ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, JUly 1980, p. 105-110 Vol. 18, No.1 0066-4804/80/07-0105/06$02.00/0 Cefroxadine (CGP-9000), an Orally Active Cephalosporin K. YASUDA,' S. KURASHIGE,2 AND S. MITSUHASHI2* Department ofMicrobiology, School ofMedicine, Gunma University, Maebashi, Gunma,2 and Medical Department, Pharmaceuticals Division, CHIBA-GEIGY Limited, Takarazuka,' Japan Cefroxadine (CGP-9000; CXD), 7,8[D-2-amino-2-(1,4-cyclohexadienyl)-aceta- mido]-3-methoxy-ceph-3-em-carboxylic acid, is a new orally active cephalosporin derivative. The spectrum of antibacterial activity of CXD is identical with that of cephalexin (CEX), but CXD was twofold more effective than CEX against Escherichia coli and Klebsiella pneumoniae. CXD was as stable to penicillinase as CEX, but it was hydrolyzed by cephalosporinase, with a relative rate of hydrolysis similar to that of CEX. The affinities of CXD and CEX to penicillin- binding proteins of E. coli were estimated; the affinity of CXD to penicillin- binding protein lBs was higher than that of CEX. Consistent with this, CXD had more intensive lytic activity than CEX. In vivo antibacterial activities of CXD and CEX were compared using systemic infections of mice with E. coli and K. pneumoniae; CXD was consistently more active than CEX. Although there are many cephalosporins water. A loopful (about 0.005 ml) of diluted culture available that are suitable for parenteral admin- was inoculated by Micro-planter (Kubota, Japan) onto istration, orally active cephalosporins are less agar plates containing a series of serial twofold dilu- tions ofa drug. MIC values were scored after overnight common. Among oral cephalosporins introduced incubation at 37°C. on the market, cephalexin (CEX) is now in wide- The effect of inoculum size was determined by spread use in the treatment of a variety of infec- means of a twofold serial agar dilution method on HI tious diseases. Cefroxadine (CXD) is the dihy- agar. The inocula were overnight cultures diluted in drophenylglycin derivative of a structurally physiological saline to 10-1, 10-3, and 10-5. modified 7-amino-cephalosporanic acid and is The 50% infective dose. Values for 50% infective structurally related to CEX, but possesses anti- dose were determined by the method of Kato et al. bacterial properties that distinguish it from (2). A bacterial culture in peptone water was diluted CEX. This paper presents the in vitro and in to 2 x 103 to 3 x 103 cells per ml with fresh peptone vivo evaluation of CXD. water. HI agar plates containing various concentra- microbiological tions of a drug were prepared, and a 0.1-ml sample of MATERIALS AND METHODS diluted bacterial suspension was spread on each plate. After overnight incubation at 37°C the number of Antibiotics. CXD, 7f?[D-2-amino-2-(1,4-cyclohex- colonies that had grown on the plate were counted. adienyl)-acetamido]-3-methoxy-ceph-3-em-carboxylic The mean growth inhibition was calculated from a acid, is an orally active cephalosporin derivative (Fig. mean number of colonies on five plates at each drug 1) which was synthesized in the Research Laborato- concentration and on five drug-free agar plates. ries, Pharmaceuticals Division, CIBA-GEIGY Lim- Bactericidal activity. An overnight culture of ited, Basel, Switzerland. Other antibiotics were com- each strain in antibiotic medium no. 3 (Difco) was mercial products. diluted to a final concentration of about 104 cells per Test strains. Strains stocked in this laboratory ml with antibiotic medium no. 3 containing a series of were used as standard stock cultures and are from the serial twofold dilutions of a drug. MICs were read after Reference Laboratory of Drug-Resistant Bacteria, incubation at 37°C for 18 h. One loopful of each culture Gunma University. They were originally isolated from tube in the MIC test series was spotted onto drug-free clinical materials. HI agar plates, and after incubation at 37°C for 18 h, Media. Heart infusion (HI) agar (Eiken), HI broth the minimal bactericidal concentrations of antibiotic (Eiken), and antibiotic medium no. 3 (Difco) were were determined as the lower concentration of drug used. Other media were peptone water and medium that prevented visible growth on HI agar plates. An- B. The former consisted of 10 g of polypeptone, 5 g of other method consisted of counting the number of NaCl, and 1 liter ofdistilled water, the latter contained viable cells at appropriate time intervals after addition 2 g of yeast extract, 10 g of polypeptone, 7 g of of drugs. Na2HPO4. 12H20, 2 g of KH2PO4, 1.2 g of glucose, and Stability to /?-lactamase. The enzyme samples 0.4 g of MgSO4- 7H20 in 1 liter of distilled water. were prepared as follows. A 1-ml brain heart infusion In vitro antibacterial activity. Minimal inhibi- culture of each strain was diluted 10-fold with medium tory concentration (MIC) of a drug was determined by B and incubated at 37°C. The cells were harvested by an agar dilution method. Overnight culture in peptone centrifugation, washed with 0.05 M phosphate buffer water was diluted to 106 cells per ml with fresh peptone (pH 7.0), and resuspended in the same buffer. The 105 106 YASUDA, KURASHIGE, AND MITSUHASHI ANTIMICROB. AGENTS CHEMOTHER. cells were disrupted in an ultrasonicator. Enzyme ac- fractionated on a sodium dodecyl sulfate-polyacryl- tivity was photometrically measured (6). All the mea- amide slab gel. The PBPs were detected by fluorog- surements were made at a substrate concentration of raphy with X-ray film (Fuji RHs). The level of [14C] 0.1 mM. The figures in Table 3 are relative values, penicillin G binding to 1A and lBs at each competitor taking the absolute rate of cephaloridine hydrolysis as concentration was quantitated by densitometry of the 100 for cephalosporinase and that of penicillin G hy- X-ray film. drolysis as 100 for penicillinase. In vivo antibacterial activity. The in vivo anti- Pseudomonas aeruginosa ML4259 Rms139+ (7) bacterial activity of CXD was determined by experi- and Klebsiella pneumoniae GN49 were used as the mental infection of mice with gram-negative bacteria. standard strains capable of producing the known types Twenty male mice (ICR strain) weighing 18 to 22 g of penicillinase, and Escherichia coli GN5482, P. were used for each dose level. The mice were chal- aeruginosa GN918 (12), Enterobacter cloacae lenged intraperitoneally with sufficient microorgan- GN7471, and Proteus morganii GN5407 were used for isms to kill all nontreated mice within 48 h. The cephalosporinase. microorganisms were grown on an HI agar plate and PBPs. The affinity of CXD and the comparative suspended in physiological saline solution. Mice in- compound, CEX, to the penicillin-binding proteins fected with E. coli ML4707 and K. pneumoniae (PBPs) was examined. Seven PBPs were detected in GN6445 were treated orally immediately after infec- E. coli JE1011 by the modified method (11) described tion and 3 h later. The total number of surviving mice by Spratt (8). CXD and CEX were used as competitors was recorded, usually 1 week after infection, and the for '4C-labeled penicillin G binding to E. coli PBPs. amount of a single dose (milligrams per kilogram) that The concentrations of competitor added to the reac- gave protection to 50% of the infected mice was esti- tion mixture were 1-, 5-, and 25-fold greater than the mated by means of a log-probit plot (3). concentration of 14C-labeled penicillin G (34 itg/ml). The binding reaction was terminated, and the proteins RESULTS of the inner membrane were selectively solubilized and An.ftnPIfi;&wrIUiho+va1l 5jM%&rU*Qeywt.ium Tha111; DIMLILUaae.twnim V1nf antibacterial activity of CXD against gram-pos- itive and gram-negative bacteria is shown in Table 1. CXD was active against both gram- positive and gram-negative organisms suscepti- -OCH3. 2H2o ble to CEX. Both antibiotics exhibited almost similar effectiveness against gram-positive or- ganisms, but CXD was more active against FIG. 1. Structure of CXD (CGP- 9000). CEX - susceptible gram - negative organisms. TABLE 1. Antibacterial activity of CXD against standard strains of bacteria MIC (g/ml)b Test organisma CXD CEX 106 lol 106 lol Staphylococcus aureus 1.56 3.13 3.13 6.25 FDA209PJC-1 S. aureus E-46 3.13 6.25 6.25 12.5 S. aureus Terajima 1.56 3.13 3.13 6.25 Escherichia coli 3.13 6.25 12.5 12.5 NIHJ-JC-2 Salmonella typhi 901 1.56 3.13 3.13 6.25 S. paratyphi 1015 1.56 3.13 3.13 6.25 S. schottmuelleri 8006 1.56 3.13 3.13 6.25 Klebsiellapneumoniae 1.56 3.13 3.13 6.25 PCI-602 Serratia marcescens >100 >100 >100 >100 L4MI 184 Proteus vulgaris OX-19 >100 >100 >100 >100 P. rettgeri IF03850 >100 >100 >100 >100 P. mirabilisIF03849 6.25 12.5 50 100 Pseudomonas aeruginosa >100 >100 >100 >100 IF03445 aThe tested strains are the standard strain stocked at the Laboratory of Bacterial Resistance, School of Medciine, Gunma University. b Overnight HI broth culture was diluted with physiological saline, and one loopful (0.005 ml) of i06 or 108 cells per ml was inoculated. VOL. 18, 1980 CEFROXADINE 107 Both CXD and CEX acted equally poorly tration of CXD required to inhibit the growth of against Serratia marcescens, Proteus species, 50% of the total number of tested E. coli strains and P. aeruginosa. (MIC50) was 3.13 to 6.25 ,ug/ml, whereas that of Antibacterial activity. The antibacterial ac- CEX was 6.25 to 12.5 ,ug/ml. The MIC50 of CXD tivity of CXD against gram-positive and gram- against K.