ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1992, p. 1894-1901 0066-4804/92/091894-08$02.00/0

Vol. 36, No. 9

Copyright © 1992, American Society for Microbiology

In Vitro and In Vivo Antibacterial Activities of E1077, a Novel Parenteral Cephalosporin with a Broad Antibacterial Spectrum KATSURA HATA,* MASAKO OTSUKI, AND TAKESHI NISHINO Department of Microbiology, Kyoto Pharnaceutical University, 5-Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607, Japan Received 3 February 1992/Accepted 3 July 1992

E1077 is a new iijectable cephalosporin with a broad spectrum of antibacterial activity against gram-positive and gram-negative bacteria, including staphylococci and Pseudomonas aeruginosa. The in vitro activities of E1077 against clinical isolates of methicillin-susceptible Staphylococcus aureus (MIC of E1077 for 90%o of the strains tested [MIC,0], 0.78 ;g/ml) and methicillin-resistant S. aureus (MIC90, 50 pLg/ml) were similar to those of cefpirome and flomoxef. Against Enterococcusfaecalis (MIC,0, 6.25 ;ag/ml), E1077 was the most active of the drugs tested and four times more active than cefpirome. The MIC90s of E1077 for streptococci, Haemophilus influenzae, and Neisseria gonorrhoeae ranged from 0.05 to 0.78 ,ug/ml; E1077 was similar in activity to cefpirome. E1077 inhibited 90% of most species of the family Enterobacteriaceae at concentrations of 100 0.39->100 6.25->100 0.20->100 3.13-> 100 0.20-> 100

6.25 50 >100 50 >100 12.5

Staphylococcus epidermidis (67)

E1077 Cefpirome Ceftazidime Cefuzonam Cefotaxime Flomoxef

0.39-100 0.20-100 0.20-> 100 0.10-> 100 0.20-> 100 0.20-100

0.78 0.78 6.25 0.78 3.13 1.56

6.25 3.13 25 3.13 12.5 12.5

Streptococcus pneumoniae (33)

E1077 Cefpirome Ceftazidime Cefuzonam Cefotaxime Flomoxef

100 0.39-100 6.25->100

Neisseria gonorrhoeae (31)

E1077 Cefpirome Ceftazidime Cefuzonam Cefotaxime Flomoxef

Haemophilus influenzae (52)

E1077

Branhamella catarrhalis (52)

0,012-0.05

0.20-0.39

0.39 0.78 12.5 0.39 1.56 0.39

3.13 6.25 >100 25 >100 >100

0.78 1.56 12.5 0.78 3.13 1.56

50 100 >100 100 >100 100

6.25 25 >100 >100 >100 >100

100 0.10->100 0.20->100

0.05 0.20 0.39 0.39 0.39 25

1.56 3.13 100 100 >100 >100

0.05-0.39 0.10-0.78 0.10-50 0.20-50 0.10-50 1.56-100

0.05 0.10 0.20 0.20 25

0.20 0.78 25 25 25 100

0.05 0.10 0.78 0.39 0.39 6.25

0.78 3.13 >100 50 50 >100 12.5 25 100 100 >100 >100

0.05-0.39 0.05-0.39 0.05-0.20

0.012-0.78 0.025-0.78 0.10-1.56 '0.006-3.13 0.012-1.56 0.05-3.13

0.012-3.13 0.025-3.13 0.05->100 0.012-50 0.012-100

0.39

E1077

0.10-50 0.10-100 0.20-100 0.39->100 0.20->100 0.39->100

1.56 1.56 1.56 3.13 3.13 25

0.05-0.78 0.05-1.56 0.05-1.56 0.05-1.56

0.05-0.20 0.20-3.13

0.05 0.10 0.10 0.20 0.05 0.39

0.20 0.39 0.20 0.78 0.20 3.13

0.05-50 0.05-25 0.05-3.13 0.025-12.5 0.025-25 0.20-0.78

0.78 0.39 0.10 0.20 0.20 0.39

12.5 3.13 0.39 0.78 1.56 0.78

0.025-3.13 0.025-6.25

0.05 0.05 0.20 0.20

0.39 0.78 25 6.25 12.5 100

Flomoxef E1077

Cefpirome Ceftazidime Cefuzonam Cefotaxime

Flomoxef E1077

Cefpirome Ceftazidime Cefuzonam Cefotaxime

Flomoxef Morganella morganii (40)

50%

0.10-100

Ceftazidime Cefuzonam Cefotaxime

Proteus vulgaris (40)

90%

Range

Flomoxef Cefpirome

Proteus mirabilis (38)

MIC (,Lg/ml)

Antibacterial

E1077

Cefpirome Ceftazidime Cefuzonam Cefotaxime Flomoxef

0.10-100 0.05-25 0.025-50 0.39->100

0.20 1.56

Continued on following page

IN VITRO AND IN VIVO ACTIVITIES OF E1077

VOL. 36, 1992

1897

TABLE 1-Continued Organism (no. of strains)

Providencia rettgeri (36)

Antibacterial agent

E1077

Cefpirome

Ceftazidime Cefuzonam Cefotaxime Flomoxef

Pseudomonas aeruginosa (50)

E1077

Cefpirome

Ceftazidime Cefuzonam Cefotaxime

Flomoxef Acinetobacter calcoaceticus (32)

E1077

Cefpirome Ceftazidime Cefuzonam Cefotaxime

Flomoxef

tions of E1077. After 3 h of incubation, morphological changes of cells were examined by differential interference microscopy for S. aureus and by phase-contrast microscopy for P. aeruginosa. Systemic infection in mice. The protective effect of E1077 against systemic infections in mice was determined as described previously (3). Male ddY strain mice weighing 18 to 20 g were used. The bacterial strains, except for Streptococ-

MIC (,ug/ml) Range

0.012-1.56 0.012-6.25 0.10-12.5 0.012-3.13 '0.006-0.78 0.05-50 0.78-25 1.56-25 0.78-25 12.5-> 100 12.5-> 100 > 100-> 100 0.39-25 0.78-50 1.56-50 3.13-100 1.56-100 3.13->100

50%

0.025 0.10 0.39 0.20 0.20 0.20

1.56 6.25 3.13 25 25 > 100

1.56 3.13 6.25 25 12.5 50

90% 0.39 1.56 3.13 1.56 0.78 12.5 6.25 12.5 6.25 100 50 > 100 12.5 12.5 25 100 50 >100

cuspneumoniae type III, were cultured overnight at 37°C in nutrient broth (Nissui). For S. pneumoniae, broth was supplemented with 10% horse serum. Challenge organisms were prepared in nutrient broth and 3% hog mucin (Orthana Kemisk Fabrik A/S) in sterile water. The mice were injected intraperitoneally with 0.5 ml of the bacterial suspension. The challenge inoculum was sufficient to kill 100% of untreated control mice within 48 h postinfection, with the exception of

FIG. 1. Differential interference micrographs of S. aureus Smith exposed to E1077 for 3 h. Panels: A, untreated control; B, 0.025 ,ug/ml; C, 0.1 pLg/ml; D, 0.39 pg/ml; E, 1.56 .g/ml; F, 6.25 ,g/ml.

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ANTIMICROB. AGENTS CHEMOTHER.

HATA ET AL.

FIG. 2. Phase-contrast micrographs of P. aeruginosa E-2 exposed to E1077 for 3 h. Panels: A, untreated control; B, 0.39 1Lg/ml; C, 1.56 ,ug/ml; D, 6.25 ,ug/ml; E, 25 ,ug/ml; F, 100 ,ug/ml.

miCe infected with Klebsiella pneumoniae KC-1 or P. aeruginosa 15846, which died within 3 days after challenge. The antibiotic doses were administered subcutaneously in a volume of 0.2 ml of normal saline 2 h after infection. At least five levels (serial twofold doses) of the test compounds were employed with 10 mice at each level, and the 7-day survival ratios were determined. The 50% effective dose (ED50) was calculated by the Litchfield-Wilcoxon method (7). RESULTS

Antibacterial activity. The antibacterial activities of E1077, cefpirome, ceftazidime, cefuzonam, flomoxef, and cefotaxime against clinical isolates are shown in Table 1. E1077 was active against staphylococci. The MIC90 of E1077 for methicillin-susceptible S. aureus strains was 0.78 ,ug/ml, which was similar to those of cefpirome, cefuzonam, and flomoxef and 1/16 to 1/4 times those of cefotaxime and ceftazidime. The MIC50 and MIC90 of E1077 for MRSA strains were 6.25 and 50 ,ug/ml, respectively. Its MIC50 was similar to that of flomoxef and eight times lower than those of cefuzonam and cefpirome, and its MIC-w was similar to those of cefpirome, cefuzonam, and flomoxef. Ceftazidime and cefotaxime were inactive against MRSA, with MIC50s of >100 ,ug/ml. The activity of E1077 was comparable to that of cefpirome and cefuzonam against Staphylococcus epidermidis. Against streptococci, E1077 was comparable to cefpirome and cefotaxime and 4 to 16 times more active than ceftazidime and flomoxef. However, E1077 was four times less active than cefuzonam against S. pneumoniae. E1077, with an MIC90 of 6.25 ,ug/ml for Enterococcus faecalis, was the most potent of the compounds tested. It

was four times more active than cefpirome. The other reference compounds did not inhibit most strains of E. faecalis at a concentration of 100 ,ug/ml or less. The MIC90s of E1077 for N. gonorrhoeae and H. influenzae were 0.05 and 0.20 ,ug/ml, respectively. E1077 was 4 to 16 times more active against these species than was flomoxef and was comparable to the other compounds tested. Against B. catarrhalis, E1077, with an MIC90 of 0.78 ,ug/ml, was four times more active than cefpirome but eight times less active than ceftazidime. In general, E1077 and cefpirome were more active against most members of the family Enterobacteriaceae than were the other compounds tested. The MIC90s of E1077 for Escherichia coli and K pneumoniae were 0.05 and 0.20 ,ug/ml, respectively. E1077 was equal to or four times more active than cefpirome against these species. The MIC90s of E1077 for Enterobacter cloacae, Enterobacter aerogenes, and Citrobacter freundii were 1.56, 0.20, and 0.78 jig/ml, respectively, and E1077 was equal to or four times more active than cefpirome and 64 to > 128 times more active than ceftazidime, cefuzonam, cefotaxime, and flomoxef against these species. Against Serratia marcescens, E1077 was comparable to cefpirome and eight or more times more active than the other reference compounds, with an MIC90 of 12.5 ,ug/ml. Against Morganella morganii and Providencia rettgeri, E1077 was equal to or 4 times more active than cefpirome and 4 to 64 times more active than ceftazidime and cefuzonam, with MIC90s of 0.39 and 0.39 ,g/ml, respectively. E1077 was similar in activity to cefpirome, ceftazidime, and cefotaxime and four times more active than cefuzonam against Proteus mirabilis. E1077 was four or more times less active than these reference compounds against Proteus vulgaris.

IN VITRO AND IN VIVO ACTIVITIES OF E1077

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1899

TABLE 2. Protective effects of E1077 and reference compounds against systemic infection in mice Organism

Staphylococcus aureus Smith

Challenge dose, CFU/mouse (no. of LD50Sa)

3.3 x 106 (260)

MIC

(F±g/ml)

ED50, mg/kg (95% confidence limits)

Cefpirome Ceftazidime Cefuzonam Flomoxef

0.39 0.78 6.25 0.39 0.39

0.300 (0.198-0.455) 0.987 (0.728-1.34) 9.17 (6.86-12.3) 3.07 (2.02-4.65) 0.613 (0.351-1.07)

Test compound

E1077

Streptococcus pneumoniae type III

6.6 x 10 (90)

E1077 Cefpirome Ceftazidime Cefuzonam Flomoxef

0.025 0.025 0.20 0.012 0.20

Escherichia coli KC-14

1.5 x 105 (90)

E1077 Cefpirome Ceftazidime Cefuzonam Flomoxef

0.025 0.05 0.20 0.10 0.05

Klebsiella pneumoniae KC-1

2.4 x 104 (26)

E1077

0.025 0.05 0.10 0.10 0.05

Cefpirome Ceftazidime Cefuzonam Flomoxef

Citrobacterffreundii B3

1.7 x 106 (22)

E1077 Cefpirome Ceftazidime Cefuzonam Flomoxef

Serratia marcescens T-55

2.1 x 106 (80)

E1077

Cefpirome Ceftazidime Cefuzonam Flomoxef Pseudomonas aeruginosa 15846

Acinetobacter calcoaceticus Ac-54

7.5 x 104 (74)

2.2 x 105 (18)

2.18 (1.23-3.86) 4.19 (2.75-6.38) 37.0 (20.2-67.9) 12.7 (10.5-15.5) 2.78 (1.79-4.31) 0.0094 (0.0071-0.0125) 0.0173 (0.0112-0.0267) 0.0369 (0.0270-0.0504) 0.0707 0.102 (0.0839-0.124) 0.247 (0.149-0.410) 0.332 (0.240-0.460) 0.939 (0.595-1.48) 0.852 (0.541-1.34) 12.3 (10.1-14.9)

0.78 1.56 100 25 100

0.152 (0.108-0.214) 0.224 (0.139-0.362) 30.6 22.5 (14.6-34.7) 16.3 (11.4-23.4)

0.78 0.39 0.39 0.78 6.25

0.187 (0.103-0.341) 0.131 (0.071-0.242) 0.207 (0.141-0.303) 0.948 (0.540-1.67) 3.35 (2.10-5.34)

Cefpirome Ceftazidime Cefuzonam Flomoxef

0.78 6.25 3.13 50 >100

2.66 (1.85-3.83) 11.7 (7.26-18.9) 5.70 (3.64-8.94) >200 >200

E1077 Cefpirome Ceftazidime Cefuzonam Flomoxef

1.56 3.13 6.25 25 50

4.88 (3.02-7.89) 9.31 (5.47-15.9) 8.90 (6.02-13.2) 97.1 (52.4-179.8) 101.9 (63.0-165.1)

E1077

a LD50, 50% lethal dose.

Against P. aeruginosa, the activity of E1077 was similar to that of ceftazidime. The MIC50 and MIC90 of E1077 for P. aeruginosa were 1.56 and 6.25 ,ug/ml, respectively. The MIC50 of E1077 was equal to or four times lower than those of ceftazidime and cefpirome. E1077 was as active as cefpirome and ceftazidime against Acinetobacter calcoaceticus, with an MIC50 of 1.56 ,ug/ml and an MIC90 of 12.5

p,g/ml. Morphological effects. The morphological changes induced by E1077 in S. aureus Smith and P. aeruginosa E-2 were examined by differential interference and phase-contrast microscopy, respectively. In S. aureus (Fig. 1), E1077 induced a little swelling at a concentration of one-fourth of the MIC and lysis above the MIC. In P. aeruginosa (Fig. 2), E1077 induced formation of filamentatous cells from nor-

mally rod-shaped cells at concentrations of one-fourth to four times the MIC. When P. aeruginosa cells were exposed to high concentrations of E1077 (25 and 100 ,ug/ml), formation of spheroplasts and bulges and cell lysis were observed. Activity against systemic infections in mice. The in vivo activity of E1077 against systemic infections in mice is shown in Table 2. E1077 showed in vivo activity against gram-positive and gram-negative bacterial infections, as reflected by its in vitro activity. The ED50 of E1077 against S. aureus was 0.300 mg/kg; E1077 was comparable to flomoxef, 3 times more effective than cefpirome, and 10 to 30 times more effective than cefuzonam and ceftazidime. Against S. pneumoniae infection, the activity of E1077 was comparable to those of cefpirome and flomoxef and about 5

1900

ANTIMICROB. AGENTS CHEMOTHER.

HATA ET AL.

to 17 times those of ceftazidime and cefuzonam. The activities of E1077 against E. coli and K pneumoniae infections, with ED50s of 0.0094 and 0.247 mg/kg, respectively, were similar to those of cefpirome and higher than those of the other reference compounds. The ED50 of E1077 against C. freundii was 0.152 mg/kg; E1077 was similar in efficacy to cefpirome and 10 to 20 times as effective as the other reference compounds. Against S. marcescens infection, the activity of E1077 (ED50, 0.187 mg/kg) was comparable to those of cefpirome and ceftazidime and about 5 to 20 times those of cefuzonam and flomoxef. E1077 was also effective against P. aeruginosa infection. The activity of E1077, with an ED50 of 2.66 mg/kg, was similar to that of ceftazidime and four times that of cefpirome, while cefuzonam and flomoxef were ineffective against P. aeruginosa infection. Against A. calcoaceticus infection, the activity of E1077 (ED50, 4.88 mg/kg) was comparable to those of cefpirome and ceftazidime and about 20 times higher than those of cefuzonam and flomoxef. DISCUSSION

E1077 is a new injectable cephalosporin, which has been described by Watanabe et al. (13), with a broad antibacterial spectrum and potent antibacterial activity. One of the antibacterial characteristics of E1077 is its activity against S. aureus and E. faecalis. The activity of E1077 against methicillin-susceptible S. aureus was comparable to those of cefpirome, cefuzonam, and flomoxef. E1077 also inhibited 50% and 90% of MRSA strains at concentrations of 6.25 and 50 ,ug/ml, respectively. Its activity was similar to that of flomoxef, which has been reported to be more active in vitro than other cephalosporins against MRSA (8). However, its activity, with an MIC90 of 50 ,ug/ml, is probably inadequate for clinical use for MRSA infections. In addition, E1077 was active against E. faecalis, which was resistant or less susceptible to most of the existing cephalosporins. The MIC90 of E1077 for E. faecalis was 6.25 ,ug/ml, and its activity was four times that of cefpirome. The other reference compounds were inactive (MIC50s, >100 ,ug/ml). Kamiya et al. have suggested that the high activity of E1077 against S. aureus and E. faecalis is provided by the introduction of a quatemary ammoniopropenyl substituent at the 3 position of the cephem nucleus (4). A second characteristic of E1077 is that E1077 is very effective against most members of the family Enterobacteriaceae. In general, the activity of E1077 was comparable to that of cefpirome and higher than those of the other reference compounds against these species. In particular, the activity of E1077 was much higher (.64 times) than those of ceftazidime, cefuzonam, cefotaxime, and flomoxef against Enterobacter and Citrobacter species, which produce high levels of chromosomal ,-lactamase (11). Cefpirome (6) and cefepime (10) have been reported to be active against these species. In this study, E1077 was four times more active than cefpirome against E. aerogenes and C. freundii. E1077 also showed higher in vivo activity against systemic infection caused by a strain of C. freundii that was resistant to the other extended-spectrum cephalosporins than did ceftazidime and cefuzonam. This activity seems to be due to the resistance of E1077 to enzymatic hydrolysis and the low affinity of the compound for 1-lactamases (13). E1077 possessed potency comparable to that of ceftazidime against P. aeruginosa, an opportunistic pathogen that is one of the major causes of life-threatening nosocomial

bacterial infections. The M'C50 and MIC90 of E1077 for P. aeruginosa were 1.56 and 6.25 ,ug/ml, respectively, and its activity was comparable to that of ceftazidime and four times higher than that of cefpirome. Watanabe et al. have reported that introduction of the aminothiadiazolyl group in the 71 side chain of the cephem nucleus enhances antibacterial activity against P. aeruginosa (14). The in vivo activity of E1077 in systemic infection clearly reflected its in vitro activity against P. aeruginosa. Thus, E1077 has a broad antibacterial spectrum covering gram-positive bacteria, including staphylococci and E. faecalis, and gram-negative bacteria, including P. aeruginosa. The in vitro study results were generally consistent with those of Watanabe et al. (13). E1077 also showed efficacy in systemic infections with various bacteria, and this was clearly reflected by its in vitro activity. Overall, E1077 is a very promising antibacterial agent for the treatment of various bacterial infections. Further studies on pharmacokinetic and toxicological behaviors are therefore warranted. ACKNOWLEDGMENT This study was financially assisted by Eisai Co., Ltd. REFERENCES 1. Committee for Revision of MIC Determination Method. 1981. Revision of minimal inhibitory concentration (MIC) determination method. Chemotherapy (Tokyo) 29:76-79. 2. Hikida, M., M. Inoue, and S. Mitsuhashi. 1986. In vitro antibacterial activity of L-105, a new cephalosporin. J. Antimicrob. Chemother. 18:585-591. 3. Hiruma, R., M. Otsuki, M. Tashima, Y. Obana, and T. Nishino. 1990. In-vitro and in-vivo antibacterial activities of E1040, a new cephalosporin with potent antipseudomonal activity. J. Antimicrob. Chemother. 26:769-781. 4. Kamiya, T., T. Naito, Y. Kai, Y. Komatsu, M. Sasho, N. Sato, T. Nakamura, S. Negi, Y. Machida, and Y. Yamauchi. 1990. Program Abstr. 30th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 447. 5. Kessler, R. E., M. Bies, R. E. Buck, D. R. Chisholm, T. A. Pursiano, Y. H. Tsai, M. Misiek, K. E. Price, and F. Leitner. 1985. Comparison of a new cephalosporin, BMY 28142, with other broad-spectrum ,3-lactam antibiotics. Antimicrob. Agents Chemother. 27:207-216. 6. Kobayashi, S., S. Arai, S. Hayashi, and K. Fujimoto. 1986. P-Lactamase stability of cefpirome (HR810), a new cephalosporin with a broad antibacterial spectrum. Antimicrob. Agents Chemother. 30:713-718. 7. Litchfield, J. T., Jr., and F. Wilcoxon. 1949. A simplified method of evaluating dose-effect experiments. J. Pharmacol. Exp. Ther. 96:99-113. 8. Murakami, K., K. Nomura, M. Doi, and T. Yoshida. 1987. Production of low-affinity penicillin-binding protein by low- and high-resistance groups of methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 31:1307-1311. 9. Neu, H. C., N. Aswapokee, P. Aswapokee, and K. P. Fu. 1979. HR756, a new cephalosporin active against gram-positive and gram-negative aerobic and anaerobic bacteria. Antimicrob. Agents Chemother. 15:273-281. 10. Phelps, D. J., D. D. Carlton, C. A. Farrell, and R. E. Kessler. 1986. Affinity of cephalosporins for P-lactamases as a factor in antibacterial efficacy. Antimicrob. Agents Chemother. 29:845848. 11. Sanders, C. C. 1987. Chromosomal cephalosporinases responsible for multiple resistance to newer 3-lactam antibiotics. Annu. Rev. Microbiol. 41:573-593. 12. Seibert, G., M. Limbert, I. Winkler, and T. Dick. 1983. Antibacterial activity in vitro and P-lactamase stability of the new

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cephalosporin HR810 in comparison with five other cephalosporins and two aminoglycosides. Infection 11:275-279. 13. Watanabe, N., R. Hiruma, and K. Katsu. 1992. In vitro evaluation of E1077, a new cephalosporin with a broad antibacterial spectrum. Antimicrob. Agents Chemother. 36:589-597. 14. Watanabe, N., K. Katsu, M. Moryama, and K. Kitoh. 1988. In

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vitro evaluation of E1040, a new cephalosporin with potent antipseudomonal activity. Antimicrob. Agents Chemother. 32:693-701. 15. Wise, R., J. H. Andrews, and K. A. Bedford. 1980. Comparison of in vitro activity of GR20263, a novel cephalosporin derivative, with activities of other beta-lactam compounds. Antimicrob. Agents Chemother. 17:884-889.

In vitro and in vivo antibacterial activities of E1077, a novel parenteral cephalosporin with a broad antibacterial spectrum.

E1077 is a new injectable cephalosporin with a broad spectrum of antibacterial activity against gram-positive and gram-negative bacteria, including st...
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