ANTIMICROBIAL AGENTS
AND
CHEMOTHERAPY, Apr. 1991,
p.
653-659
Vol. 35, No. 4
0066-4804/91/040653-07$02.00/0 Copyright © 1991, American Society for Microbiology
In Vitro and In Vivo Evaluation of Ro 09-1428, a New Parenteral Cephalosporin with High Antipseudomonal Activity M. ARISAWA,1* Y. SEKINE,' S. SHIMIZU,2 H. TAKANO,3 P. ANGEHRN,4 AND R. L. THEN4 Department of Chemotherapy and Biochemistry, Nippon Roche Research Center, Kamakura 247,1 Nippon Pharmaceutical Development Institute Co., Ltd., Hokkaido 054,2 and Sankei Pharmaceutical Co., Ltd., Tokyo 101,3 Japan, and Pharmaceutical Research Department, F. Hoffmann-La Roche AG, Basel 4002, Switzerland4 Received 17 September 1990/Accepted 7 February 1991
Ro 09-1428, a new parenteral cephalosporin with a catechol moiety attached at position 7 of the cephalosporin ring, showed high in vitro activity against Escherichia coli, Kkbsielia pneumoniae, Proteus mirabilis, Proteus vulgaris, and Streptococcus pyogenes, with MICs for 90% of strains tested (MIC9s) of c0.39 ,ug/ml. MorganeUa morganii, Providencia reftgeri, Citrobacterfreundii, Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae were inhibited with MIC90s of 100
0.05 100 0.05->100 0.2->100 0.1->100
0.025 0.2 0.39 0.2
Serratia marcescens (26)
Ro 09-1428 Ceftriaxone Ceftazidime Cefuzonam
0.1->100 0.1-50 0.2->100 0.2-100
0.78 6.25 1.56 3.13
25 25 6.25 25
Enterobacter cloacae (27)
Ro 09-1428 Ceftriaxone Ceftazidime Cefuzonam
0.1-12.5 0.1-100 0.2-50 0.1-100
0.39 0.2 0.39 0.39
6.25 12.5 6.25 12.5
Haemophilus influenzae (23)
Ro 09-1428 Ceftriaxone Ceftazidime Cefuzonam
0.2-1.56 s0.006-0.025 0.05-0.39 100
12.5 6.25 25
6.25 50 25 50
Pseudomonas aeruginosa (54)
Ro 09-1428 Ceftriaxone Ceftazidime Cefuzonam
0.012-3.13 1.56->100 0.39-50 6.25->100
0.05 50 3.13 25
0.39 >100 50 >100
Staphylococcus aureus (49) (methicillin susceptible)
Ro 09-1428 Ceftriaxone Ceftazidime Cefuzonam
3.13-6.25 1.56-6.25 3.13-12.5 0.39-0.78
3.13 1.56 6.25 0.39
3.13 3.13 12.5 0.78
Staphylococcus epidermidis (27)
Ro 09-1428 Ceftriaxone Ceftazidime Cefuzonam
0.78-50 0.39-25 3.13-25
3.13 3.13 12.5 0.78
12.5 12.5 25 1.56
Acinetobacter calcoaceticus (26)
0.1-3.13
0.39 -:0.006 0.1 0.012 1.56
3.13
0.1 0.1
3.13 100 >100 25
0.78 0.012
0.39 0.025
Continued on following page
656
ARISAWA ET AL.
ANTIMICROB. AGENTS CHEMOTHER. TABLE 1-Continued Atboi
Organism
(no. of isolates)
MIC
Range
(,ug/ml) 50%
90%
Streptococcus pyogenes (26)
Ro 09-1428 Ceftriaxone Ceftazidime Cefuzonam
0.2-0.39 64 FLg/ml), while it was extremely active in DST agar (MIC, 0.25 ,ug/ml). The influence of supplemental blood, phosphate, and ferric ion was tested. Sheep blood did not markedly increase the MICs determined in DST agar and Mueller-Hinton medium. Although supplementing with 15 mM phosphate did not influence the activity of Ro 09-1428 in DST agar, a fourto eightfold enhancement of activity was detected for E. coli and P. aeruginosa on Mueller-Hinton agar. Influence of ferric ion was extreme for P. aeruginosa but not for other species tested; the MIC of Ro 09-1428 for P. aeruginosa increased 64 times when 20 ,uM Fe3+ was included. Ferric iron (up to 1 mM) did not affect the growth rate of two P. aeruginosa strains. Generation times of the strains were 27 and 26 min in Mueller-Hinton medium, almost equal to the generation time of 29 min of the strains determined in the iron-supplemented (1 mM) medium. Analysis of DST and Mueller-Hinton agar by X-ray fluorescence showed marked differences in the amount of chloride (4 versus 15%), potassium (1.3 versus 0.1%), iron (100 versus 18 ppm), and zinc (31 versus 10 ppm). Effects of inoculum on in vitro activity. Inoculum effect on MIC was also tested for two to four strains (each) of ceftazidime-resistant and -susceptible strains from P. aeruginosa, E. cloacae, and C. freundii and five strains (each) of TABLE 2. Stability of Ro 09-1428 against hydrolysis by ,B-lactamases Relative rate of hydrolysisa Enzyme source
E. cloacae 908 R K. oxytoca 1082 E C. freundii 1203 P. vulgaris 1028 ,-c E. coli TEM-1 E. coli TEM-2 a
Ro 09-1428
Ceftriaxone
Ceftazidime
0.0042 0.032 0.0048 0.26 64 64 >32 >32 >32 32
VOL. 35, 1991
A NEW PARENTERAL CEPHALOSPORIN, Ro 09-1428
TABLE 6. Affinity for PBPs
TABLE 4. Activities of Ro 09-1428 against ceftazidime-
resistant strains MIC (,ug/ml)
Organism (no. of strainsa)
Antibiotic
Geometric
Range
mean b
C. freundii (19)
Ro 09-1428 Ceftazidime
0.2->100 12.5->100
1.3 67
E. cloacae (7)
Ro 09-1428
Ceftazidime
0.39-12.5 12.5->100
1.9 28
S. marcescens (5)
Ro 09-1428 Ceftazidime
12.5->100 12.5-50
66 44
P. aeruginosa (8)
Ro 09-1428 Ceftazidime
0.05-0.2 12.5-50
0.10 25
a
b
Ceftazidime-resistant strains from Table 1 included. MIC of >100 Lg/ml is assumed to be 200 ,ug/ml.
E. coli producing TEM-5 was much more susceptible than K. pneumoniae producing the same enzyme. S. marcescens with TEM-3 was resistant to Ro 09-1428 at a concentration of 66 ,ug/ml, whereas two TEM-3-producing strains of E. coli and Enterobacter aerogenes were moderately susceptible, with MICs of 2 and 4 ,ug/ml, respectively. A large difference in susceptibility between Ro 09-1428 and ceftazidime was observed with P. aeruginosa isolates that overproduce type Id ,-lactamase. To further study this, MICs of Ro 09-1428 and ceftazidime were determined for a larger number of ceftazidime-resistant strains whose ,-lactamase types were not determined. Ceftazidime-resistant strains of C. freundii, E. cloacae, or P. aeruginosa were susceptible to Ro 09-1428, with the largest difference in susceptibility between Ro 09-1428 and ceftazidime observed for P. aeruginosa (Table 4). However, all ceftazidime-resistant S. marcescens strains were resistant to Ro 09-1428, with MICs of .12.5 ,ug/ml. Inhibition of B-lactamases. The inhibitory activities of Ro 09-1428 for ,-lactamases from various strains were determined in order to compile affinity profiles of the compoundenzyme interaction (Table 5). The inhibition profile obtained was similar to that of other cephalosporins, viz, high activity toward P-lactamases of class C, which preferentially hydrolyze cephalosporins, and low activity toward ,-lactamases of class A. Ro 09-1428 closely resembled ceftazidime in showing a much lower affinity for cephalosporin hydrolyzing ,-lactamases than ceftriaxone and cefotaxime.
657
IC50 (,ugmI)
Organism
PBP
S. aureus Schoch
1 2 3 4
0.1 0.28 0.1 10
1.0 >100
E. coli W3110
la lb 2 3 4 5 6
5.0 10 5.0 0.05 100 100 54
1.7 6.0 >100 0.64 >100 >100 >100
0.13 1.6 0.3 0.23 0.5 >100 5
P. aeruginosa K799/WT
la lb 3 4 4' 5
0.1 3.1 0.01 0.5 50 100
0.08 9.7 0.03 4.2 >100 >100
0.17 0.62 0.17 0.10 >100 >100
Ro 09-1428
Ceftazidime
0.1-1 0.39
Ampicillin
32 0.016 0.06 2 32 0.25 2
>32 0.5 0.25 4 32
(0.026-0.048) (0.025-0.060) (0.007-0.019) (0.017-0.046)
AND
CHEMOTHERAPY, Apr. 1991,
p.
653-659
Vol. 35, No. 4
0066-4804/91/040653-07$02.00/0 Copyright © 1991, American Society for Microbiology
In Vitro and In Vivo Evaluation of Ro 09-1428, a New Parenteral Cephalosporin with High Antipseudomonal Activity M. ARISAWA,1* Y. SEKINE,' S. SHIMIZU,2 H. TAKANO,3 P. ANGEHRN,4 AND R. L. THEN4 Department of Chemotherapy and Biochemistry, Nippon Roche Research Center, Kamakura 247,1 Nippon Pharmaceutical Development Institute Co., Ltd., Hokkaido 054,2 and Sankei Pharmaceutical Co., Ltd., Tokyo 101,3 Japan, and Pharmaceutical Research Department, F. Hoffmann-La Roche AG, Basel 4002, Switzerland4 Received 17 September 1990/Accepted 7 February 1991
Ro 09-1428, a new parenteral cephalosporin with a catechol moiety attached at position 7 of the cephalosporin ring, showed high in vitro activity against Escherichia coli, Kkbsielia pneumoniae, Proteus mirabilis, Proteus vulgaris, and Streptococcus pyogenes, with MICs for 90% of strains tested (MIC9s) of c0.39 ,ug/ml. MorganeUa morganii, Providencia reftgeri, Citrobacterfreundii, Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae were inhibited with MIC90s of 100
0.05 100 0.05->100 0.2->100 0.1->100
0.025 0.2 0.39 0.2
Serratia marcescens (26)
Ro 09-1428 Ceftriaxone Ceftazidime Cefuzonam
0.1->100 0.1-50 0.2->100 0.2-100
0.78 6.25 1.56 3.13
25 25 6.25 25
Enterobacter cloacae (27)
Ro 09-1428 Ceftriaxone Ceftazidime Cefuzonam
0.1-12.5 0.1-100 0.2-50 0.1-100
0.39 0.2 0.39 0.39
6.25 12.5 6.25 12.5
Haemophilus influenzae (23)
Ro 09-1428 Ceftriaxone Ceftazidime Cefuzonam
0.2-1.56 s0.006-0.025 0.05-0.39 100
12.5 6.25 25
6.25 50 25 50
Pseudomonas aeruginosa (54)
Ro 09-1428 Ceftriaxone Ceftazidime Cefuzonam
0.012-3.13 1.56->100 0.39-50 6.25->100
0.05 50 3.13 25
0.39 >100 50 >100
Staphylococcus aureus (49) (methicillin susceptible)
Ro 09-1428 Ceftriaxone Ceftazidime Cefuzonam
3.13-6.25 1.56-6.25 3.13-12.5 0.39-0.78
3.13 1.56 6.25 0.39
3.13 3.13 12.5 0.78
Staphylococcus epidermidis (27)
Ro 09-1428 Ceftriaxone Ceftazidime Cefuzonam
0.78-50 0.39-25 3.13-25
3.13 3.13 12.5 0.78
12.5 12.5 25 1.56
Acinetobacter calcoaceticus (26)
0.1-3.13
0.39 -:0.006 0.1 0.012 1.56
3.13
0.1 0.1
3.13 100 >100 25
0.78 0.012
0.39 0.025
Continued on following page
656
ARISAWA ET AL.
ANTIMICROB. AGENTS CHEMOTHER. TABLE 1-Continued Atboi
Organism
(no. of isolates)
MIC
Range
(,ug/ml) 50%
90%
Streptococcus pyogenes (26)
Ro 09-1428 Ceftriaxone Ceftazidime Cefuzonam
0.2-0.39 64 FLg/ml), while it was extremely active in DST agar (MIC, 0.25 ,ug/ml). The influence of supplemental blood, phosphate, and ferric ion was tested. Sheep blood did not markedly increase the MICs determined in DST agar and Mueller-Hinton medium. Although supplementing with 15 mM phosphate did not influence the activity of Ro 09-1428 in DST agar, a fourto eightfold enhancement of activity was detected for E. coli and P. aeruginosa on Mueller-Hinton agar. Influence of ferric ion was extreme for P. aeruginosa but not for other species tested; the MIC of Ro 09-1428 for P. aeruginosa increased 64 times when 20 ,uM Fe3+ was included. Ferric iron (up to 1 mM) did not affect the growth rate of two P. aeruginosa strains. Generation times of the strains were 27 and 26 min in Mueller-Hinton medium, almost equal to the generation time of 29 min of the strains determined in the iron-supplemented (1 mM) medium. Analysis of DST and Mueller-Hinton agar by X-ray fluorescence showed marked differences in the amount of chloride (4 versus 15%), potassium (1.3 versus 0.1%), iron (100 versus 18 ppm), and zinc (31 versus 10 ppm). Effects of inoculum on in vitro activity. Inoculum effect on MIC was also tested for two to four strains (each) of ceftazidime-resistant and -susceptible strains from P. aeruginosa, E. cloacae, and C. freundii and five strains (each) of TABLE 2. Stability of Ro 09-1428 against hydrolysis by ,B-lactamases Relative rate of hydrolysisa Enzyme source
E. cloacae 908 R K. oxytoca 1082 E C. freundii 1203 P. vulgaris 1028 ,-c E. coli TEM-1 E. coli TEM-2 a
Ro 09-1428
Ceftriaxone
Ceftazidime
0.0042 0.032 0.0048 0.26 64 64 >32 >32 >32 32
VOL. 35, 1991
A NEW PARENTERAL CEPHALOSPORIN, Ro 09-1428
TABLE 6. Affinity for PBPs
TABLE 4. Activities of Ro 09-1428 against ceftazidime-
resistant strains MIC (,ug/ml)
Organism (no. of strainsa)
Antibiotic
Geometric
Range
mean b
C. freundii (19)
Ro 09-1428 Ceftazidime
0.2->100 12.5->100
1.3 67
E. cloacae (7)
Ro 09-1428
Ceftazidime
0.39-12.5 12.5->100
1.9 28
S. marcescens (5)
Ro 09-1428 Ceftazidime
12.5->100 12.5-50
66 44
P. aeruginosa (8)
Ro 09-1428 Ceftazidime
0.05-0.2 12.5-50
0.10 25
a
b
Ceftazidime-resistant strains from Table 1 included. MIC of >100 Lg/ml is assumed to be 200 ,ug/ml.
E. coli producing TEM-5 was much more susceptible than K. pneumoniae producing the same enzyme. S. marcescens with TEM-3 was resistant to Ro 09-1428 at a concentration of 66 ,ug/ml, whereas two TEM-3-producing strains of E. coli and Enterobacter aerogenes were moderately susceptible, with MICs of 2 and 4 ,ug/ml, respectively. A large difference in susceptibility between Ro 09-1428 and ceftazidime was observed with P. aeruginosa isolates that overproduce type Id ,-lactamase. To further study this, MICs of Ro 09-1428 and ceftazidime were determined for a larger number of ceftazidime-resistant strains whose ,-lactamase types were not determined. Ceftazidime-resistant strains of C. freundii, E. cloacae, or P. aeruginosa were susceptible to Ro 09-1428, with the largest difference in susceptibility between Ro 09-1428 and ceftazidime observed for P. aeruginosa (Table 4). However, all ceftazidime-resistant S. marcescens strains were resistant to Ro 09-1428, with MICs of .12.5 ,ug/ml. Inhibition of B-lactamases. The inhibitory activities of Ro 09-1428 for ,-lactamases from various strains were determined in order to compile affinity profiles of the compoundenzyme interaction (Table 5). The inhibition profile obtained was similar to that of other cephalosporins, viz, high activity toward P-lactamases of class C, which preferentially hydrolyze cephalosporins, and low activity toward ,-lactamases of class A. Ro 09-1428 closely resembled ceftazidime in showing a much lower affinity for cephalosporin hydrolyzing ,-lactamases than ceftriaxone and cefotaxime.
657
IC50 (,ugmI)
Organism
PBP
S. aureus Schoch
1 2 3 4
0.1 0.28 0.1 10
1.0 >100
E. coli W3110
la lb 2 3 4 5 6
5.0 10 5.0 0.05 100 100 54
1.7 6.0 >100 0.64 >100 >100 >100
0.13 1.6 0.3 0.23 0.5 >100 5
P. aeruginosa K799/WT
la lb 3 4 4' 5
0.1 3.1 0.01 0.5 50 100
0.08 9.7 0.03 4.2 >100 >100
0.17 0.62 0.17 0.10 >100 >100
Ro 09-1428
Ceftazidime
0.1-1 0.39
Ampicillin
32 0.016 0.06 2 32 0.25 2
>32 0.5 0.25 4 32
(0.026-0.048) (0.025-0.060) (0.007-0.019) (0.017-0.046)
