial Activities of SM-216601, a New Broad-Spectrum Parenteral Carbapenem

Yutaka Ueda,* Katsunori Kanazawa, Ken Eguchi, Koji Takemoto, Yoshiro Eriguchi, and Makoto Sunagawa

Sumitomo Pharmaceuticals Research Division, 3-1-98 Kasugade-naka, Konohana, Osaka 554-0022, Japan

Received 15 April 2005/ Returned for modification 24 May 2005/ Accepted 22 July 2005


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ABSTRACT
 
SM-216601 is a novel parenteral 1ß-methylcarbapenem. In agar dilution susceptibility testing, the MIC of SM-216601 for 90% of the methicillin-resistant Staphylococcus aureus (MRSA) strains tested (MIC90) was 2 µg/ml, which was comparable to those of vancomycin and linezolid. SM-216601 was also very potent against Enterococcus faecium, including vancomycin-resistant strains (MIC90 = 8 µg/ml). SM-216601 exhibited potent activity against penicillin-resistant Streptococcus pneumoniae, ampicillin-resistant Haemophilus influenzae, Moraxella catarrhalis, Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis, with MIC90s of less than 0.5 µg/ml, and intermediate activity against Citrobacter freundii, Enterobacter cloacae, Serratia marcescens, and Pseudomonas aeruginosa. The therapeutic efficacy of SM-216601 against experimentally induced infections in mice caused by S. aureus, E. faecium, E. coli, and P. aeruginosa reflected its in vitro activity and plasma level. Thus, SM-216601 is a promising candidate for nosocomial bacterial infections caused by a wide range of gram-positive and gram-negative bacteria, including multiresistant pathogens.


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INTRODUCTION
 
The emergence of multiresistant gram-positive cocci such as methicillin-resistant staphylococci, vancomycin-resistant enterococci, and penicillin-resistant Streptococcus pneumoniae (PRSP) has reduced the value of antibacterial chemotherapy in recent years (3, 16). Resistance to ß-lactam antibiotics is a particularly serious concern because of the advantages of ß-lactams over other classes of antibacterials. A broad antibacterial spectrum, bactericidal activity, and an excellent safety profile make them suitable for empirical treatment of a variety of bacterial infections, especially for pediatric and immunocompromised patients in hospitals. Therefore, there is an urgent need for new ß-lactams effective against resistant pathogens.

We previously reported that a 2-(4-arylthiazol-2-ylthio)-1ß-methylcarbapenem, SM-17466, and its derivatives exhibited potent activity against methicillin-resistant Staphylococcus aureus (MRSA) corresponding to a high affinity for bacterial penicillin-binding protein 2a (PBP2a) (22, 28). SM-17466 showed a higher level of activity against Enterococcus faecium than existing carbapenems, prompting us to search for new carbapenems with potent activity against MRSA and vancomycin-resistant E. faecium (VRE) (25). Consequently, we identified SM-197436, SM-232721, and SM-232724, having a dihydropyrrole or tetrahydropyridine ring instead of the pyridium moiety of SM-17466 at the C-2 side chain, with improved activity against E. faecium (26). In addition, our observation that the dihydropyrrole derivative SM-197436 was more active against gram-negative bacteria than SM-232721 and SM-232724 suggested the possibility of developing novel broad-spectrum carbapenems (34). We examined the structure-activity relationships of a series of 2-(4-tetrahydropyridinylthiazol-2-ylthio)-1ß-methylcarbapenems and 4-dihydropyrrolyl thiazole analogs with regard to their antibacterial activities especially against gram-negative bacteria and their convulsant activity in mice and selected SM-216601 as a promising candidate for a broad-spectrum carbapenem (Fig. 1) (33).



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  		FIG. 1. Chemical structure of SM-216601.

In this study, we investigated the activity of SM-216601 against various clinical isolates in comparison with vancomycin, linezolid, and several other ß-lactam antibiotics. A time-kill study of SM-216601 for key resistant pathogens, such as MRSA, VRE, PRSP, ampicillin-resistant Haemophilus influenzae, and Pseudomonas aeruginosa was also done. In addition, the efficacy of SM-216601 against systemic infections caused by methicillin-susceptible S. aureus (MSSA), MRSA, Escherichia coli, and P. aeruginosa and experimental E. faecium subcutaneous abscesses was evaluated in mice.

(Part of this work was presented in abstract form at the 44th Interscience Conference on Antimicrobial Agents and Chemotherapy, 30 October to 2 November 2004, Washington, D.C. [abstr. F-330, p. 197].)


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MATERIALS AND METHODS
 
Organisms. Most of the clinical isolates used in this study were collected from different patients in various hospitals in Japan from 2001 to 2002. Twenty strains of clinical isolates of VRE were obtained from various institutions or hospitals in the United States and Europe. The ß-lactamase-producing organisms were from our bacterial collections. All isolates were maintained in glycerol broth at –80°C.

Antimicrobial agents. SM-216601, meropenem, and linezolid were synthesized in the laboratories of the Sumitomo Pharmaceuticals Research Division. Imipenem and cilastatin were prepared from Tienam (Banyu Pharmaceutical Co., Ltd., Tokyo, Japan). Panipenem was prepared from Carbenin (Sankyo Co., Ltd., Tokyo, Japan). Benzyl[14C]penicillin (PCG) was purchased from Amersham International, Plc. (Buckinghamshire, United Kingdom). The other antimicrobial agents were obtained from commercial sources.

Susceptibility testing. MICs were determined by the twofold serial agar dilution method with Mueller-Hinton agar (MHA; Nippon BD Company Ltd., Tokyo, Japan) unless otherwise specified. Susceptibility testing was performed with MHA supplemented with 5% defibrinated horse blood for streptococci and with 5% Fildes enrichment (BBL Microbiology Systems, Cockeysville, Md.) for H. influenzae. Brucella HK agar (Kyokutoh Seiyaku, Tokyo, Japan) supplemented with 5% defibrinated horse blood was used for the culture of anaerobic bacteria. The final inocula comprised approximately 104 and 106 CFU per spot for aerobic bacteria and anaerobic bacteria, respectively. Agar plates were incubated at 35°C for 18 to 24 h. Incubation was carried out anaerobically in GasPak jars (BBL) for anaerobes and in an atmosphere of 5% CO2 for streptococci, H. influenzae, and Moraxella catarrhalis. The MIC was defined as the lowest drug concentration that completely prevented visible growth.

Time-kill assay. The bactericidal activities of the drugs against each of the strains of S. aureus, E. faecium, S. pneumoniae, H. influenzae, and P. aeruginosa tested were assessed by conducting time-kill assays (21). The test organisms (about 105 CFU/ml) were precultured at 35°C for 1 h and consequently treated with the drugs at one-fourth, one, and four times the MIC with shaking at 35°C. Aliquots were removed 1, 2, 4, 6, 8, and 24 h after drug addition. An undiluted aliquot (50 µl) and 10-fold serial dilutions of the aliquot were plated onto 25 ml of agar for determining viable counts. The limit of viable counts was 20 CFU/ml.

Affinities for bacterial PBPs. Affinities for PBP2a of MRSA SP9099-9H (penicillinase-free, homologous resistant strain), PBP5 of E. faecium TL-3273 (ampicillin- and vancomycin-resistant strain), and PBPs of P. aeruginosa NCTC10490 and E. coli K-12 c600 were determined by a competition assay with PCG as previously reported (7, 23, 25, 35). Briefly, 100 µg of the bacterial membrane fraction was pretreated with drugs for 30 min at 30°C (MRSA) or 37°C (VRE) and then incubated with PCG for another 30 min, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For gram-negative bacteria, 300 µg of the membrane fraction was pretreated with drugs at 30°C for 10 min, followed by incubating with PCG for another 10 min. The reaction mixture was incubated at room temperature for 20 min after the addition of 2 µl of a solution containing 3 volumes of 20% sodium sarcosine and 1 volume of 180-mg/ml unlabeled PCG and centrifuged at room temperature for 30 min. The supernatant was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Binding affinity was measured by a BAS2000 Image Analyzer (Fuji Photofilms) and expressed as the 50% inhibitory concentration (IC50), i.e., the concentration (in micrograms per milliliter) that inhibited radiolabeling with PCG by 50% compared with the control.

Resistance to hydrolysis by DHP-I. The resistance of SM-216601 to hydrolysis by dehydropeptidase I (DHP-I) was determined with purified mouse renal DHP-I (8) and recombinant human DHP-I, which was prepared as reported by Adachi et al. (1). The activity of DHP-I was spectrophotometrically determined by measuring the hydrolysis of glycyldehydrophenylalanine as a substrate (6). The velocity of hydrolysis of carbapenems in the presence of mouse DHP-I (4.7 mU/ml) or human DHP-I (85 mU/ml) was expressed in micromoles per minute per unit. The relative rate of hydrolysis was also calculated as a ratio against the hydrolysis rate for imipenem, which was assigned a value of 1.00.

In vivo experiments. Three-week-old male slc:ICR mice weighing 11 to 13 g were purchased from Japan SLC, Inc. (Shizuoka, Japan), and adapted to standardized environmental conditions (temperature, 23 ± 2°C; humidity, 55% ± 10%) for 1 week before the experiments. All animal procedures were performed in accordance with the institution's guidelines for the humane handling, care, and treatment of research animals. SM-216601, imipenem, and meropenem were used as a mixture with an equal dose of cilastatin, a DHP-I inhibitor, in the animal experiments unless specified otherwise. All statistical analyses described below were performed with Statistical Analysis System (SAS) version 8.01 for Windows (SAS Institute, Inc., Cary, N.C.).

(i) Experimental bacterial septicemia in mice. Mice were administered 200 mg of cyclophosphamide per kg of body weight subcutaneously 4 days before infection. Overnight cultures of S. aureus Smith (MSSA) and SP-12249 (MRSA) in MHA were harvested and suspended with 8% gastric mucin (Difco) in phosphate-buffered saline. A 0.2-ml aliquot of the bacterial suspension was administered intraperitoneally to each of the cyclophosphamide-pretreated mice. Two hours after infection, groups of 10 mice were injected subcutaneously with a single dose of antibiotics. For E. coli SP-6088 and P. aeruginosa IFO3451, bacterial inoculation of normal mice was conducted as described above. Consequently, groups of 10 mice were subcutaneously injected with a single dose of antibiotics 2 h after infection with E. coli and with three doses of antibiotics 1, 2, and 3 h after infection with P. aeruginosa, respectively. The 50% effective dose (ED50) (in milligrams per kilogram of body weight) and 95% confidence intervals were calculated by the probit method from survival rates 7 days after infection.

(ii) Experimental E. faecium subcutaneous abscesses in mice. Each mouse was injected beneath the loose skin of the left groin with 0.5 ml of a bacterial diluent of E. faecium TL-3273 (approximately 108 CFU). SM-216601 and linezolid were administered subcutaneously 1 and 3 h after infection (six mice per group). The abscesses were excised 72 h after infection, at which time their formation could be confirmed visibly, and viable cell counts of the number of bacteria per abscess were made in duplicate by using standard plating procedures. Statistical analysis of the difference between drug-treated groups and a control group was performed by using Dunnett's test for multiple comparisons of significance.

(iii) Determination of plasma drug levels. A dose of 20 mg of antibiotic per kg was intravenously injected into three mice in each group. A sample of heart blood was obtained 5, 15, 30, 60, and 90 min after drug administration. The level of biologically active ß-lactams in plasma was determined by the bioassay method with Bacillus subtilis ATCC 6633 for SM-216601 and imipenem or with E. coli NIHJ for meropenem and ceftazidime as the indicator organism. Standard curves were made for the antibiotics in pooled mouse serum. The disk diffusion bioassay was performed in triplicate with 50 µl of serum from ß-lactam-administered mice with a standard solution. The levels of vancomycin and linezolid in plasma were determined by high-pressure liquid chromatography-UV detection. Standard curves were generated by linear regression. Samples with unknown concentrations of drugs in serum were calculated from the equation of the line. The pharmacokinetic parameters were calculated according to the moment analysis.


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RESULTS
 
In vitro antimicrobial activity. The comparative in vitro activities of SM-216601 and other antibiotics against 396 strains of gram-positive and gram-negative clinical isolates are given in Table 1. SM-216601 was highly active against both ß-lactam-susceptible and ß-lactam-resistant staphylococci. Notably, the MIC at which 90% of the strains are inhibited (MIC90) among 30 isolates of MRSA was 2 µg/ml and comparable to those of vancomycin (1 µg/ml) and linezolid (2 µg/ml). Twelve strains of MRSE were also susceptible to SM-216601, with MICs ranging from 0.125 to 1 µg/ml.


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  		TABLE 1. Antibacterial activities of SM-216601 against clinical isolates

Penicillin-susceptible streptococci were also highly susceptible to SM-216601, the highest MIC being ≤0.016 µg/ml. The MIC90 of SM-216601 against a total of 14 isolates of PRSP was 0.25 µg/ml, which was twofold less than that of panipenem but comparable to that of imipenem and 16-fold and 32-fold more potent than penicillin G and flomoxef, respectively.

The MICs of SM-216601 against 12 isolates of E. faecalis ranged from 1 to 2 µg/ml, which were similar to those of imipenem, ampicillin, vancomycin, and linezolid and superior to those of meropenem and flomoxef. The MICs of SM-216601 against 32 isolates of E. faecium, including 20 vancomycin-resistant strains, ranged from 0.5 to 16 µg/ml and were lower than those of the other ß-lactams tested. The MIC50 and MIC90 of SM-216601 were 4 and 8 µg/ml, which were twofold and fourfold higher than those of linezolid, respectively. The susceptibility of E. faecium to SM-216601 was not influenced by the vancomycin resistance phenotype (data not shown). Thus, SM-216601 proved to be the most active ß-lactam antibiotic tested against those two enterococci.

SM-216601 exhibited strong activity against E. coli, K. pneumoniae, H. influenzae, M. catarrhalis, and Proteus mirabilis, with MIC90s of less than 0.5 µg/ml, which were roughly comparable to those of imipenem or panipenem. The activity of SM-216601 against ampicillin-resistant H. influenzae (MIC90, 0.25 µg/ml) was particularly notable. The MIC90s against Serratia marcescens, Enterobacter cloacae, and Citrobacter freundii ranged from 4 to 16 µg/ml, which were higher than those of other carbapenems but comparable to those of flomoxef. In addition, SM-216601 showed marginal activity against P. aeruginosa: the MIC90 (32 µg/ml) was two- to eightfold higher than those of other carbapenems. In terms of its effect on anaerobic bacteria, SM-216601 showed strong activity against peptostreptococci and B. fragilis and was similar in potency to the other carbapenems tested.

Time-kill assays. In time-kill assays, SM-216601 and quinupristin-dalfopristin caused a 2-log reduction in the CFU of S. aureus SP-12249 (MRSA) at the MIC and at four times the MIC (4 µg/ml) until 4 h, whereas the killing rates of vancomycin were relatively low and linezolid showed almost no bactericidal activity at any concentration tested. After 24 h of incubation, no bacterial cell was observed when SM-216601 and vancomycin were applied at four times the MIC, while the other two agents could not eradicate MRSA over the highest concentrations (Fig. 2A). For vancomycin- and ampicillin-resistant E. faecium TL-3273, 8 µg/ml of SM-216601 caused a 4-log reduction after 24 h; its killing activity was the greatest among the compounds tested (Fig. 2B). SM-216601 and the other carbapenems tested were highly bactericidal against S. pneumoniae 181. A four-log reduction by those carbapenems at concentrations at the MIC and 1/4x the MIC was achieved at 6 h. The bactericidal activity of vancomycin was slightly weaker than that of any carbapenem. Ampicillin exhibited a potent killing effect similar to that of the carbapenems, but its effective concentration was much higher (Fig. 2C). SM-216601 caused a time-dependent reduction in viable cells of H. influenzae 231, which is a ß-lactamase-negative, ampicillin-resistant strain, at the MIC and four times the MIC; its killing kinetics was similar to those of ampicillin and meropenem, but the MIC of SM-216601 was the lowest of the three ß-lactams. Meanwhile, cefotaxime and imipenem were less bactericidal than SM-216601. Notably, imipenem did not show sufficient killing activity, even at four times the MIC (Fig. 3A). Although the MIC of SM-216601 against P. aeruginosa IFO3451 was two- to eightfold higher than the MICs of other carbapenems, SM-216601 achieved a time-dependent decrease in the number of viable cells at concentrations above the MIC. The killing activity of SM-216601 was similar to those of existing carbapenems (Fig. 3B).



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  		FIG. 2. Bactericidal activities of SM-216601 and reference compounds against (A) S. aureus SP-12249 (MRSA), (B) E. faecium TL-3273, and (C) S. pneumoniae 181 (PRSP).



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  		FIG. 3. Bactericidal activities of SM-216601 and reference compounds against (A) H. influenzae 231 (ß-lactamase negative, ampicillin resistant) and (B) P. aeruginosa IFO3451.

Affinity for bacterial PBPs. The IC50s of SM-216601 and imipenem for PBP2a of MRSA strain SP-9099-9H and for PBP5 of vancomycin- and ampicillin-resistant E. faecium TL-3273 were determined by using a competition assay with PCG (Table 2). The MIC of SM-216601 for both strains was 2 µg/ml and correlated well with the corresponding IC50s, 1 µg/ml against MRSA and 2 µg/ml against E. faecium. A comparison of the IC50s and corresponding MICs of SM-216601 and imipenem revealed that the improvement in the anti-MRSA and anti-E. faecium activities of SM-216601 could be ascribed to the activity against those low-affinity PBPs.


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  		TABLE 2. Binding affinities of SM-216601 and imipenem for PBP2a of S. aureus SP-9099-9H and PBP5 of E. faecium TL-3273

The binding affinities of SM-216601 and meropenem for the PBPs of E. coli K-12 c600 and P. aeruginosa NCTC10490 are shown in Table 3. As for E. coli, SM-216601 exhibited potent binding affinities for the PBPs, except for PBP4; the IC50s of PBP2 (0.18 µg/ml) and PBP5/6 (0.54 µg/ml) showed a good correlation with the MIC (0.25 µg/ml). In accordance with a previous study (24), meropenem strongly bound PBP2 and PBP4 but not PBP5/6 of E. coli. The IC50s of SM-216601 for PBP1, -2, -3, and -4 of P. aeruginosa were two- to threefold higher than those of meropenem, but SM-216601 exhibited a higher affinity for PBP 5/6 than did meropenem. Thus, an interesting difference in affinity for gram-negative bacterial PBPs was observed between the two carbapenems. However, the affinities of SM-216601 and meropenem for the PBPs of P. aeruginosa could not fully explain the differences among the MICs of the carbapenems tested.


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  		TABLE 3. Binding affinities of SM-216601 and meropenem for gram-negative bacterial PBPs

Antimicrobial activity against ß-lactamase-producing bacteria. As shown in Table 4, SM-216601 maintained potent activity against E. coli, Providencia rettgeri, E. cloacae, and Morganella morganii producing cephalosporinase (group 1 ß-lactamase). The MICs of SM-216601 were higher than those of imipenem for other cephalosporinase-producing strains but still lower than those of flomoxef, even with a large inoculum of bacteria (108 CFU/ml). In addition, SM-216601 exhibited good activity against E. coli and K. pneumoniae producing extended-spectrum ß-lactamases. However, there was no activity against carbapenemase (group 3 ß-lactamase) producers, consisted with the results obtained with imipenem and meropenem.


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  		TABLE 4. Activities of SM-216601 against various ß-lactamase-producing gram-negative bacteria

Resistance to hydrolysis by DHP-I. In advance of animal studies, the resistance of SM-216601 to enzymatic hydrolysis was tested with renal DHP-I from mice and recombinant human DHP-I. The rate of SM-216601 hydrolysis by mouse DHP-I was 0.803 µmol/min/U, and that by human DHP-I was 0.029 µmol/min/U. These DHP-I susceptibilities were similar to those of meropenem (0.820 µmol/min/U by mouse DHP-I and 0.027 µmol/min/U by human DHP-I) but significantly different from those of imipenem (0.499 and 0.082 µmol/min/U, respectively). Since, in contrast to human DHP-I, SM-216601 was more susceptible to mouse DHP-I than imipenem was, we coadministered equal amounts of cilastatin with SM-216601 and other carbapenems for the following in vivo studies to avoid a species-specific effect by DHP-I on the metabolism of carbapenems in mice.

In vivo efficacy against gram-positive and gram-negative bacteria. The therapeutic efficacy of SM-216601 (with cilastatin) against systemic infections with MSSA and MRSA in immunosuppressed mice was determined (Table 5). The MICs of SM-216601, vancomycin, linezolid, and imipenem against S. aureus strain Smith were 0.016, 1, 2, and 0.016 µg/ml, respectively. The ED50 of SM-216601 against infection with the strain was 0.09 mg/kg, which was comparable to that of imipenem and much lower than the ED50 of vancomycin or linezolid. Meanwhile, SM-216601 showed in vitro activity against the MRSA SP-12249 strain comparable to those of vancomycin and linezolid, with an MIC of 1 µg/ml. The ED50 of SM-216601 against MRSA infection was 2.89 mg/kg, equivalent to the values for vancomycin and linezolid. In this model, imipenem showed little protective activity (ED50 = 14.93 mg/kg), which seemed to be consistent with its low level of in vitro activity. These results suggested that the in vivo efficacy of SM-216601 against these MSSA and MRSA strains was consistent with the corresponding level of in vitro activity. SM-216601 also had potent efficacy against gram-negative bacterial infections in mice (Table 6). The ED50s of SM-216601 against systemic infection with E. coli SP-6088 and P. aeruginosa IFO3451 were between those of meropenem and ceftazidime, regardless of the MICs of SM-216601, which were higher than those of meropenem or ceftazidime. Thus, the difference in in vivo efficacy between SM-216601 and ceftazidime was not reflected in their in vitro efficacies, even considering the favorable pharmacokinetics of SM-216601 in mice indicated by a comparison of the concentration of the drug 5 min after administration (C5 min) and its elimination half-life (t1/2) to those of ceftazidime, which might be expected to result in a prolonged time above the MIC of SM-216601 (see Table 8).


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  		TABLE 5. Activity of SM-216601 against S. aureus systemic infection in immunosuppressed micea


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  		TABLE 6. Activities of SM-216601 against E. coli and P. aeruginosa systemic infections in micea


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  		TABLE 8. Pharmacokinetic parameters of SM-216601 in micea

We next determined the efficacy of SM-216601 against E. faecium by using a subcutaneous abscess mouse model (Table 7). The strain of E. faecium used in this model, TL-3273, exhibited reduced susceptibility to ampicillin and vancomycin (MICs, 128 µg/ml and 256 µg/ml, respectively) but was susceptible to SM-216601 and linezolid (both MICs, 2 µg/ml). This strain formed visible abscesses in mice at 3 days after subcutaneous inoculation of approximately 108 CFU of bacteria, and the number of organisms in abscesses of untreated mice ranged from 107 to 108 CFU in multiple experiments. SM-216601 dose dependently reduced bacterial numbers in abscesses following two subcutaneous injections of 7.5, 20, and 40 mg/kg/dose and caused a more-than-2-log reduction at the highest dose. Linezolid also caused a 0.5-log reduction of bacterial numbers at 40 mg/kg/dose. Its activity at 40 mg/kg/dose was weaker than even that of SM-216601 at 7.5 mg/kg/dose.


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  		TABLE 7. Activity of SM-216601 against experimental E. faecium TL-3273 subcutaneous abscesses in micea

Pharmacokinetics in mice. The pharmacokinetics of SM-216601 administered intravenously at a dose of 20 mg/kg are compared with those of imipenem, vancomycin, and linezolid (experiment 1) and those of meropenem and ceftazidime (experiment 2) in Table 8. The C5 mins of SM-216601, imipenem, vancomycin, and linezolid were 74.6, 53.8, 37.8, and 26.0 µg/ml, respectively. The area under the plasma concentration-time curve (AUC) for SM-216601 was 2,054 µg min/ml, the value being 1.2- to 2.7-fold higher than the values of the reference agents tested. The t1/2 of SM-216601 was 17.8 min, whereas those of imipenem, vancomycin, and linezolid were 7.7, 16.5, and 54.9 min, respectively. SM-216601 exhibited a notably higher C5 min and AUC than those of meropenem and ceftazidime. In addition, the t1/2 of SM-216601 (20.8 min) was approximately twofold longer than those of meropenem (10.1 min) and ceftazidime (11.6 min).


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DISCUSSION
 
A new parenteral carbapenem, SM-216601, exhibited improved activity against methicillin-resistant staphylococci and E. faecium, distinguishing it from existing carbapenems. The notably high affinities of SM-216601 for PBP2a of MRSA and PBP5 of ampicillin-resistant E. faecium suggest the importance of the C-2 side chain containing a lipophilic thiazole moiety for binding to these low-affinity PBPs and consequently for sufficient antibacterial activities.

SM-216601 was highly active against E. coli, K. pneumoniae, M. catarrhalis, and ampicillin-resistant H. influenzae, which are frequently isolated as causative bacteria from patients with a variety of nosocomial infections, especially respiratory infections. In addition, SM-216601 was less active against E. cloacae, C. freundii, S. marcescens, and P. aeruginosa than were other carbapenems, but the activity was still comparable to or higher than that of the cephalosporin flomoxef.

We previously reported that the anti-gram-negative bacterial activities of a series of 2-thiazolcarbapenem derivatives, including SM-216601, were correlated with their physicochemical properties, which were well known to affect the outer membrane permeability of the carbapenems (33). For instance, increasing the lipophilicity of the C-2 side chain tended to reduce the antibacterial activities against E. cloacae, C. freundii, S. marcescens, and P. aeruginosa, which were bacterial species possessing low outer membrane permeability. Although a substantial difference in PBP affinity was observed between SM-216601 and meropenem in this study (Table 3), it is not likely that this explains the difference in the MICs among carbapenems tested against E. coli and P. aeruginosa. Thus, these observations suggest that the outer membrane permeability of SM-216601 affects its anti-gram-negative bacterial activity.

It is well known that carbapenems mainly pass through the outer membrane of P. aeruginosa via a specific porin channel, the outer membrane D2 protein (OprD) (29, 31), and that the uptake of carbapenems by OprD is interfered with by basic amino acids in the culture medium (30), which thus results in a reduction in the antipseudomonal activity (19). In this context, we compared the MICs of SM-216601 against the P. aeruginosa PAO1 strain, which has been extensively studied in previous reports (9), between MHA and nutrient agar (NA). As a result, the MIC of SM-216601 in NHA was 16 µg/ml while the MIC was 1 µg/ml (16-fold decrease) in NA, whose basic amino acid content was lower than that of MHA. In addition, the degree of MIC change of SM-216601 in NA was more pronounced than those of imipenem (twofold decrease), meropenem (eightfold decrease), and panipenem (fourfold decrease). These unpublished observations may suggest the involvement of OprD in the uptake of SM-216601 by P. aeruginosa. Furthermore, meropenem but not imipenem has been reported to be a substrate of certain efflux systems in the outer membrane of P. aeruginosa, and overexpression of efflux pumps results in reduced susceptibility to meropenem (17). Due to the structural similarities of SM-216601 to meropenem, such as the presence of the 1ß-methyl group and a relatively weak basic C-2 side chain, the possible involvement of an efflux system(s) in its antipseudomonal activity cannot be excluded.

The in vivo efficacies of SM-216601 in a murine septicemia model of MRSA and a subcutaneous abscess model of E. faecium seemed to reflect its potent in vitro activities against both bacterial strains tested. The efficacies of SM-216601 were stronger than those of linezolid, although these agents had the same MICs. In contrast to the bacteriostatic activities of linezolid against those strains, the bactericidal activity of SM-216601 might contribute to such high in vivo efficacies. The ED50s of SM-216601 against murine systemic infections with E. coli and P. aeruginosa were higher than those of meropenem, and the difference between the two carbapenems may be consistent with their in vitro activities. On the other hand, SM-216601 was more potent than ceftazidime in those models although its MICs were higher than those of ceftazidime. As shown in Table 8, the better pharmacokinetics of SM-216601 than that of ceftazidime in mice could be an explanation. However, the significant difference between the in vivo efficacies of meropenem and ceftazidime, their MICs and pharmacokinetic parameters in mice being similar, suggests that another factor(s) superior to cephalosporins, such as the time above the MIC necessary to exhibit antibacterial effects in mice, might have a positive impact on the efficacies of both SM-216601 and meropenem.

Since the 1990s, nosocomial infections with multiresistant gram-positive bacteria have been recognized as a serious problem because the treatment against such pathogens has been limited (4). In addition, since multiresistant gram-positive bacteria often cause polymicrobial infections with gram-negative bacteria, superinfection is a concern for the long-term treatment of narrow-spectrum antibacterials such as glycopeptides or an oxazolidinone. In this context, combination therapy with vancomycin or linezolid and broad-spectrum agents such as expanded-spectrum cephalosporins have been used for empirical therapy in severe cases of nosocomial infections in which the involvement of multiresistant gram-positive pathogens is suspected. However, the risk of adverse reactions and undesired progression of drug resistance due to the overuse of narrow-spectrum or bacteriostatic agents is considered a major drawback of this kind of treatment (2b). Thus, monotherapy with a highly tolerated agent effective against both multiresistant gram-positive bacteria and major gram-negative bacteria is highly desirable. Although a number of new cephalosporin and carbapenem compounds targeting MRSA have been disclosed (11, 27), the development of a broad-spectrum ß-lactam still appears challenging. An increase in the anti-MRSA activity of ß-lactam compounds often results in a reduction in the level of activity against gram-negative bacteria, with few exceptions (2a, 10, 12-15, 20, 32, 34). In addition, improving the activity against E. faecium is also difficult and only a few ß-lactams have been reported to be effective against E. faecium (18, 25, 34).

SM-216601 showed an antibacterial activity which extends to MRSA and VRE, in comparison with existing carbapenems. Moreover, this carbapenem exhibited better pharmacokinetics than imipenem and meropenem in mice. Similar pharmacokinetics were observed in rats, dogs, and cynomolgus monkeys (data not shown), suggesting a long-acting pharmacokinetic profile of SM-216601 in humans. In conclusion, SM-216601 should be a promising candidate as a broad-spectrum ß-lactam antibiotic for the treatment of nosocomial infections by a number of gram-positive and gram-negative bacteria, especially multiresistant pathogens, including MRSA and VRE.


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ACKNOWLEDGMENTS
 
We thank Y. Sumita for suggestions and acknowledge the excellent technical assistance of K. Urasaki and Y. Hirai.


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FOOTNOTES
 
* Corresponding author. Mailing address: Discovery Research Laboratories II, Sumitomo Pharmaceuticals Research Division, 3-1-98 Kasugade-naka, Konohana, Osaka 554-0022, Japan. Phone: 81-6-8466-5271. Fax: 81-6-6466-5491. E-mail: yutakau{at}sumitomopharm.co.jp. Back


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Antimicrobial Agents and Chemotherapy, October 2005, p. 4185-4196, Vol. 49, No. 10
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.10.4185-4196.2005
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