ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1978, p. 784-790 0066-4804/78/0013-0784$02.00/0 Copyright 0 1978 American Society for Microbiology

Vol. 13, No. 5

Printed in U.S.A.

SK&F 75073, New Parenteral Broad-Spectrum Cephalosporin with High and Prolonged Serum Levels PAUL ACTOR,* JOSEPH V. URI, IHOR ZAJAC, JOSEPH R. GUARINI, LILLIAN PHILLIPS, DONALD H. PITKIN, DAVID A. BERGES, GEORGE L. DUNN, JOHN R. E. HOOVER, AND JERRY A. WEISBACH Research and Development Division, Smith Kline & French Laboratories, Philadelphia, Pennsylvania 19101 Received for publication 28 October 1977

SK&F 75073, a new parenteral cephalosporin, was found to have broad in vitro and in vivo antibacterial activity including isolates usually resistant to cephalothin and cefazolin. This activity included indole-positive Proteus and Enterobacter species and some Serratia isolates. Proteus mirabilis strains were particularly susceptible, as were Haemophilus influenzae and Neisseria species. The activity of SK&F 75073 against gram-positive bacteria was poorer than that of the control cephalosporins. This cephalosporin is highly bound to serum proteins, and a loss in in vitro activity was observed in the presence of serum. Parenteral administration of SK&F 75073 to experimental animals (mice, dogs, squirrel monkeys) resulted in high and prolonged serum levels when compared with cefazolin and other injectable cephalosporins. This favorable serum profile was reflected in the excellent protection observed in mice infected with pathogenic bacteria.

A number of new parenteral cephalosporin and cephamycin derivatives with improved use potential over the available marketed products are currently under development. These compounds are reported to have a broader spectrum of antibacterial activity and good stability to a large number of f-lactamases produced by bacterial isolates (2, 7, 9, 11, 12, 14, 15). One such compound is SK&F 75073 [7-D-mandelamido3-(1-sulfomethyltetrazole-5-ylthiomethyl) -3-cephem-4-carboxylic acid, disodium salt] a parenteral cephalosporin with broad antibacterial activity and high and extended serum levels (Fig. 1). This cephalosporin is one of a series of interesting compounds resulting from the attachment of an acidic substituent to the 1-position of the tetrazole ring of 3-tetrazolethiomethyl-cephalosporins (D. A. Berges, G. L. Dunn, J. R. E. Hoover, S. J. Schmidt, G. W. Chan, J. J. Taggart, C. M. Kinzig, F. R. Pfeiffer, P. Actor, C. S. Sachs, J. V. Uri, and J. A. Weisbach, Program Abstr. Intersci. Conf. Antimicrob. Agents Chemother. 16th, Chicago, Ill., Abstr. no. 87, 1976). Although structurally related to cefamandole, SK&F 75073 has unique microbiological and pharmacokinetic properties that distinguish it from cefamandole. Initial observations on antimicrobial activity and serum levels in experimental animals with SK&F 75073 have been presented previously and are now reported in detail (P. Actor, J. V. Uri, I. Zajac, J. R. Guarini, C. S.

Sachs, L. Phillips, D. A. Berges, G. L. Dunn, J. R. E. Hoover, and J. A. Weisbach, Program Abstr. Intersci. Conf. Antimicrob. Agents Chemother. 16th, Chicago, Ill., Abstr. no. 86, 1976). MATERIALS AND METHODS Cephalosporins. SK&F 75073 and cefamandole were synthesized in the Smith Kline & French Laboratories. Cephalothin (Eli Lilly & Co.) and cefazolin (Smith Kline & French Laboratories) were used as commercial preparations. Cefoxitin was kindly supplied by Merck & Co., Inc. Animals. Male albino Swiss mice weighing 18 to 21 g were used for the serum level and efficacy studies. Squirrel monkeys (0.8 to 1.1 kg) and female beagle dogs (9 to 11 kg) were employed for serum level studies.

Efficacy tests: (i) in vitro activity. The minimum inhibitory concentrations (MICs) were determined by the agar dilution method as previously reported (1). The surface of the agar was inoculated with the aid of a Steers apparatus (13). After overnight incubation at 37°C, the MICs were read as the lowest concentration of antibiotic completely inhibiting growth. For determination of effect of serum on antibiotic activity, pooled human serum was added in a final concentration of 50% to Muelier-Hinton broth. The MIC determinations were carried out using microtiter methodology (6). The MICs for the inoculum size experiments were also carried out in Mueller-Hinton broth using the microtiter apparatus. The test medium employed for the staphylococci and the gram-negative bacterial species was Mueller784

VOL. 13, 1978 I

SK&F 75073, NEW BROAD-SPECTRUM CEPHALOSPORIN

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FIG. 1. Chemical structure of SK&F 75073.

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(BBL). The MICs for Streptococcus pyand Streptococcus pneumoniae were determined in Todd Hewitt agar (BBL) supplemented with N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid (HEPES) buffer 0.05 M and laked sheep erythrocytes (2.5%). The Neiserria species were tested on MuellerHinton agar containing: HEPES buffer, 0.05 M; IsoVitaleX, 0.5% (BBL); Fildes enrichment, 2.5% (BBL); dextrose, 1%; and laked sheep erythrocytes, 2.5%. Haemophilus influenzae strains were tested on Mueller-Hinton agar containing: HEPES buffer, 0.05 M; yeast, 0.5%; nicotinamide adenine dinucleotide (Sigma); IsoVitaleX, 1%; dextrose, 1%; and hemin, 10 mg/ml. The test medium for Bacteroides fragilis was Schaedler medium (BBL). The inoculum size employed for the organisms tested in the agar dilution test ranged from 104 to i0" organisms applied directly to the surface of the agar. The inoculum used for the microtiter MIC test ranged from 104/ml to 108/ml as indicated in the text. The organisms employed were for the most part clinical isolates from the Smith Kline & French culture collection. (ii) In vivo efficacy. Cultures of test organisms were diluted in 5% hog gastric mucin and injected intraperitonealy to produce uniformly lethal mouse infections. Unless otherwise noted, test antibiotics were administered subcutaneously at 1 and 5 h after infection. Final survival percentages for groups of 10 mice each, obtained after 3 days of observation, were used to estimate the 50% effective dose (ED50) and the 50% lethal dose (LD50) values. The ED50 and LD50 values were determined by the method of Litchfield and Wilcoxon (8). Pharmacokinetic studies. Serum levels and urinary recovery in mice, dogs, and squirrel monkeys were determined after parenteral administration (3). Mice and squirrel monkeys were injected with 20 mg of drug per kg subcutaneously and intramuscularly, respectively. Dogs were given an intramuscular dose of 10 mg/kg. Sequential blood samples were obtained from dogs and squirrel monkeys, whereas samples from mice were obtained by sacrifice of duplicate pooled groups of 10 mice each. The blood samples were allowed to clot, and the serum obtained was stored at -20°C prior to assay. Urinary antibiotic recovery during the 4-h period after dosing was determined in duplicate groups of 10 mice. Dog urine samples were collected over a 6-h period. All urine samples were stored at -20°C. Antibiotic concentration in serum and urine was determined by disk plate assay with Bacillus subtilis ATCC 6633 as the indicator organism. For serum assays, standard curves were prepared by plotting dose-response data for each antibiotic by using appropriate pooled sera as diluent for the standard. For urine assays, standard curves were ogenes

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RESULTS AND DISCUSSION MICs. SK&F 75073 was found to have a broad spectrum of in vitro activity that includes all the bacterial organisms that are susceptible to the cephalosporins available commercially or under development. The MICs for SK&F 75073, cefamandole, cefazolin, and cephalothin against 217 clinical isolates are shown in Table 1. The activity of SK&F 75073 against the gram-positive cocci was generally poorer than that of the other cephalosporins (two- to eightfold) but well within the achievable blood levels obtained in experimental animals (Fig. 2 to 4). Cephalothin tended to show the best activity against Staphylococcus aureus isolates followed by cefazolin, cefamandole, and SK&F 75073. On the other hand, the gram-negative bacteria were highly susceptible to SK&F 75073, and the activity of this antibiotic was equal to or superior to that of cefamandole, cefazolin, and cephalothin against these gram-negative organims. SK&F 75073 was particularly active against Neisseria species. The activity of SK&F 75073 against H. influenzae including ampicillin-resistant strains was excellent. Of the 27 strains of H. influenzae examined, all were susceptible to SK&F 75073 at 1.6 ,ug/ml. At this antibiotic concentration (1.6 ,ug/ml), 88% of the isolates were susceptible to cefamandole, and 4% were susceptible to cefazolin. H. influenzae showed a biphasic response to ampicillin, with the seven resistant strains having a MIC ranging from 12.5 to 100 ,ug/ml. Against the three most common gram-negative pathogens, Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis, SK&F 75073 was found to be more active than the control cephalosporins. P. mirabilis isolates were particularly susceptible to SK&F 75073, with 90% of the strains inhibited at a MIC of 0.4 ,ug/ml as compared with 6.3 ,ug/ml for cefamandole. SK&F 75073 showed somewhat less in vitro activity than cefamandole against isolates of indole-positive Proteus and Enterbacter species. None of the cephalosporins tested showed significant activity against Pseudomonas aeruginosa or B. fragilis. Effect of inoculum size and serum on in vitro activity. Cefamandole has been reported to show a marked loss in activity against indolepositive Proteus and Enterobacter species when the in vitro inoculum size was increased by a factor of 100 or more over that usually employed in MIC determinations (4, 5, 10). A series of experiments was carried out to examine this

786

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400 9 50 >400 25 3.1 25 Proteus vulgaris 6 6.3 200 12.5 25 200 6.3 Proteus rettgeri 10 6.3 200 50 100 400 1.6 Enterobacter cloacae >400 7 1.6 6.3 200 200 400 Enterobacter aerogenes >400 6 1.6 200 200 400 Enterobacter liquefaciens 6.3 X a(A) Inoculum of 6.5 104 colony-forming units (CFU)/ml for S. aureus, 1.2 x 105 CFU/ml for Proteus spp., andb 9 x 106 CFU/ml for Enterobacter spp. (B) Inoculum of 6.5 x 106 CFU/ml for S. aureus, 1.2 x 107 CFU/ml for Proteus spp., and 8 x 108 CFU/ml for Enterobacter spp. c Pen G, Penicillin G. Organism

TABLE 3. Activity of SK&F 75073, cefamandole, and cefazolin against experimental infections in mice ED60 (mg/kg)'

Organism

Staphylococcus aureus 674 (Pen G susceptible)" Staphylococcus aureus HH127 (Pen G resistant) Escherichia coli 12140 Escherichia coli HH33779 Klebsiella pneumoniae 4200 Klebsiellapneumoniae 1200 Proteus mirabilis 442 Proteus mirabilis 416 Proteus morgani 179 Enterobacter cloacae HH31254 Haemophilus influenzae 133 (ampicillin resistant) aTotal subcutaneous dosage (1 and 5 h after challenge). b Pen G, Penicillin G.

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1.0 0.7 6.3 15.5 6.3 3.1 10.7 4.8 200 84 29

SK&F 75073, NEW BROAD-SPECTRUM CEPHALOSPORIN

VOL. 13, 1978

TABLE 4. Effect of a single subcutaneous dose of SK&F 75073 and cefamandole administered to mice before challenge with E. coli Dosage time (h) before challenge 1 4

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tective in this experiment at the three time intervals examined, whereas cefamandole was 16 times less active when given at 1 h and failed to protect at the highest level administered at 4 and 6 h prior to infection. Pharmacokinetics in experimental animals. The initial pharmacokinetic studies were carried out in mice given a subcutaneous dose of 20 mg of SK&F 75073, cefamandole, or cefazolin per kg. Serum concentrations of samples taken at intervals up to 2 h after dosing were assayed microbiologically. Urine, collected over a 4-h period, was assayed and examined by chromatographic procedures to detect microbiologically active metabolites. The serum levels are shown in Fig. 2. The peak serum level of SK&F 75073 was 111 ,ug/ml as compared with 54 ug/ml for cefazolin and 34 ,ig/ml for cefamandole. At 2 h after administration, the comparative serum levels for SK&F 75073, cefazolin, and cefamandole were 68, 2.8, and

SK&F 75073, new parenteral broad-spectrum cephalosporin with high and prolonged serum levels.

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1978, p. 784-790 0066-4804/78/0013-0784$02.00/0 Copyright 0 1978 American Society for Microbiology Vol. 13...
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