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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 1987, p. 1085-1092 Vol. 31, No. 7 0066-4804187/071085-08$02.00/0 Copyright ©3 1987, American Society for Microbiology In Vitro and In Vivo Antibacterial Activities of CS-807, a New Oral Cephalosporin YUKIO UTSUI,l* MATSUHISA INOUE,2 AND SUSUMU MITSUHASHI1 Episome Institute, 2220 Kogure, Fujimi-mura, Seta-gun, Gunma 371-01,1 and Laboratory of Drug Resistance in Bacteria, School of Medicine, Gunma University, 39-22, Showamachi 3-chome, Maebashi, Gunma 371,2 Japan Received 8 December 1986/Accepted 21 April 1987 CS-807 is a new oral prodrug of R-3746, a cephalosporin derivative, with potent in vitro and in vivo antibacterial activity against both gram-positive and gram-negative bacteria. The susceptibility of about 1,200 clinical isolates to R-3746 was determined by the agar dilution method. Ninety percent or more of pathogens such as Staphylococcus aureus, streptococci, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, indole-positive and indole-negative Proteus spp., Providencia rettgeri, and Haemophilus influenzae were inhibited at concentrations ranging from .0.01 to 1.56 ,ug/ml. Furthermore, at a concentration of 3.13 ,ug/ml, 50% or more of Staphylococcus epidermidis, Morganella morganii, Citrobacterfreundii, and Serratia marcescens strains were also inhibited. Pseudomonas aeruginosa and Xanthomonas maltophilia were resistant to R-3746. The activity of R-3746 was scarcely influenced by several growth conditions. R-3746 was highly resistant to hydrolysis by Il-lactamases derived from various species of bacteria. Killing-curve studies demonstrated bactericidal activity of R-3746 at concentrations above the MIC. R-3746 showed high affinity for penicillin- binding proteins 1, 3, and 4 of Staphylococcus aureus and 1A, 1l$s, and 3 ofEscherichia coli. Systemic infections in mice caused by various pathogens, including j3-lactamase-producing strains, responded well to therapy with oral doses of CS-807. Various oral cephem antibiotics having relatively broad MATERIALS AND METHODS antibacterial spectra have been developed and used world- wide (2, 3, 15, 18, 19, 34). The effectiveness, however, of the Chemicals. CS-807 and R-3746 were prepared by Sankyo oral antibiotics in the treatment of bacterial infections is Co., Ltd., Tokyo, Japan. The other antibiotics were gifts: somewhat limited because of their poor activity against penicillin G, amoxicillin, and methicillin from Banyu Phar- gram-negative bacilli, in particular against opportunistic maceutical Co., Ltd., Tokyo, Japan; cephaloridine from pathogens belonging to the Enterobacteriaceae or Pseudo- Nihon Glaxo Co., Ltd., Tokyo, Japan; cefaclor from monadaceae. Shionogi & Co., Ltd., Osaka, Japan; cefixime from Fujisawa Recently, new oral cephem antibiotics such as cefixime Pharmaceutical Co., Ltd., Osaka, Japan; and T-2588 and (7), T-2588 (T. Yasuda, A. Yotsuji, S. Okamoto, and S. T-2525 from Toyama Chemical Co., Ltd., Tokyo, Japan. All Mitsuhashi, Program Abstr. 24th Intersci. Conf. Antimicrob. antibiotics were prepared daily with distilled water to the Agents Chemother., abstr. no. 224, 1984), and ME-1207 (M. desired concentrations. '4C-labeled penicillin G (potassium Inoue, A. Tamura, T. Yoshida, R. Okamoto, K. Atsumi, K, 6-phenyl [1-_4C]acetamidopenicillanate; 54 mCi/mmol) was Nishihata, and S. Mitsuhashi, 25th ICAAC, abstr. no. 582, purchased from Amersham International, Buckinghamshire, 1985) have been developed, and clinical efficacy studies of United Kingdom. cefixime and T-2588 are in progress. Those compounds have Bacterial strains. Standard strains stocked at our labora- the characteristics of much higher in vitro and in vivo tories and clinical isolates of various species collected from antibacterial activity than any other oral antibiotics, such as several hospitals in a wide area of Japan were used. cephalexin, cefadroxil, cefroxadine, cefradine, cefatrizine, Determination of MICs and MBCs. MICs were determined and cefaclor, against gram-negative bacteria, including op- by a twofold serial agar dilution method. As media, unless portunistic pathogens, and of enhanced resistance to various otherwise specified, sensitivity test broth (STB) and sensi- types of ,-lactamase. tivity disk agar (SDA) (both from Nissui Seiyaku Co., Ltd., CS-807 is a new oral prodrug of R-3746; its proper chem- Tokyo, Japan) were used for culture and MIC determina- ical name is 1-(isopropoxycarbonyloxy)ethyl(6R, 7R)-7-[2- tions, respectively. For streptococci, defibrinated horse (2-amino-4-thiazolyl)-(Z)-2-(methoxyimino)acetamido]-3- blood (Nippon Bio-Supp. Center, Tokyo Japan) was addcd methoymethyl-8-oxo-5-thia-1-azabicyclo[4,2,0]-oct-2-ene-2- to the agar medium to a final concentration of 5%. For carboxylate. Chemical structures of CS-807 and R-3746 are Haemophilus influenzae, brain-heart infusion broth (BHIB) shown in Fig. 1. and agar (BHIA) (both from Difco Laboratories, Detroit, The present report deals with the in vitro antibacterial Mich.) supplemented with 10 ptg of hemin and 5 Fxg of activity of R-3746 together with the therapeutic effect of 1-NAD per ml were used. For Bacteroides fragilis, Gifu CS-807 on systemic infections in mice compared with anaerobic medium broth and agar (Nissui Seiyaku) were cefaclor, amoxicillin, cefixime, and T-2588 (T-2525). used. The other media were STB containing 0.4% potassium nitrate for Pseudomonas aeruginosa and BHIB for culture of streptococci. Overnight cultures of test strains were diluted with a buffered saline-0.01% gelatine solution to a final concentration of approximately 106 CFU/ml. One spot (10 * Corresponding author. LI) of the diluted cultures, corresponding to approximately 5 1085 1086 UTSUI ET AL. ANTIMICROB. AGENTS CHEMOTHER. COOR (PBPs) were assayed by the method of Spratt (29) with some H2N tL 0>Ns CH2OCH3 modifications. Experiments to determine the binding affinity of 3-lactam antibiotics for PBPs were performed by prein- N ,-C O N H - 5 cubation of the membrane fractions with nonradioactive drugs at 30°C for 10 min before addition of '4C-labeled penicillin G. Fluorographs of PBPs were analyzed quantita- tively with a densitometer (model CS-910; Shimadzu Corp., C H 3 Kyoto, Japan), and the I50 (concentration required to inhibit CS-807 R=-CHO COO CH(CH3)2 4C-labeled penicillin G binding by 50%) was calculated. Protective effect. Test organisms for systemic infections in R-3 746 R = - Na mice were cultured in HIB containing 5% horse serum FIG. 1. Chemical structures of CS-807 and R-3746. (Streptococcus pneumoniae 2132 and Streptococcus pyo- genes 1412) or in BHIB (other organisms) at 370C for 18 h. Subcultures, after being suitably diluted with fresh HIB or x 104 CFU, was applied to 10-ml agar layers containing BHIB, were suspended in the same amount of 8% gastric various concentrations of antibiotics with an inoculator mucin (Difco Laboratories), except E. coli ML4707. Four- (Microplanter; Sakuma Seisakusho, Tokyo, Japan). The week-old ICR male mice weighing 19 to 21 g were infected plates were incubated at 37°C for 18 h, whereas those to intraperitoneally with 0.2-ml portions of bacterial suspen- which Bacteroidesfragilis was spotted were incubated for 24 sion, corresponding'to 2 to 10 times the minimal lethal dose. h at 37°C under anaerobic conditions. The MIC was defined Under these conditions, untreated mice died within 48 h. as the lowest concentration of antibiotic which prevented Immediately after infection, 10 mice for each dose level were visible growth of bacteria. treated orally with 0.1 ml of a single-dose regimen (six dose MBCs were determined by the twofold dilution method in levels or more) of test drugs which were diluted with 0.5% heart infusion broth (HIB; Eiken Chemical Co., Ltd., To- carboxymethylcellulose solution by intragastric tube. The kyo, Japan) with a final inoculum of 5 x 105 CFU/ml. After 50% effective dose (ED50) was calculated by' the Litchfield- 18 h of incubation at 37°C, the lowest drug concentration Wilcoxon probit method (22) according to the survival rates which allowed no visible growth was defined as the MIC, at 5 days after infection. and the MBC was defined as the lowest concentration which yielded no colony formation after incubation overnight at RESULTS 37°C on drug-free SDA plates to which one spot (about 10 ,ul) from clear subcultures after MIC determinations was ap- Susceptibility of clinical isolates. The susceptibility of clin- plied. ical isolates to R-3746 and the reference drugs is listed in Killing-curve studies. Overnight cultures in HIB were Table 1. The lowest concentration of R-3746 at which 90% of diluted I00-fold with fresh HIB, and 9.9-ml portions of the the clinical isolates were inhibited- (MIC%0) ranged from diluted samples were incubated at 37°C for 2 h'with shaking <0.01 to 1.56 pLg/ml for methicillin-susceptible Staphylococ- until they reached ca. 5 x i05 CFU/ml. Portions (0.1 ml) of cus aureus, Streptococcus pneumoniae, Streptococcus antibiotics were added to the cultures at various concentra- pyogenes, and several members of the Enterobacteriaceae tions around the MIC determined by the'broth dilution such as Escherichia coli, Klebsiella pneumoniae, Klebsiella method. Subcultures were further incubated at 37°C'with oxytoca, Proteus mirabilis, Proteus vulgaris, Proteus shaking. Samples (0.1 ml) were removed at 0, 1, 2, 4, 6, and inconstans, Providencia rettgeri, and H. influenzae. Ap- 24 h, diluted 10-fold serially with 0.9% physiological saline proximately 50% of Staphylococcus epidermidis, Morga- solution, and plated on SDA to determine the CFU remain- nella morganii, Citrobacter freundii, and Serratia marces- ing. cens strains were inhibited at concentrations ranging from Stability to j-lactamase. Various types of P-lactamase (14) 0.39 to 3.13 xg/ml. The MIC%0s for these organisms were 50, used' in this study were purified by the methods reported 12.5, >100, and 25 ,ug/ml, respectively. R-3746 had rather previously (5, 6, 11-13, 16, 25-27, 31) and stored at -70°C weak antibacterial activity against Enterococcus faecalis, until assay.