In vitro and in vivo activities of SCE-2787, a new parenteral cephalosporin with a broad antibacterial spectrum.

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, JUlY 1992, p. 1358-1366 0066-4804/92/071358-09$02.00/0 Copyright © 1992, American Society for Microbiology

Vol. 36, No. 7

In Vitro and In Vivo Activities of SCE-2787, a New Parenteral Cephalosporin with a Broad Antibacterial Spectrum TOMOYUKI IWAHI,* KENJI OKONOGI, TOSHIYUKI YAMAZAKI, SHIKIKO SHIKI, MASAHIRO KONDO, AKIO MIYAKE, AND AKIRA IMADA

Research and Development Division, Takeda Chemical Industries, Ltd., 2-17-85, Jusohonmachi, Yodogawa-ku, Osaka 532, Japan Received 18 October 1991/Accepted 15 April 1992

SCE-2787, a new cephalosporin having a condensed azolium moiety in the 3 position and an aminothiadiazolyl group in the 7j side chain, was evaluated for its in vitro and in vivo activities in comparison with those of ceftazidime, flomoxef, cefpirome, and E1040. Against methicillin-susceptible strains of Staphylococcus aureus and Staphylococcus epidermidis, SCE-2787 was more active than ceftazidime and E1040 and was as active as flomoxef and cefpirome, with MICs for 90% of strains tested (MIC90s) being 1.56 ,ug/ml or less. SCE-2787 was also active against Pseudomonas aeruginosa, for which the MIC90 was 6.25 ,ug/ml, which was lower than that of cefpirome and comparable to that of ceftazidime. SCE-2787 was marginally active against methicillin-resistant strains of staphylococci and Enterococcus faecalis, although its MIC90s were the lowest among those of the antibiotics tested. The activities of SCE-2787 against Streptococcus species, most members of the family Enterobacteriaceae, and Haemophilus influenzae exceeded those of ceftazidime and flomoxef and were comparable to those of cefpirome. Furthermore, MIC90s of SCE-2787 were significantly lower than those of ceftazidime for ceftazidime-resistant isolates of Citrobacterfreundii and Enterobacter cloacae. SCE-2787 was resistant to hydrolysis by various types of 13-lactamases, including the Bush group 1 13-lactamases, and had low affinities for these enzymes, with Km or Ki values of > 100 ,uM. The in vitro activity of SCE-2787 was reflected in its efficacy in mouse protection tests. Thus, SCE-2787 appears to be a promising cephalosporin that should be further evaluated in clinical trials. During the past decade, a number of cephalosporins with broad spectra of activity and resistance to hydrolysis by ,-lactamases have been developed. However, the clinical usefulness of these antibiotics has been limited because of their rather weak activities against Staphylococcus species and Pseudomonas aeruginosa (14) and the rapid development of resistance in some gram-negative bacilli, such as Citrobacter, Enterobacter, and Serratia species (11, 22). In the course of modifications of cephalosporins aimed at improving their activity, SCE-2787, 713-[2-(5-amino-1,2,4thiadiazol 3 -yl) 2(Z) methoxyiminoacetamido] 3 (imidazo[1 ,2-b]pyridazinium 1 -yl)methyl-3 -cephem -4-carboxylate, inner salt hydrochloride, has been found to be highly active against not only members of the family Enterobacteriaceae, including strains resistant to ceftazidime, but also staphylococci and Pseudomonas aeruginosa (12). Its chemical structure (Fig. 1) is characterized by a condensed azolium moiety in the 3 position and an aminothiadiazolyl group in the 7, side chain of a cephalosporin. In this report, we describe the in vitro and in vivo activities of SCE-2787 in comparison with those of ceftazidime (17), flomoxef (25), cefpirome (23), and E1040 (26). We also report the interaction of SCE-2787 and the reference 1-lactam antibiotics with various types of 3-lactamases, including the Bush group 1 ,B-lactamases. (A part of this work was presented at the 29th Interscience Conference on Antimicrobial Agents and Chemotherapy, Houston, Tex., 17 to 20 September 1989 [7].) -

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MATERIALS AND METHODS Antibiotics. SCE-2787, cefpirome, and E1040 were synthesized in the Research and Development Division, Takeda Chemical Industries Ltd., Osaka, Japan. Other antibacterial agents were purchased commercially, as follows: ceftazidime, Nippon Glaxo Co., Ltd., Tokyo, Japan; flomoxef and cephaloridine, Shionogi and Co., Ltd., Osaka, Japan; methicillin, Banyu Pharmaceutical Co., Ltd., Tokyo, Japan; and benzylpenicillin (Meiji Seika Kaisha, Ltd., Tokyo, Japan). Organisms. The clinical isolates used in this study were kindly supplied by five clinical laboratories in Japan between 1987 and 1988 and were maintained in our laboratory. Laboratory standard strains were obtained from our culture collection. Determination of MICs and MBCs. Mueller-Hinton broth (BBL Microbiology Systems, Cockeysville, Md.) or Mueller-Hinton agar (BBL) was used for all assays except the following. For Streptococcus species and Corynebactenium diphtheriae, Mueller-Hinton broth or Mueller-Hinton agar was supplemented with 10% horse serum. Haemophilus influenzae was tested with brain heart infusion (BHI; Difco Laboratories, Detroit, Mich.) supplemented with 5% Fildes enrichment (Difco). Bordetella pertussis was tested with Iso-Sensitest agar (Oxoid, Basingstoke, United Kingdom) supplemented with 5% bovine blood. For anaerobes, GAM agar (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) was used. Trypticase soy agar (BBL), nutrient agar (Eiken Chemical Co., Ltd., Tokyo, Japan), BHI agar (Difco), and heart infusion agar (Difco) were also used to examine the effect of culture medium. Bacterial suspensions for inocula were prepared by diluting overnight cultures of the organisms to be tested in the fresh broth medium to give the desired concentrations. The MICs by the microbroth dilution method were deter-

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Corresponding author. 1358

VOL. 36, 1992

IN VITRO AND IN VIVO ACTIVITIES OF SCE-2787

H2N>S SN N

CONHT N

NOCH3

O

s

C

CH2-N'

NG .HCI *OE

FIG. 1. Chemical structure of SCE-2787.

mined with a MIC 2000 titration system (Dynatech Laboratories, Inc., Alexandria, Va.) as described previously (6). Volumes of 1.5 RI of the bacterial suspensions containing about 103 CFU were inoculated into 0.1 ml of broth medium containing twofold serial dilutions of an antibiotic in microtiter wells, since preliminary studies indicated that the inoculation of 103 CFU, rather than 104 CFU gives results that are more comparable to the agar dilution MICs. Thus, the inoculum levels used in the broth microdilution tests were 1 order of magnitude lower than the recommendations of the National Committee for Clinical Laboratory Standards (13). The MICs by the agar dilution method were determined with an automatic agar dilution system (Dainippon Seiki Co., Ltd., Kyoto, Japan). Ten-milliliter agar layers containing twofold serial dilutions of an antibiotic in petri dishes were automatically prepared, and 5-,ul volumes of the bacterial suspensions containing about 104 CFU were applied onto the surfaces of the agar plates with an inoculumreplicating apparatus (Micro-planter; Sakuma Seisakusyo, Tokyo, Japan) that was incorporated into the system. The MIC was defined as the lowest concentration of antibiotic that inhibited development of visible growth in the broth or on the agar after 20 h of incubation at 37°C. MBCs were determined by slight modifications of the method of Pearson et al. (19). Briefly, about 5 x 105 CFU of organisms to be tested were inoculated into 0.8 ml of Trypticase soy broth (BBL) containing twofold serial dilutions of an antibiotic in glass tubes. After the MIC was determined, the cultures were diluted 10-fold with 0.9% saline, and 0.1-ml volumes of the dilutions were spread on antibiotic-free Trypticase soy agar plates. The MBC was defined as the lowest concentration of antibiotic that gave >99.9% reduction of the inoculum. 13-Lactamase assays. The ,-lactamases from various bacterial strains were purified as reported previously (6). Each of the enzyme preparations contained only one kind of ,-lactamase, as indicated by isoelectric focusing. The hydrolysis rates of antibiotics were determined by the spectrophotometric assay at a substrate concentration of 200 puM in 50 mM phosphate buffer (pH 7.0) at 30°C with a Gilford Response II spectrophotometer (CIBA Corning Diagnostic Co., Oberlin, Ohio). Enzyme activities were calculated from the following differences in molar extinction coefficients (AE; in molar-' centimeters-') between the antibiotics and the corresponding hydrolyzed compounds at the wavelengths indicated in parentheses: SCE-2787, 7,050 (265 nm);

ceftazidime, 8,640 (260 nm); flomoxef, 8,950 (270 nm); cefpirome, 8,720 (260 nm); cephaloridine, 9,830 (260 nm); benzylpenicillin, 800 (230 nm). Km was also determined spectrophotometrically. Ki was determined by assessing microiodometrically the abilities of antibiotics to inhibit the hydrolysis of ampicillin for penicillinases or cephalothin for cephalosporinases as described previously (18). Kinetic parameters were estimated from Lineweaver-Burk plots.

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Mouse protection tests. Most bacterial strains were cultured overnight at 37°C in BHI. Streptococcus species and Haemophilus influenzae were cultured overnight at 37°C on Trypticase soy agar supplemented with 10% bovine blood and in BHI supplemented with 5% Fildes enrichment, respectively. All strains except Streptococcus pneumoniae were suspended in 8% mucin (Difco). Streptococcus pneumoniae was suspended in BHI. Four-week-old Slc:ICR male mice (weights, 19 to 23 g) were infected intraperitoneally with 0.5 ml of the bacterial suspension. The challenge inocula corresponded to 30 to 100 times the number of bacteria required to kill 50% of the control mice. Under these conditions, all of the untreated mice died within 2 days. Five mice were treated subcutaneously at each dose level with 0.2 ml of a single-dose regimen of test antibiotics immediately after infection. Five dose levels or more were tested for an antibiotic. All experiments were repeated at least three times. The 50% effective dose was calculated by the probit method (3) from the survival rate recorded on day 5 after infection.

RESULTS Antibacterial spectrum. The activity of SCE-2787 against various species of clinical isolates was compared with those of the reference ,B-lactam antibiotics by the broth microdilution method (Table 1). The MICs of SCE-2787 required to inhibit 90% of organisms tested (MIC90s) for methicillinsusceptible Staphylococcus aureus (0.78.pg/ml) and methicillin-susceptible Staphylococcus epidermidis (1.56 ,g/ml) were 8- to 16-fold lower than those of ceftazidime and E1040 and comparable to those of flomoxef and cefpirome. SCE2787 was active against methicillin-resistant isolates of Staphylococcus aureus, with an MIC90 of 25 ,ug/ml, but for methicillin-resistant isolates of Staphylococcus epidermidis, the MIC90 of SCE-2787 was 3.13 ,ug/ml. Against Streptococcus species, SCE-2787 exhibited a potent activity, with MIC90s of 0.1 ,ug/ml or less, which was 4- to 32-fold lower than those of ceftazidime and flomoxef and similar to those of cefpirome. The MIC90 of SCE-2787 for Enterococcus faecalis was 25 p,g/ml. The MIC90s of SCE-2787 for Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Providencia rettgeri, and Providencia stuartii were .0.2 ug/ml; SCE-2787 was generally two- to eightfold more active than ceftazidime and flomoxef and was as active as cefpirome. The MIC90s of SCE-2787 for Citrobacter freundii and Enterobacter cloacae were 0.2 and 6.25 pug/ml, respectively, which were 8- to 64-fold lower than those of ceftazidime and flomoxef and comparable to those of cefpirome. SCE-2787 was also as active as ceftazidime and cefpirome against Proteus mirabilis and Serratia marcescens, with MICgOs of 0.2 and 3.13 p,g/ml, respectively. However, SCE2787 was the least active against Proteus vulgaris, with an MIC90 of 12.5 p,g/ml. The MIC90 of SCE-2787 for Morganella morganii was 0.78 pug/ml, which was eightfold lower than those of ceftazidime and flomoxef but fourfold higher than that of cefpirome. Against Pseudomonas aeruginosa, the MIC90 (6.25 ,ug/ml) of SCE-2787 was four- to eightfold lower than that of cefpirome and was equal to that of ceftazidime, although it was slightly higher than that of E1040. SCE-2787 was also active against Haemophilus influenzae, with an MIC90 of 0.2 ,g/ml; it was eightfold more active than flomoxef and was as active as ceftazidime and

cefpirome.

The MICs of SCE-2787 for laboratory strains of other aerobes, as determined by the agar dilution method, were as

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ANTIMICROB. AGENTS CHEMOTHER.

IWAHI ET AL.

TABLE 1. Comparative activities of SCE-2787 and reference antibiotics against aerobic bacteria Organism (no. of strains)

Staphylococcus aureus MSb (81)

(LWg/ml)a

.nii.cMIC Aftibiotic Range 50% SCE-2787 Ceftazidime Flomoxef

Cefpirome

E1040 Methicillin

MRb (30)

SCE-2787 Ceftazidime Flomoxef

Cefpirome

E1040 Methicillin

Staphylococcus epidermidis MS (35)


Cefpirome E1040

Methicillin MR (20)


Cefpirome E1040 Methicillin

Streptococcus pyogenes (50)


Cefpirome Streptococcus pneumoniae (48)


Cefpirome Enterococcus faecalis (46)


Cefpirome Escherichia coli (50)


Cefpirome E1040

Citrobacterfreundii (61)


Cefpirome E1040

Klebsiella pneumoniae (50)

SCE-2787 Ceftazidime

Flomoxef Cefpirome Klebsiella oxytoca (43)


Cefpirome

0.39-3.13 3.13-25 0.2-0.78 0.39-1.56 1.56-25 0.78-6.25

0.78-100 12.5->100 1.56-100 0.78-100 6.25-> 100 12.5-> 100 0.1-1.56 1.56-50 0.2-6.25 0.1-3.13 1.56-50 0.2-6.25 0.78-6.25 25-> 100 3.13-100 0.78-50 6.25-100 25-> 100

0.78 6.25 0.39 0.78 12.5 1.56

25 100 12.5 25 50 100

90%

0.78 12.5 0.39 0.78 12.5 3.13

25 >100 50 100 100 >100

0.39 6.25 0.39 0.39 6.25 3.13

1.56 25 3.13 3.13 25 6.25

1.56 50 25 6.25 12.5 >100

3.13 >100 100 50 25 >100

0.0125-0.025 0.1-0.39 0.1-0.2 0.006-0.0125

0.0125 0.2 0.1 0.0125

0.025 0.2 0.2 0.0125

0.0125-1.56 0.1-25 0.025-12.5 0.006-0.78

0.05 0.2 0.2 0.025

0.1 3.13 0.39 0.1

1.56-> 100 3.13-> 100 0.78-> 100 0.39-> 100

12.5 >100 100 12.5

25 >100 >100 50

0.05-0.2 0.05-0.39 0.025-1.56 0.025-0.39 0.05-0.39

0.05 0.1 0.05 0.05 0.1

0.2

0.05-3.13 0.1-25 0.05->100 0.025-3.13 0.1-3.13

0.05 0.39 0.2 0.1 0.1

0.2 12.5 25 0.2 0.39

0.025-0.78 0.1-3.13 0.025-3.13 0.025-3.13

0.05 0.2 0.05 0.05

0.1 0.39 0.1 0.1

0.05-0.39 0.05-0.39 0.025-0.78 0.025-1.56

0.05 0.2 0.05 0.05

0.05 0.39 0.1 0.1

0.1 0.2 0.1 0.1

Continued on following page

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1361

TABLE 1-Continued Organism (no. of strains)

Antibiotic

MIC (p.g/ml)a

Enterobacter cloacae (60)

SCE-2787 Ceftazidime Flomoxef Cefpirome E1040

Range 0.025-12.5 0.025->100 0.05->100 0.025-25 0.1-25

Serratia marcescens (49)

SCE-2787 Ceftazidime Flomoxef Cefpirome

0.1-12.5 0.1-12.5 0.39-> 100 0.05-6.25

Proteus vulgaris (52)


0.1-25 0.025-0.78

Proteus mirabilis (50)

50%

90%

0.05 0.2 1.56 0.1 0.2

6.25 50 >100 6.25 1.56

0.39 0.78 50 0.78

3.13 3.13 >100 3.13

0.1-12.5

0.2 0.05 0.2 0.2

12.5 0.2 0.39 3.13


0.1-12.5 0.025-0.39 0.1-1.56 0.05-12.5

0.2 0.05 0.2 0.1

0.2 0.1 0.2 0.2

Morganella morganii (49)


0.1-1.56 0.05-25 0.2-6.25 0.025-0.39

0.2 0.1 1.56 0.05

0.78 6.25 6.25 0.2

Providencia rettgeri (47)


0.006-0.2 0.025-3.13 0.025-1.56 0.006-0.2

0.05 0.1 0.1 0.1

0.1 0.78 0.39 0.2

Providencia stuartii (30)


0.0125-0.78

0.05 0.1 0.1 0.1

0.2 0.39 0.2 0.39

0.78 0.78 >100 3.13 0.78

6.25 6.25 >100 25 1.56

0.1 0.1 0.78 0.1

0.2 0.1 1.56 0.1

Pseudomonas aeruginosa (90)

Haemophilus influenzae (49)

0.1-0.78

0.0125-3.13

0.025-6.25 0.0125-1.56


0.1-25 0.1-100 >100 0.2->100


0.05-0.2 0.05-0.2

0.1-6.25

0.39-1.56 0.05-0.1

a Determined by the broth microdilution method with an inoculum of ca. 103 CFU. 50% and 90%, MICs for 50 and 90% of the strains tested, respectively. b MS, methicillin susceptible (MIC, 100 12.5-> 100 0.2-3.13 0.1-3.13

1.56 100 100 0.78 0.78


0.05-0.78

0.1

0.2

0.2-0.78 0.39-50 0.05-0.78 0.1-1.56

0.39 6.25 0.05 0.1

0.39 25 0.1 0.2


0.39-12.5 25-> 100 50-> 100 0.39-50 0.2-25

3.13 100 > 100 1.56 0.78

12.5 > 100 > 100 12.5 3.13

SCE-2787 Ceftazidime Cefpirome E1040

0.39-12.5 0.78-6.25 0.78-50 0.39-12.5

1.56 3.13 6.25 1.56

6.25 6.25 12.5 3.13

SCE-2787 Ceftazidime Cefpirome E1040

6.25-> 100 12.5-> 100 25-> 100 1.56-50

50 100 100 6.25

> 100

> 100 3.13 1.56

>100 > 100 > 100 25

a Determined by the agar dilution method with an inoculum of ca. 104 CFU per spot. 50% and 90%, MICs for 50 and 90% of the strains tested, respectively. CAZ-S, ceftazidime susceptible (MIC, 12.5 pLg/ml).

b

than those for the ceftazidime-susceptible isolates of that species, and in most isolates a cross-resistance between SCE-2787 and ceftazidime was observed. Effect of assay conditions. The MICs of SCE-2787 for three strains each of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Eschenrchia coli, Klebsiella pneumoniae, Citrobacter freundii, Enterobacter cloacae, Serratia marcescens, and Pseudomonas aeruginosa did not differ by more than twofold when they were determined by the agar dilution method on Mueller-Hinton agar, Trypticase soy agar, nutrient agar, BHI agar, and heart infusion agar. The activity of SCE-2787 against the same strains on Mueller-Hinton agar was little altered by the change in pH from 6 to 8 and by the addition of heat-inactivated horse serum at final concentrations of 20 to 40%. The effects of these factors with SCE-2787 were similar to those with ceftazidime and cefpirome. The MICs and MBCs of SCE2787 for five strains each of Staphylococcus aureus, Staphylococcus epidennidis, Enterococcus faecalis, Escherichia coli, Enterobacter cloacae, and Pseudomonas aeruginosa were determined by the broth dilution method. The MBCs of SCE-2787 for all the strains tested were identical to or at most fourfold higher than the MICs, with the exception of

those for two strains of Staphylococcus aureus, one strain of Staphylococcus epidennidis, one strain of Enterococcus faecalis, and two strains of Enterobacter cloacae, which were relatively resistant to the killing actions of all the cephalosporins tested. The bactericidal effects (MBCs/ MICs) with SCE-2787 were similar to those with ceftazidime and cefpirome (data not shown). P-Lactamase studies. The susceptibilities of SCE-2787 and the reference P-lactam antibiotics to hydrolysis by 11 commonly encountered 1-lactamases were examined by determining their relative rates of hydrolysis (Table 3). SCE-2787 was highly resistant to hydrolysis by plasmid-mediated penicillinases (Bush group 2) and chromosomal cephalosporinases (Bush group 1) from Enterobacter cloacae, Citrobacter freundii, and Pseudomonas aeruginosa, as were ceftazidime and cefpirome. However, SCE-2787, like cefpirome, was hydrolyzed to some extent by some types of 1-lactamases. The comparative stabilities of SCE-2787 and cefpirome varied depending on the type of 1-lactamase; SCE-2787 was hydrolyzed at a lower rate than cefpirome was by the plasmid-mediated group 2d (OXA-1) 1-lactamase and the chromosomally mediated penicillinase (group 2b') from Klebsiella oxytoca, but it was hydrolyzed at a higher

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TABLE 3. Susceptibilities of SCE-2787 and reference antibiotics to hydrolysis by ,-lactamases Group'

Common name

Relative rate of hydrolysis (%) oft:

Source

SCE-2787

Citrobacterfreundii GN1706 Enterobacter cloacae TN1282 Serratia marcescens TN81 Pseudomonas aeruginosa U31

1 1 1 1

2a 2b 2b' 2c 2d

TEM-1 PSE-1 OXA-1

2e 2e

CAZ

FMOX

CPR

PCG

8.08 24.8 9.18 57.3

0.06 0.05 0.17 0.10

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