Pseudomonal Activity

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1980, p /80/ /08$0.00/0 Vol. 17, No. 5, a New Broad-Spectrum Cephalosporin with Anti- Pseudomonal Activity CYNTHIA H. O'CALLAGHAN,* P. ACRED, P. B. HARPER, D. M. RYAN, SUSAN M. KIRBY, AND S. M. HARDING Glaxo Group Research Limited, Greenford, Middlesex, England UB6 OHE is a new broad-spectrum injectable cephalosporin which is stable to most 8i-lactamases. Its in vitro activities were of the same order as those of cefotaxime against most gram-negative bacteria, were clearly inferior to cefotaxime against Staphylococcus aureus, but were significantly more active against Pseudomonas aeruginosa. Against the 5 strains used, was significantly more active than any of the other agents tested: piperacillin, azlocillin, gentamicin, amikacin, and carbenicillin. protected mice against experimental infections with P. aeruginosa more effectively than other f-lactam antibiotics; its general effectiveness in this test was comparable with gentamicin. Studies on human volunteers showed that it produces high, long-lasting blood levels, with much of the antibiotic being recovered in the urine. Intramuscular and intravenous injections were well tolerated by the volunteers, and there were no untoward side effects. Although the cephalosporins have been used in clinical medicine for many years, the usefulness of the currently available members of the group is impaired by limitations such as restricted antibacterial spectrum, susceptibility to,8-lactamases from gram-negative organisms, and metabolic degradation in the body. Compounds such as cefuroxime (), cefotiam (7), and cefotaxime (1,5) have overcome some of these problems, but further improvements were desirable. For example, cefuroxime has good resistance to,8-lactamases from many gram-negative organisms and is stable to metabolic degradation, but its intrinsic antibacterial activity is not as high as that of cefotiam. The spectrum of cefotiam is rather restricted by a degree of susceptibility of the compound to many,b-lactamases., with good enzyme resistance and very high intrinsic antibacterial activity, suffers from metabolic degradation which reduces its activity in the body. To a large extent the newly developed (6R, 7R)-7-[(Z)- - (-aminothiazol--yl)-- (-carboxyprop-- yloxyimino)acetamido]-3- (pyridinium- 1 -ylmethyl)ceph-3-em--carboxylate; see Fig. 1) does not have these limitations. This report summarizes the results of studies designed to evaluate this new agent with respect to its in vitro, in vivo, and human pharnacokinetic properties. MATERIALS AND METHODS Anmtibiotics. was prepared in our own laboratories and used as the monosodium salt in all experiments. Cephaloridine and piperacillin were also 876 synthesized in our own laboratories. was kindly donated by Hoechst A.G., Frankfurt. The other antibiotics used were commercially available. Bacterial isolates. The organisms used were mainly clinical isolates, identified to species by the API system (API Laboratory Products, Farnborough, England). Some isolates have been used over a period of time because they possess stable properties of special interest. All isolates were stored in sealed vials in liquid nitrogen. These stock suspensions were used to inoculate plates of Casitone agar, which were then incubated for 18 h at 37 C. Casitone broth (10 ml) was inoculated from these plates to provide the log-phase cultures used for the minimal inhibitory concentration (MIC) tests. MIC determinations. MICs were determined by parallel tests in serial twofold dilutions of freshly prepared standard antibiotic solutions in Casitone agar, supplemented for fastidious organisms as necessary. Plates were inoculated with a replicate inoculating device (Denley Instruments Ltd., Bolney, Sussex). Each point inoculation contained approximately 105 or 107 colony-forming units (CFU) of a 6-h log-phase culture in Casitone broth. The MIC, in micrograms per milliliter, was read after 18 h of incubation at 37 C as the lowest concentration that inhibited visible growth. Antibiotic assay. Concentrations in serum and urine were determined by large-plate microbiological assay, using Oxoid Antibiotic Agar no. with 3 g of sodium citrate added per liter, seeded with Bacillus subtilis 190E at ph 7. Urine samples for assay, diluted 1:10 or to a greater extent dependent upon concentration with 0.05 M phosphate buffer (ph 6), were assayed against standard solutions prepared in the same phosphate buffer. Undiluted urine samples were assayed against standard solutions prepared in undiluted urine from

2 VOL. 17, s N J-C- C-NH- S- ~N /\CH3 %'CH \O- -COOH codc CH3 FIG. 1. Chemical structure of, (6R, 7R)- 7- [(Z)--(-aminothiazol--yl)--(-carboxyprop--ylox- yimino)acetamido]-3-(pyridinium-1-ylmethyl)ceph-3- em--carboxylic acid. the species under test. Similarly, serum samples were assayed undiluted against standards prepared in serum from the species under test. Twofold doubling dilution series from 100 to 0.63 itg/ml were used for preparation of the various standard solutions. Serum binding determinations. was mixed with serum to give a 5-,ug/ml solution, which was then subjected to ultrafiltration using Amicon Centriflo membrane cones (CF5, 5,000 molecular weight cut-off). The amount of compound appearing in the ifitrate was assayed microbiologically, and the percent binding was calculated. Stability to,a-lactamases. Crude enzyme preparations were made as previously described (3). Stability studies were done by the spectrophotometric method (3). Experimental chemotherapy. Protection tests on mice were done as previously described (6) in Charles River (Harefield) albino female mice (18 g), but using 1.5% dried bakers' yeast suspension to potentiate virulence. Organisms used in the protection test were maintained on nutrient agar slopes at C (Proteus mirabilis 1315E; Klebsiella aerogenes [Curtis]; Serratia sp. 3999; Pseudomonas aeruginosa 3 and ; Staphylococcus aureus 853E, 630E, and 663E) or suspended in 10% skim milk over liquid nitrogen (Escherichia coli 05E, Proteus morganii 1356E, Enterobacter cloacae 010E, P. aeruginosa SM and SM5). Infecting inocula. The infecting inocula were prepared as follows. (i) Organisms maintained on nutrient agar slopes. Fresh overnight cultures grown on nutrient agar slopes at 37 C were washed off and diluted to the required number of organisms with physiological saline. (ii) Organisms maintained over liquid nitrogen. Ampoules containing the organisms were removed from liquid nitrogen refrigeration, thawed at 37 C for 30 min, and diluted to the required number of organisms in physiological saline. The challenge suspensions contained 10 to 100 times the 50% lethal dose of the infecting organisms. The strains used were clinical isolates with proven virulence to mice. Five or 10 animals were used at each of the serial fourfold dose concentrations of antibiotic tested. Antibiotic was given subcutaneously (0. ml per animal) at 1 and 5 h after challenge, except for the severe infections with the highly virulent P. aeruginosa strains, when the, NEW ANTI-PSEUDOMONAL CEPHALOSPORIN 877 mice were dosed at 1, 3, and 5 h after challenge. The median effective dose was calculated by logit transformation from the numbers of animals surviving on day 5. Toxicity testing. Toxicity tests on mice, rats, rabbits, and dogs have revealed no adverse effects; these results will be reported in detail elsewhere. In particular, no adverse effects were found on the kidney or liver in rats given repeated doses of, even at high levels. It was therefore considered safe to proceed to studies on human volunteers. Human volunteer studies. The monosodium salt of was given to 15 male volunteers aged 19 to 6 years. Nine received more than one dose of drug. Each dose-route combination was given to three volunteers; the same three had 50 and 500 mg intramuscularly (i.m.) with a 3-week interval, three had 50 mg intravenously (i.v.) and 750 mg i.m. with a 3-week interval, and three received 500 mg i.v. on consecutive days, with 1 g of probenecid by mouth on day. Physical examination and laboratory tests before each study were undertaken to exclude volunteers with abnormalities, those with positive prick tests to common allergens, and those with a history of antibiotic allergy. The studies were approved by an ethical committee, and written informed consent was obtained from each volunteer. Sterile filtered aqueous solutions of drug were prepared immediately before use, and the dose administered was calculated as the free acid. Injections i.m. were given deep into the lateral aspect of the right thigh in 1,, 3, and ml for the 15-, 50-, 500-, and 750-mg doses, respectively. All i.v. injections were given as a bolus over 1 min in 10 ml of aqueous solution. Blood samples throughout each study day were taken via an indwelling winged needle. Antibiotic activity in blood and urine was estimated by microbiological assay; full hematological and biochemical tests and urinalysis were performed before and on one or two occasions after each dose on each volunteer. Creatinine clearance was measured in each volunteer on the day of dosing, to enable calculation of the drugcreatinine clearance ratio. A best-fit concentration time curve was calculated from the observed serum levels and the timed urinary recoveries from each volunteer. A one-compartment open system model appeared adequate to characterize the i.m. data, as did a two-compartment model for the i.v. data. From each curve thus derived, various pharmacokinetic parameters were calculated. Bioautographs. Urine samples were investigated for biologically active metabolites by chromatography on Whatman no. 1 paper buffered to ph 6 with 0.05 M phosphate buffer in n-propanol-water (7:3, vol/vol). After development overnight, the papers were dried and placed on nutrient agar seeded with B. subtilis 190E. RESULTS Antibacterial activity. The in vitro activity of was compared with that of cefotaxime at two inoculum levels. was highly active against a wide range of commonly encountered gram-negative pathogens (Table 1), with an order of activity similar to that of cefotaxime against most species; it was, however, more active against P. aeruginosa. is active against Streptococcus pneumoniae, al-

3 878 O'CALLAGHAN ET AL. ANTIMICROB. AGENTS CHEMOTHER. though cefotaxime is some 10 times more active. has less activity against S. aureus, cefotaxime being 8- to -fold more active. Increase in inoculum size from 105 to 107 CFU often affects the in vitro activity of cephalosporins. This was judged to be significant when the MIC increased by more than one doubling dilution step. Neither compound showed a significant inoculum effect against the gram-positive organisms. Against the gram-negative strains, was less active than cefotaxime against 7 isolates, of equal activity against 1, and more active against 1, when tested at the lower inoculum level. However, when the inoculum was increased to 107 CFU, was less active against only isolates, of equal activity against 1, and more active against 6. The range of inoculum effect shown by was 3- to -fold against 5 of the isolates, whereas the inoculum effect of cefotaxime varied from 3- to 50-fold against 13 isolates. The geometric mean MICs, MIC range, and associated data (Table ) against 33 assorted strains from 1 bacterial species confirmed the high broad-spectrum activity of. Organisms for use in these screens represent a selection of the more resistant isolates seen in the clinic. Despite this, MIC ranges were comparatively narrow, and concentrations required to kill even 90% of isolates usually fell within clinically achievable limits. Despite the poorer activity of against S. aureus, 90% of the isolates were inhibited by 8,ug/ml even at the higher inoculum level. Bacteroides fragilis strains proved to be the most resistant group, with a the geometric mean MIC of 10.9,ug/ml at 105 CFU/ml. In this instance, the spread of MICs was comparatively wide, and although 8,tg/ml was sufficient to kill 50% of isolates at both inoculum levels, more than 15,tg/ml was required to kill 90% of these isolates. The high activity of against strains of P. aeruginosa is an important feature of the antibacterial spectrum. The results in Table 3 show that was the most active antipseudomonal compound tested. gave the lowest geometric mean MIC values at both inocula, was least affected by increase in inoculum size, and showed the smallest MIC range; a concentration of,ug/ml was sufficient to kill 90% of isolates at the higher inoculum level. The other compounds tested failed to show this combination of properties. They were generally less active and showed a wider MIC range, and several were inoculum sensitive. The activities of the two aminoglycosides would have appeared TABLE 1. Antibacterial activity of and cefotaxime MIC (jug/ml) at bacterial inoculum: Strain 105 CFU 107 CFU 105 CFU 107 CFU Staphylococcus aureus 663E Streptococcus pneumoniae <0.03 <0.03 Escherichia coli 851E < E. coli (TEM+) 1193Ea Salmonella typhimurium 80E Shigella sonnei 08Ea Enterobacter cloacae 119Ea 8 E. cloacae 1083E Citrobacter freundii 56a Providencia stuartii 6D386a Serratia marcescens 13E" Serratia liquefaciens 691a < Klebsiella aerogenes 1371Ea Proteus mirabilis 31E < Proteus vulgaris 135Ea Proteus rettgeri 538a Proteus morganii 531a 8 Pseudomonas aeruginosa 1191Ea 8 31 Haemophilus influenzae 1886Ea <0.03 <0.03 Bordetella pertussis 87E Bacteroides fragilis 0Ea 8 Neisseria gonorrhoeae 83381a <0.03 <0.03 <0.03 <0.03 Citrobacter diversus 06Ea Acinetobacter calcoaceticus 81a 8 31 B-Lactamase producer.

4 VOL. 17, 1980 TABLE., NEW ANTI-PSEUDOMONAL CEPHALOSPORIN 879 In vitro activity of against 51 clinical isolates MIC (pg/mi) at inoculum: Organism 105 CFU 107 CFU (no. of strains) For % of strains: For % of strains: Mean (range) Mean (range) Staphylococcus aureus 6.8 (-15) (-15) 8 8 (5) Streptococcus spp.a (3) 0.6 (0.06-) (0.06-) Escherichia coli () 0.19 (0.06-1) (0.13-1) Klebsiella spp. (5) 1.1 (0.5-) 1 (1-) Enterobacter cloacae 0.1 (0.06-) (0.5-8) () Serratia spp. (1) 0.19 (0.06-1) (0.13-) Proteus vulgaris (1) 0.13 (0.06-) (0.13-) 1 Citrobacter/Providencia 0.71 ( ) (0.5-15) 15 Spp. (3) Salmonella spp. () 0. (0.13-1) (0.5-1) Bacteroides fragilis (5) 10.9 (0.5->15) (-15) 8 15 Haemophilus influenzae 0.0 (SO ) (C ) (1) Bordetella pertussis (9) 1.5 ( ) ( ) a Excluding fecal streptococci. TABLE 3. In vitro activity of against 5 isolates of P. aeruginosa MIC (pg/ml) at inoculum: 105 CFU 107 CFU Compound For % of strains: For % of strains: Mean (range) Mean (range) (1-8) 3.8 (1-6) 11 (0.-6) (0.5->15) 6 Piperacillin.5 (1-31) (0.5->15) 8 3 Azlocillin. (<0.06-) 8 0 (1->15) Carbenicillin 38.5 ( ) (0.5->15) (0.1->15) (0.5->15) 8 Amikacin 9.3 (0.->15) (1>15) 6 higher had they been tested in nutrient broth, but the activity of the compounds measured by the agar dilution test correlated well with their in vivo activity (see Table 5). Resistance to f-lactamases. The rates of hydrolysis of and cefotaxime were calculated relative to the rate of hydrolysis of cephaloridine at the same time by the same enzyme preparation; cephaloridine hydrolysis was arbitrarily assigned a value of 100 for comparison purposes. Table shows that both GR 063 and cefotaxime were highly resistant to the fl-lactamases from S. aureus and many gram-negative organisms. Both compounds had some susceptibility to OXA-1, an R factor-mediated enzyme which attacks the isoxazolyl penicillins. was much more susceptible than to the class 1 enzyme from Citrobacter diversus, and this was clearly reflected in its poor MIC for the producing organism at the high inoculum level. Serum binding. has a low level of serum binding in vitro, with mean values of 13, 19, 11, 0, 8, and 17% for mice, rats, rabbits, dogs, monkeys, and humans, respectively, derived from three measurements in each species. Protective effects in mice. The results of protection tests in mice challenged with seven different fb-lactamase-producing strains of Enterobacteriaceae and four strains of P. aeruginosa are given in Table 5. In general, the activities of, cefotaxime, and gentamicin were similar, although cefotaxime had significantly poorer activity against the strain of Proteus vulgaris, where its MIC was also poor; cefotaxime was also less active against the Serratia strain for which it had good in vitro activity.

5 880 O'CALLAGHAN ET AL. ANTIMICROB. AGENTS CHEMOTHER. Enzyme TABLE. Resistance of to a range of (3-lactamases, compared with cefotaxine and cephaloridine R-factor (R) Rate of hydrolysis or chromosomal (C) Cephaloridine' Penicillin G' TEM-1 R TEM- R SHV-1 R OXA-1 R OXA- R OXA-3 R Citrobacter diversus R Kl C P99 C Staphylococcus aureus R aarbitrarily attributed values for comparison purposes. had activity equivalent to gentamicin against three of the four strains of P. aeruginosa tested; it was considerably more effective than azlocillin, cefotaxime, and carbenicillin against all four. Although the in vitro activity of against S. aureus is not high, it protected mice challenged with three strains of S. aureus with an effectiveness equal to that of cloxacillin, but less than that of methicillin (Table 6). Human volunteer studies. The average serum level/time curves for the i.m. injections of are shown in Fig. A; the i.v. serum level curves are shown in Fig. B. Some pharmacokinetic parameters for both dose routes are given in Table 7. The compound gave good longlasting serum levels after i.m. injection, with a half-life of approximately 100 min. The peak serum levels increased approximnately arithmetically with increasing dose and occurred between 1 and 1.5 h after dosing. One i.m. dose of 750 mg maintained serum concentrations in excess of 8,Ag/ml for 6 h. Injection i.v. gave an ultimate half-life of about 1.8 h, and 500 mg maintained serum concentrations in excess of 8,ug/ml for.6 h. The areas under the serum curve did not increase proportionally to the dose. The apparent volume of distribution was of the order of 1 liters, and the renal clearance averaged 66 ml/min. The urinary recoveries ranged from 50 to 87% with no evidence of microbiologicaly active metabolites when the urines were examined by chromatography followed by bioautograph. After probenecid, the levels were higher, but there was no effect on the rate of excretion of the drug. It therefore appears that there is little or no net tubular secretion of, which accounts for its longer half-life of over 1.5 h. Although it appears that there were dose-related increases in ultimate half-life and volume of distribution in these preliminary experiments, each group contained only three volunteers, and the differences observed were not considered significant. The injections were well tolerated, with only transient pain after i.m. administration, and there was no pain or local complications after i.v. dosing. Laboratory investigations on each volunteer showed no drug-related changes. DISCUSSION is a parenteral cephalosporin antibiotic with a broad spectrum of antibacterial activity. It has very good activity against most species of gram-negative bacteria and is highly resistant to the f8-lactamases they produce, being comparable with cefotaxime in most instances. However, the spectrophotometric method used here appeared to be insufficiently sensitive to detect small differences between the,b-lactamase stabilities of highly enzyme-resistant compounds; for this purpose more stringent tests such as incubation for several hours with heavy cell suspensions () will be needed. Cefotaxiime proved to be less stable to some B8-lactamases, showed an inoculum effect in vitro against some organisms, and it is deacetylated in vivo to less active products (R. Bax, L. White, D. Reeves, R. Ings, M. Bywater, and H. A. Holt, Program Abstr. Intersci. Conf. Antimicrob. Agents Chemother. 19th, Boston, Mass., abstr. no. 88, 1979; L. 0. White, H. A. Holt, D. S. Reeves, M. J. Bywater, and R. P. Bar, 19th ICAAC, abstr. no. 881, 1979). In contrast, GR 063 has high enzyme stability and an insignificant inoculum effect in vitro, together with stability in the animal body; these properties would all contribute to good activity in vivo. Its performance against the gram-negative pathogens in vivo was as good as predicted from in vitro MICs. is less active in vitro against staphylococci than against gram-negative organisms;

6 VOL. 17, 1980, NEW ANTI-PSEUDOMONAL CEPHALOSPORIN 881 OrganigMa E. coli 05 P. mirabilis 1315 P. morganii 1375 P. vulgaris 1356 E. cloacae 010 K. aerogenes Curtis Serratia sp P. aeruginosa SM P. aeruginosa SM5 P. aeruginosa 3 P. aeruginosa TABLE 5. Protecting activity of against fatal experimental infections in mice Infecting inoculum (CFU per mouse) Compound 1.1 x x 10l 1. x x 10.3 x x 10" 1.3 x X x x x 105 a All organisms produce f3-lactamases. bed50, 50% effective dose (milligrams per kilogram). C At 107 CFU per inoculum. d 80% survival at 3.1 mg/kg, lowest dose level. Cefotaximne Azlocillin Carbenicillin Azlocillin Carbenicillin Azlocillin Carbencillin Azlocillin Carbenicillin ED50b (95% confidence limits) 0.3 (0.-0.7) 0.1 ( ) 1.0 (0.5-.0) 3.1 ( ) 1. (0.-.9) 6.3 ( ) <3. ld.6 (1.1-5.) 0.9 (0.5-.0) 3.5 ( ) 3.0 ( ) 1. (0.7-.3) 0.1 ( ) 0.6 ( ) 0. (0.-0.7) 1.9 (0.5-.0) 5. ( ). ( ) 0. (0.-0.9) 5. (.9-10.). (1.-.8) 1.6 (0.-5.7) 33.0 (8.5-17) 3.6 ( ) 10.3 (1.-1) 10 (3.0-1,06).7 ( ) 100 (3.8-0) 6.3 (1.8-.6) 73.0 (5.7-87) >00.5 ( ) > (1.8-.6) 5.0 (1.3-7) > (1.3-7) 73.0 (5.7-87).6 ( ) 73.0 (5.7-87) 100 (7.6-36) MICC (pg/ml) however, its level of activity against these orga- effective as cloxacillin in protecting mice exper- imentally infected with staphylococci, despite a 6-fold MIC disadvantage. In contrast to its poor nisms cannot be attributed to susceptibility to staphylococcal,b-lactamase (Table ). It was as

7 CONK O'CALLAGHAN ET AL. ANTIMICROB. AGENTS CHEMOTHER. TABLE 6. activity against acute, fatal S. aureus infections in mice S. aureus nfecting ED50 b MICC straina Stredna (CFU per mouse) (cofidnc limts MIC inoculum Compound(cnieelmts #/) x ( ) Cloxacillin 1.6 (9.8-6.) 0.5 Methicillin 10.7 (.9-1.0) 630. x (6.-6.) Cloxacillin. ( ) 0.5 Methicillin 7. ( x ( ) Cloxacillin 5. (.9-10.) 0.5 Methicilhin 1.8 (0.-5.0) a All organisms produce -lactamases. b ED5o, 50% effective dose (milligrans per kilogram). c At 107 CFU per inoculum. g E E ' 0-30 ' E 0' X 10- E Time (mins) O Time (mins) FIG.. (A) Serum levels of after i.m. administration of (0) 750 mg, (A) 500 mg, or (U) 50 mg to human volunteers. (B) Serum levels of after i.v. administration of (E) 500 mg, (A) 50 mg, or (0) 15 mg to human volunteers. anti-staphylococcal activity, has good activity against most strains of streptococci, apart from group D. B An interesting feature of the spectrum of GR 063 is its high activity in vitro and in vivo against P. aeruginosa. Unlike many other antipseudomonal,b-lactam antibiotics, the MICs of against P. aeruginosa do not increase markedly with increase in inoculum level. In protection tests in mice, the performance of GR 063 was in general equivalent to that of gentamicin and markedly superior to those of cefotaxime, azlocilhin, and carbenicillin. was well tolerated when given i.m. to human volunteers, and there was no evidence of metabolic break-down. Further experiments in volunteers have shown in that 90% of GR 063 is generally recovered from urine in the 0- to 1-h period; excretion after h is negligible. The concentrations of obtained in body fluids after either i.v. or i.m. injection exceeded the MICs of almost every organism tested for long periods; a 750-mg dose given i.m. produced serum levels that exceeded Jg/ml for h. The MICs of against 97% of the specially assembled mixed collection of multiply resistant isolates were Lg/ml or less, and 81% of the organisms were susceptible to,ug/ ml or less. At least 90% of the isolates in a similar collection of strains of P. aerugunosa were susceptible to,ug/ml or less. The broad antibacterial spectrum of GR 063, coupled with its low serum binding, good pharnacokinetics in humans, and high recovery of unchanged antibiotic in urine, suggests that it should be a highly effective therapeutic agent, particularly against difficult infections. The injections were well tolerated, and toxicological examination has revealed no side effects, so that should provide a safe and much needed alternative to the aminoglycosides.

8 VOL. 17, 1980 TABLE 7., NEW ANTI-PSEUDOMONAL CEPHALOSPORIN 883 Computer-calculated mean human parameters after parenteral a Peak Peak Ultimate Area under Time above Apparent Renal Route Dose concn time half-ife the curve 8 isg/ml vol of clearance g (Ag/mi) (h) (h) (pg/ml per h) (h) distribution (ml/min) i.m i.v a Results are standardized to 70 kg to allow for differences in body weight. ACKNOWVLEDGMENTIS We thank our colleagues Elena Beniacova, Angela Collard, Linda Curtis, Ursula Mason, David Monsey, Iain Simpson, Michael Sowa, John Thornton, Christine Watts, and Victoria Wiggins for their collaboration and expert assistance. LITERATURE CITED 1. Heymes, R., A. Lutz, and E. Schrinner Experimental evaluation of HR 756 a new cephalosporin derivative: pre-clinical study. Infection 5: O'Callaghan, C. H., and P. W. Muggleton The action of cephaloridine and cloxacillin or methicillin against,6-lactamase-producing gram-negative bacteria. J. Gen. Microbiol. 8: O'Callaghan, C. H., P. W. Muggleton, and G. W. Ross Effects of,-lactamase from gram-negative organisms on cephalosporins and penicillins, p Antimicrob. Agents Chemother O'Callaghan, C. H., R. B. Sykes, D. M. Ryan, R. D. Foord, and P. W. Muggleton Cefuroxime-a new cephalosporin antibiotic. J. Antbiot. Jpn. 9: Ochiai, M., 0. Aki, A. Morimoto, T. Okada, and Y. Matsushita New cephalosporin derivatives with high antibacterial activities. Chem. Phann. Bull. 5: Ryan, D. M., C. H. O'Callaghan, and P. W. Muggleton Cefuroxime, a new cephalosporin antibiotic: activity in vivo. Antimicrob. Agents Chemother. 9: Tsuchiya, K., M. Kida, M. Kondo, H. Ono, M. Takeuchi, and T. Nishi SCE-963, a new broad spectrum cephalosporin: in vitro and in vivo antibacterial activities. Antimicrob. Agents Chemother. 1: