Vol. 34, No. 4ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1990, p. 555-5610066-48041901040555-07$02.00/0Copyright © 1990, American Society for Microbiology

Antimicrobial Activity and Stability to 1-Lactamase of BMY-28271,a New Oral Cephalosporin Ester

HIROYUKI MATSUI,'* MASAKI HIRAOKA,2 MATSUHISA INOUE,3 AND SUSUMU MITSUHASHI'Episome Institute, 2220 Kogure, Fujimi-mura, Seta-gun, Gunma 371-01,1 Preclinical Research Laboratories,

Bristol-Myers Research Institute, Ltd., I Futagoyama, Sakazaki, Kota-cho, Nukata-gun, Aichi 444_0J,2and Laboratory ofDrug Resistance in Bacteria, School of Medicine, Gunma University,

39-22, Showa-machi, Maebashi, Gunma 37J,3 Japan

Received 17 July 1989/Accepted 21 December 1989

BMY-28271, the acetoxyethyl ester of BMY-28232, 7-[(Z)-2-(2-aminothiazol-4-yl)-2-hydroxyiminoaceta-mido]-3(Z)-propen-l-yl-3-cephem-4-carboxylic acid, is a new oral cephalosporin. BMY-28232 has a widelyexpanded spectrum with high activity against gram-positive and gram-negative bacteria. BMY-28232 is farmore active than cefixime or cefteram against Staphylococcus aureus and Staphylococcus epidermidis. Againstgram-negative bacteria, the activity of BMY-28232 was comparable to or somewhat weaker than that ofcefixime or cefteram. BMY-28232 was a poor substrate for various P-lactamases. Orally administeredBMY-28271 had a good therapeutic effect on systemic infections with S. aureus and some gram-negativebacteria in mice. Oral BMY-28271 was efficacious against S. aureus Smith infection: the efficacy of BMY-28271was 80 to 90 times higher than that of cefixime or cefteram.

Many cephalosporins with broad antibacterial spectrahave been used clinically for the treatment of a wide range ofbacterial infections. Recently, there has been great progressin the development of oral cephalosporins, but each clini-cally available cephalosporin has weaknesses. Cefaclor isactive against gram-positive and some gram-negative bacte-ria but is hydrolyzed by various f-lactamases (13). Cefiximeand cefteram (T-2525) (2, 12, 14) have good activity againstgram-negative bacteria and streptococci and are not hydro-lyzed readily by various P-lactamases, but these compoundshave relatively low activities against staphylococci (2, 6, 12,14).BMY-28232, the parent compound of BMY-28271, is

active against gram-positive and gram-negative bacteria andis relatively resistant to hydrolysis by various 1-lactamases(16). The present study deals with the in vitro antibacterialactivity and P-lactamase stability of BMY-28232 and the invivo antibacterial activity of BMY-28271 in comparison withthose of cefixime and cefteram.

MATERIALS AND METHODSAntibiotics. BMY-28232 and BMY-28271 (the acetoxy-

ethyl ester of BMY-28232) were synthesized at the TokyoResearch Center of Bristol Myers Research Institute, Ltd.,Tokyo, Japan.- The other antimicrobial agents used in thisstudy were obtained from the indicated sources: cefixime,Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan; cefteram(T-2525, Ro 19-5247) and cefteram pivoxil (T-2588) (2, 12,14), Toyama Chemical Co., Ltd., Tokyo, Japan; cefaclor,Shionogi Pharmaceutical Co., Ltd., Osaka, Japan; benzyl-penicillin, Meiji Seika Kaisha, Ltd., Tokyo, Japan; cephalo-ridine, Nihon Glaxo Co., Ltd., Tokyo, Japan; and methicil-lin, Banyu Pharmaceutical Co., Ltd., Tokyo, Japan.

Organisms. The bacterial strains used in this study wererecent clinical isolates from various hospitals in Japan. The3-lactamase-producing strains used in this study were main-

tained at the Episome Institute. All strains were stored at-800C.

* Corresponding author.

Determination of MICs. MICs were determined by thetwofold agar dilution method. The media used for precultureand MIC determination were as follows: brain heart infusionbroth (Difco Laboratories, Detroit, Mich.) and sensitivitydisk agar (SDA; Nissui Seiyaku Co., Ltd., Tokyo, Japan)supplemented with 5% defibrinated horse blood for strepto-cocci; brain heart infusion broth and SDA supplementedwith 5% Fildes enrichment (Difco) for Haemophilus influ-enzae; sensitivity test broth (STB; Nissui Seiyaku) supple-mented with 0.4% KNO3 and SDA for Pseudomonas spp.;GAM broth and agar (Nissui) for anaerobes; and STB andSDA for the other microorganisms. Organisms were precul-tured for 18 h at 37°C and suitably diluted in buffered saline(pH 7.0) containing 0.01% gelatin. Approximately 104 CFUof bacteria per spot were inoculated onto drug-containingagar plates with a multiloop inoculator (Microplanter; Sa-kuma Seisakusho Co., Ltd., Tokyo, Japan). Plates wereincubated for 18 h at 37°C, except for obligate anaerobes,which were incubated for 48 h. Haemophilus influenzae andNeisseria gonorrhoeae were incubated in a candle jar, andanaerobes were incubated in an anaerobic jar. The MIC wasdefined as the lowest drug concentration which preventedvisible growth of bacteria.

Hydrolysis by 13-lactamases. The various types of P-lactam-ases (1, 11) used in this study were purified and stored at-80°C at the Episome Institute (3-5, 9, 18, 19). Hydrolysisof the drugs was assayed by a spectrophotometric method(17) at a substrate concentration of 100 ,uM. The suscepti-bility of the drugs to hydrolysis by various P-lactamases wasexpressed as the rate of hydrolysis relative to that ofcephaloridine or benzylpenicillin, which was set at 100. Forthe 3-lactamases of Enterobacter cloacae GN7471 (10),Citrobacter freundii GN7391 (15), and Proteus vulgarisGN7919 (7), Km and Vmax values were determined fromhydrolytic rates at various substrate concentrations by useof the Lineweaver-Burke plot.

Bactericidal activity. An overnight shaking culture of or-ganisms in STB at 37°C was inoculated into freshly preparedSTB, and the culture was shaken at 37°C for 2 h. When theorganisms had grown to a density of approximately 105

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TABLE 1. In vitro antibacterial activity of BMY-28232 against clinical isolates

Organism MIC (4m1)a(no. of strains) Antibiotic

Range 50% 90%Methicillin-susceptible Staphylococcus aureus (54) BMY-28232 0.39-0.78 0.78 0.78

Cefixime 6.25-25 12.5 25Cefteram 3.13-12.5 6.25 6.25

Methicillin-susceptible Staphylococcus epidermidis (30) BMY-28232 0.025-3.13 0.10 0.39Cefixime 3.13-100 6.25 50Cefteram 0.39-12.5 1.56 6.25

Methicillin-resistant Staphylococcus epidermidis (39) BMY-28232 0.05->100 6.25 >100Cefixime 12.5->100 100 >100Cefteram 3.13->100 25 100

Streptococcus pyogenes (99) BMY-28232 <0.006-0.025 0.013 0.025Cefixime 0.05-0.20 0.10 0.10Cefteram <0.006-0.013 0.006 0.013

Streptococcus pneumoniae (44) BMY-28232 <0.006-0.20 0.10 0.10Cefixime 0.025-0.78 0.20 0.20Cefteram <0.006-0.05 0.013 0.025

Clostridium perfringens (16)" BMY-28232 0.39-12.5 3.13 12.5Cefixime 3.13->100 12.5 >100Cefteram 1.56->100 3.13 100

Escherichia coli (99) BMY-28232 0.05-6.25 0.20 0.39Cefixime 0.013-12.5 0.20 0.39Cefteram 0.05-1.56 0.39 0.78

Shigella spp. (102) BMY-28232 0.025-0.39 0.10 0.20Cefixime 0.013-1.56 0.20 0.78Cefteram 0.05-0.78 0.20 0.78

Salmonella spp. (100) BMY-28232 0.10-0.78 0.20 0.39Cefixime 0.025-0.78 0.10 0.39Cefteram 0.39-1.56 0.39 1.56

Klebsiella pneumoniae (97) BMY-28232 0.025-0.78 0.10 0.10Cefixime 0.013-0.78 0.025 0.05Cefteram 0.05-3.13 0.20 0.39

Klebsiella oxytoca (50) BMY-28232 0.025-1.56 0.05 0.20Cefixime <0.006-0.78 0.013 0.05Cefteram 0.025-3.13 0.20 0.39

Proteus mirabilis (100) BMY-28232 0.05-0.39 0.10 0.20Cefixime <0.006-0.025 0.013 0.013Cefteram 0.05-0.78 0.10 0.20

Proteus vulgaris (50) BMY-28232 0.10-12.5 1.56 6.25Cefixime <0.006-0.78 0.013 0.05Cefteram 0.05-0.78 0.20 0.39

Morganella morganii (37) BMY-28232 0.10-50 1.56 12.5Cefixime <0.006-50 0.39 12.5Cefteram 0.05-25 0.10 6.25

Providencia stuartii (81) BMY-28232 <0.006-0.78 0.05 0.20Cefixime <0.006-1.56 0.013 0.20Cefteram 0.025-6.25 0.39 3.13

Providencia rettgeri (51) BMY-28232 <0.006-3.13 <0.006 0.10Cefixime <0.006-1.56 <0.006 0.05Cefteram <0.006-12.5 0.025 0.39

Citrobacterfreundii (88) BMY-28232 0.20->100 0.78 >100Cefixime 0.10->100 1.56 >100Cefteram 0.39->100 1.56 >100

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TABLE 1-Continued

Organism MIC (Lg/ml)a(no. of strains) Antibiotic

Range 50o 90o

Enterobacter cloacae (100) BMY-28232 0.10->100 6.25 >100Cefixime 0.025->100 3.13 >100Cefteram 0.10->100 1.56 >100

Enterobacter aerogenes (10) BMY-28232 0.20-100 0.39 1.56Cefixime 0.39->100 0.39 0.78Cefteram 0.39-50 0.39 0.78

Serratia marcescens (121) BMY-28232 0.78->100 12.5 >100Cefixime 0.10->100 3.13 >100Cefteram 1.56->100 12.5 >100

Pseudomonas cepacia (40) BMY-28232 0.39-25 3.13 12.5Cefixime 0.20-50 1.56 3.13Cefteram 1.56-100 12.5 25

Acinetobacter calcoaceticus (35) BMY-28232 3.13-50 6.25 25Cefixime 3.13->100 25 100Cefteram 25->100 50 >100

Haemophilus influenzae, penicillinase negative (84) BMY-28232 0.05-0.39 0.10 0.10Cefixime 0.013-0.05 0.025 0.025Cefteram <0.006-0.05 0.013 0.025

Haemophilus influenzae, penicillinase positive (32) BMY-28232 0.05-0.39 0.10 0.20Cefixime 0.013-0.20 0.025 0.05Cefteram 0.013-0.05 0.013 0.025

Branhamella catarrhalis (38) BMY-28232 0.10-0.78 0.20 0.39Cefixime 0.025-0.39 0.10 0.39Cefteram 0.05-1.56 0.20 1.56

Neisseria gonorrhoeae (24) BMY-28232 <0.006-0.78 <0.006 0.025Cefixime <0.006-0.013 <0.006 <0.006Cefteram <0.006-1.56 0.013 0.025

Bacteroides fragilis (35)" BMY-28232 313->100 25 >100Cefixime 1.56->100 100 >100Cefteram 3.13->100 50 >100

a 50o and 90%o, MICs for 50 and 90%o of strains tested, respectively.b Inoculum of 106 CFU per spot.

CFU/ml, antibiotics were added to the culture at concentra-tions ranging from one-fourth to four times the MIC deter-mined by the agar dilution method. Samples were taken atintervals and inoculated onto drug-free heart infusion agarplates; the numbers of colonies were counted after 18 h ofincubation at 37°C.

Determination of oral in vivo activity. The in vivo antibac-terial activities of the drugs were determined against sys-temic infections in mice. Ten 4-week-old male STD:ddYmice (weight, 19 to 21 g) were used for each dose of drug. Anovernight culture on heart infusion agar at 37°C was sus-pended in physiological saline for Escherichia coli ML4707and Klebsiella pneumoniae GN6445 or in 5% gastric mucinfor Staphylococcus aureus Smith and Proteus mirabilisGN4754. A 0.5-ml portion of the bacterial suspension wasinoculated intraperitoneally into each mouse. Under theseconditions, all untreated mice died within 5 days. The drugswere suspended in 0.5% carboxymethyl cellulose and weregiven to the mice as a single oral dose immediately afterinfection. The 50% effective dose was calculated by theProbit method (8) from the number of mice that survived ateach dose level 5 days after bacterial challenge.

RESULTS

Antimicrobial activity against clinical isolates. The in vitroantimicrobial activity of BMY-28232 against gram-positiveand gram-negative organisms was compared with those ofcefixime and cefteram (T-2525, Ro 19-5247). Table 1 showsthe concentrations of antibiotics required to inhibit thegrowth of 50 and 90%o (MIC%0) of the total number of strainstested. The MIC90s of BMY-28232 against gram-positiveorganisms such as methicillin-susceptible Staphylococcusaureus, methicillin-susceptible Staphylococcus epidermidis,Streptococcus pyogenes, and Streptococcus pneumoniaeranged from 0.025 to 0.78 ,ug/ml. BMY-28232 was 32- to128-fold more active than cefixime and 8- to 16-fold moreactive than cefteram against these staphylococci. AlthoughBMY-28232 was active against some strains of methicillin-resistant Staphylococcus epidermidis, most of the methicil-lin-resistant staphylococci were resistant to BMY-28232 andthe other two agents. BMY-28232 was active (MIC90s, 0.025and 0.10 ,ug/ml, respectively) against Streptococcus pyo-genes and Streptococcus pneumoniae. The MICs of BMY-28232 against Enterococcus faecalis were distributed in a

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TABLE 2. Activity of BMY-28232 against ,-lactamase producing bacteria

MIC ([Lg/ml),3-Lactamase-producing bacteriaa

BMY-28232 Cefixime Cefteram

Nonproducing hostsStaphylococcus aureus MS15009 0.20 3.13 0.78Escherichia coli ML4901 0.20 0.39 0.39

Penicillinase producersStaphylococcus aureus MS15009(pI258) (2a) 0.39 6.25 3.13Escherichia coli ML4901(Rms212) (2b) 0.20 0.20 0.39Escherichia coli ML4901(Rms213) (2d) 0.20 0.39 1.56Escherichia coli ML4901(Rtel6) (2d) 0.39 0.39 0.39Escherichia coli ML4901(Rmsl49) (2c) 0.20 0.20 0.39Escherichia coli ML4901 TEM-2 (2b) 0.39 0.39 0.39Escherichia coli ML4901 SHV-1 (2b) 0.20 0.20 0.39

Cephalosporinase producersEscherichia coli ML4901(pMS161) (Enterobacter cloacae; 1) 1.56 3.13 0.78Escherichia coli ML4901(pMS182) (Proteus vulgaris; 2e) 0.39 0.39 0.78Escherichia coli ML4901(pMS185) (Citrobacterfreundii; 1) 3.13 12.5 3.13Escherichia coli ML4901(pMS500) (Escherichia coli; 1) 1.56 6.25 0.78

CephalosporinasesEscherichia coli GN14930 (chromosomal, 1) 6.25 25 1.56Enterobacter cloacae GN7467 (chromosomal, 1) 50 50 50Providencia rettgeri GN4430 (chromosomal, 1) <0.006 <0.006 0.05Morganella morganii GN5307 (chromosomal, 1) 0.20 0.20 0.10Serratia marcescens GN14932 (chromosomal, 1) 50 6.25 3.13Pseudomonas aeruginosa GN10362 (chromosomal, 1) >100 >100 >100Proteus vulgaris GN4413 (chromosomal, 2e) 100 1.56 3.13Klebsiella oxytoca GN10650 (chromosomal) 1.56 0.10 3.13Pseudomonas cepacia GN11164 (chromosomal) 1.56 0.39 6.25

a Staphylococcus aureus MS15009 and Escherichia coli ML4901 were ,3-lactamase-nonproducing host strains. Enzyme types and the classification of Bush (1)are given in parentheses.

wide range (1.56 to >100 ,uglml), as were those of cefiximeand cefteram. Enterococcus faecium and Clostridium dif-ficile were not susceptible to BMY-28232 or the other twoagents. BMY-28232 had an MIC90 of 12.5 ,ug/ml againstClostridium perfringens, which was lower than those of theother two compounds tested.BMY-28232 was active against various species of gram-

negative bacteria such as Escherichia coli, Shigella spp.,Salmonella spp., Klebsiella spp., Proteus mirabilis, Provi-dencia stuartii, Providencia rettgeri, Enterobacter aero-genes, Haemophilus influenzae, Branhamella catarrhalis,and Neisseria gonorrhoeae. The MIC90s against these or-ganisms ranged from 0.025 to 1.56 uig/ml. BMY-28232 wasless active against Proteus vulgaris (MIC90, 6.25 ,ug/ml) thanit was against cefixime (MICg, 0.05 ,ug/ml) or cefteram(MIC90, 0.39 ,ug/ml). Against Morganella morganii, BMY-28232, with an MIC90 of 12.5 ,ug/ml, showed activity com-parable to those of cefixime and cefteram. Citrobacterfreundii, Enterobacter cloacae, and Serratia marcescensshowed broad MIC distributions against these agents, andmore than 10% of the isolates were highly resistant toBMY-28232 and the other two agents. BMY-28232 had anMIC90 of 12.5 ,ug/ml against Pseudomonas cepacia andshowed activity intermediate between those of cefixime andcefteram. BMY-28232 was more active against Acinetobac-ter calcoaceticus than it was against cefixime and cefteram.BMY-28232 had poor activity against Pseudomonas aerugi-nosa, Xanthomonas maltophilia, and Bacteroides fragilis(MIC90s, >100 jig/ml), which was similar to the activities ofcefixime and cefteram.

Antimicrobial activity against i8-lactamase-producing

strains. The MICs of BMY-28232, cefixime, and cefteram for,B-lactamase-producing organisms are shown in Table 2. Theactivity of BMY-28232 against Staphylococcus aureus andEscherichia coli was not affected by the production ofvarious types of penicillinases. The activity of BMY-28232against an Escherichia coli strain which produced chromo-somal cephalosporinases of Escherichia coli, Enterobactercloacae, and Citrobacterfreundii was 8- to 16-fold less thanthat against the host strain without these enzymes. Theseenzymes reduced the activity of BMY-28232 to the sameextent as they did the activity of cefixime. Against anEscherichia coli strain which produced Proteus vulgarisenzyme, these agents were as active as they were against thehost strain. BMY-28232 was fourfold more active thancefixime and fourfold less active than cefteram againstEscherichia coli GN14930. Providencia rettgeri GN4430 andMorganella morganhi GN5307 were equally susceptible tothese agents. BMY-28232 was 8- to 64-fold less active thanthe two other agents against Serratia marcescens GN14932and Proteus vulgaris GN4413. The activity of BMY-28232against Klebsiella oxytoca GN10650 and Pseudomonas ce-pacia GN11164 was 4- to 16-fold lower than that of cefiximeand was equal to or somewhat higher than that of cefteram.Enterobacter cloacae GN7467 and Pseudomonas aerugi-nosa GN10362 had poor susceptibilities to these agents.

Hydrolysis by the I-lactamases. The relative hydrolysisrates of BMY-28232 by P-lactamases are shown in Table 3.Hydrolysis of BMY-28232 by several types of penicillinasescould not be detected. Although BMY-28232, cefixime, andcefteram were poor substrates for cephalosporinases, slowhydrolysis of these agents was observed for the enzymes

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TABLE 3. Hydrolysis of BMY-28232 by various 1-lactamases

E-Lactamase Relative hydrolysis rate of":Enzyme source classclassa BMY-28232 Cefixime Cefteram

PenicillinaseEscherichia coli ML4901(Rms212) Type I (2b) <0.1 0.1 <0.1Escherichia coli ML4901(Rms2l3) Type II (2d) 0.3 <0.1 21.1Escherichia coli ML4901(Rtel6) Type III (2d) 0.1 <0.1 0.1Escherichia coli ML4901(Rms433) Type IV (2c) <0.1 <0.1 <0.6Staphylococcus aureus MS15009(pI258) Type V (2a) <0.1 <0.1 <0.1

CephalosporinaseEscherichia coli GN5482 (1) <0.1 <0.1 0.6Serratia marcescens GN10857 (1) 1.9 4.1 3.4Pseudomonas aeruginosa GN10362 (1) 0.8 0.3 1.7

OxyiminocephalosporinaseKlebsiella oxytoca GN10650 0.8 0.2 5.7Pseudomonas cepacia GN11164 4.1 1.8 35.5a Based on the classification of Mitsuhashi and Inoue (11). The classification of Bush (1) is given in parentheses.b Relative to the hydrolysis rate for cephaloridine (cephalosporinase) or benzylpenicillin (penicillinase and oxyiminocephalosporinase), which was taken as 100.

from Serratia marcescens GN10857 and Pseudomonasaeruginosa GN10362. BMY-28232 was hydrolyzed by anoxyiminocephalosporinase from Pseudomonas cepaciaGN11164 and was hydrolyzed more slowly by the enzymefrom Klebsiella oxytoca GN10650 than by that from Pseu-domonas cepacia. Because the cephalosporinase-producingstrains of Citrobacter freundii and Enterobacter cloacaewere resistant to BMY-28232 and the other two agents, thehydrolytic kinetics were determined for the enzymes fromCitrobacter freundii GN7391 and Enterobacter cloacaeGN7471 and compared with that for an oxyiminocephalos-porinase from Proteus vulgaris GN7919 (Table 4). BMY-28232, similar to cefixime and cefteram, was hydrolyzedvery slowly by these cephalosporinases from Citrobacterfreundii GN7391 and Enterobacter cloacae GN7471. TheVm., values of these agents for the Citrobacter freundiienzyme were smaller than 0.02% of those of cephaloridine,and those for Enterobacter cloacae enzyme were smallerthan 0.2% of those of cephaloridine. BMY-28232 was morehydrolyzable than cefixime by the oxyiminocephalospori-nase from Proteus vulgaris GN7919: VmaxlKm values ofBMY-28232, cefixime, and cefteram were 0.066, 0.030, and0.121, respectively.

Bactericidal activity. The killing curve of BMY-28232against Staphylococcus aureus FDA 209P JC-1 is shown inFig. 1. BMY-28232 displayed more bactericidal activity atlower concentrations than cefixime and cefteram did. BMY-28232, cefixime, and cefteram reduced the viable cell countscomparably and inhibited the growth of cells after 24 h ofincubation at 0.78, 100, and 12.5 pLg/ml, respectively.

Oral in vivo antibacterial activity. The protective effect ofBMY-28271, the acetoxyethyl ester of BMY-28232, against

systemic infections in mice is shown in Table 5. The 50%effective dose of BMY-28271 against Staphylococcus aureus

Smith infection was 0.84 mg/kg. The activity of BMY-28271was 80 to 90 times greater than that of cefixime or cefteramand was slightly inferior to that of cefaclor. The activity ofBMY-28271 against Escherichia coli ML4707 infections wastwo to four times greater than that of cefixime or cefteram.Against Klebsiella pneumoniae GN6445 and Proteus mirab-ilis GN4754, the activity of BMY-28271 was nearly equal tothat of cefteram and about one-eighth that of cefixime.

DISCUSSION

Recently, several new oral cephalosporins, such as ce-fixime and cefteram, have been developed. These antibioticshave highly potent activities against various species ofgram-negative bacteria and streptococci. BMY-28271 is an

orally administered acetoxyethyl ester of BMY-28232. It hadexcellent activity against methicillin-susceptible staphylo-cocci, Escherichia coli, Shigella spp., Salmonella spp.,Klebsiella spp., Proteus mirabilis, Providencia spp., Hae-mophilus influenzae, Branhamella catarrhalis, and Neis-seria gonorrhoeae. Against gram-negative bacteria, BMY-28232 had activity roughly comparable to those of cefiximeand cefteram. These results of antibacterial activities againstclinical isolates were similar to those reported by Tomatsu etal. (16). The characteristic of this drug was that it hadactivity superior to those of cefixime and cefteram againstmethicillin-susceptible staphylococci. BMY-28232 was 32-to 128-fold more active than cefixime and 8- to 16-fold moreactive than cefteram against these organisms. BMY-28232also showed bactericidal activity against Staphylococcus

TABLE 4. Hydrolysis kinetic parameters for cephalosporinases from Enterobacter cloacae GN7391,Enterobacter cloacae GN7471, and Proteus vulgaris GN7919

BMY-28232 Cefixime Cefteram Cephaloridine3-Lactamase origin KK. V1 a K. Vma K. Va K. Vma(>IM) max (>M) max (M) max (ILM) max

Citrobacterfreundii GN7391 13.1 0.00876 5.75 0.00302 6.80 0.0191 221 100Enterobacter cloacae GN7471 11.3 0.0375 8.48 0.0505 11.8 0.130 154 100Proteus vulgaris GN7919 142 9.37 449 13.3 619 74.9 78.0 100

a Values are relative to the V___ rate for cephaloridine, which was taken as 100.

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A B C

Time (hr) Time (hr) Time (hr)FIG. 1. Bactericidal activity in STB of BMY-28232, cefixime, and cefteram for Staphylococcus aureus FDA 209P JC-1. Viable cells counts

were determined after exposure of Staphylococcus aureus to BMY-28232 (A), cefixime (B), and cefteram (C) at one-fourth (0), one (A), andfour (l) times the MIC (MIC, 0.78, 100, and 12.5 ,ug/ml, respectively). 0, Control.

aureus. BMY-28232 is a poor substrate for penicillinases,and its antimicrobial activity was not affected by the produc-tion of those enzymes. Although the hydrolysis of BMY-28232 by cephalosporinases of Escherichia coli, Serratiamarcescens, and Pseudomonas aeruginosa was slow or notdetected, species that produced high levels of the cephalos-porinases were resistant to BMY-28232. Results of experi-ments with Escherichia coli strains carrying cloned ceph-alosporinases of Enterobacter cloacae, Citrobacterfreundii,and Escherichia coli showed that these cephalosporinasesreduce the antimicrobial activities of BMY-28232 and theother two agents. Hydrolysis of BMY-28232 by the ceph-alosporinases from Enterobacter cloacae and Citrobacterfreundii was not detected by Tomatsu et al. (16). Wedetermined the hydrolysis kinetic parameters of BMY-28232and the other two agents for the enzymes from Enterobactercloacae GN7471 and Citrobacterfreundii GN7391. Actually,these agents were hydrolyzed by the enzymes from Entero-bacter cloacae and Citrobacterfreundii. Thus, species thatproduce high levels of cephalosporinases can be resistant tothese agents. BMY-28232 was hydrolyzed by an oxyimi-

nocephalosporinase from Proteus vulgaris GN7919 morerapidly than cefixime was and somewhat more slowly thancefteram was. This stability of BMY-28232 for the Proteusvulgaris 3-lactamase could not explain sufficiently the loweractivity of BMY-28232 against clinical isolates of Proteusvulgaris than those of cefixime and cefteram. Other studiesthat examine permeability and affinity to penicillin-bindingproteins and other types of ,B-lactamases are needed toexplain the low susceptibility of Proteus vulgaris to BMY-28232.

Orally administered BMY-28271 had excellent therapeuticeffects against systemic infections in mice infected withStaphylococcus aureus and gram-negative bacteria. The invivo activity of BMY-28271 was comparable to or somewhatmore active than that of cefteram against Escherichia coliML4707, Klebsiella pneumoniae GN6445, and Proteus mi-rabilis GN4754. The therapeutic efficacy of orally adminis-tered BMY-28232 against Staphylococcus aureus Smith wascomparable to that of cefaclor and was far superior to thoseof cefixime and cefteram pivoxil. These in vivo activityresults were similar to those reported by Tomatsu et al. (16).

TABLE 5. Oral in vivo antibacterial activity of BMY-28271 against systemic infection in mice

Organism Inoculum Mucin AnMibioticCml ED50C 95% ConfidenceOrganism ~~~~(CFU/mouse)a ui ntboi (jig/ml), (mg/kg) limit (mg/kg)

Staphylococcus aureus Smith 1.8 x 107 (11) + BMY-28271 0.39 0.84 0.40-3.5Cefixime 12.5 77 56-110Cefteramd 3.13 68 45-110Cefaclor 0.78 0.13 0.10-0.2

Escherichia coli ML4707 4.6 x 107 (40) - BMY-28271 0.20 3.1 1.0-7.2Cefixime 0.20 12 7.3-22Cefteramd 0.20 8.5 5.1-14

Klebsiella pneumoniae GN6445 3.0 x 107 (10) - BMY-28271 0.10 8.1 5.1-13Cefixime 0.05 0.99 0.60-1.5Cefteramd 0.10 14 8.7-22

Proteus mirabilis GN4754 6.8 x 107 (20) + BMY-28271 0.20 6.6 4.2-10Cefixime <0.006 0.70 0.42-1.1Cefteramd 0.20 8.2 5.0-13

a Values in parentheses are 50%o lethal doses.b The MICs were determined by the agar dilution method. The MICs of BMY-28271 and cefteram pivoxil were measured by using BMY-28232 and cefteram.cED50, 50% effective dose.d Cefteram pivoxil was used.

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BMY-28271, A NEW ORAL CEPHALOSPORIN ESTER 561

In conclusion, the in vitro properties of BMY-28232against gram-negative bacteria were almost the same asthose of cefixime and cefteram. The unusual feature ofBMY-28271 (BMY-28232) was its potent activity in vitro andin vivo against gram-positive bacteria, especially againstmethicillin-susceptible staphylococci, against which cefix-ime and cefteram were less active. The data in this reportsuggest that BMY-28271 is worthy of further study andclinical evaluation.

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