ANTImcboBL AozNrs AND CHMoTHzURPY, July 1976, p. 132-138

Vol. 10, No. 1 Printed in U.S.A.

Copyright 0 1976 American Society for Microbiology

Laboratory Evaluation of 3-(5-Tetrazolyl)Penam, a New Semisynthetic Beta-Lactam Antibacterial Agent with Extended Broad-Spectrum Activity ARTHUR R. ENGLISH,* JAMES A. RETSEMA,

AND

JOHN E. LYNCH

Medical Research Laboratores, Pfizer Inc., Groton, Connecticut 06340 Received for publication 3 March 1976

In the new agent 3-(5-tetrazolyl)penam, hereafter referred to as CP-35,587, the carboxyl function at C3 in the penicillin nucleus has been replaced with the 5tetrazolyl moiety. Marked changes in spectrum and resistance to gram-negative (3-lactamases, particularly with regard to Klebsiella pneumoniae isolates, were conferred by this modification. The anti-Klebsiella activity clearly distinguishes the antibacterial spectrum of CP-35,587 from any known broad-spectrum penicillin. Compared to orally active cephalosporins, the spectrum advantage of CP35,587 encompasses Enterobacter, Serratia marcescens, Citrobacter, Providencia, Haemophilus influenzae, and Streptococcus faecalis, both in vitro and in murine infections produced by many of the above-named microorganisms. Thus, CP-35,587 combines and extends the antibacterial activity of broad-spectrum penicillins and orally active cephalosporins. propriate antibiotic dilutions were made in sterile bacteriological medium. Experimental animals. Male and female mice used in these experiments were purchased from Blue Spruce Farms in Altamont, N.Y. The average weight of the mice used in the chemotherapeutic studies was 13 g; the average weight of those used in pharmacokinetic studies was 25 g. Dogs used in pharmacokinetic studies were purebred beagles. MICs. Minimum inhibitory concentrations (MIC) of the antibiotics were determined by a standard dilution method in brain heart infusion agar in which the multiple inoculator described by Steers et al. was used (3, 10). The standard inoculum contained approximately 20,000 organisms. Incubation was for 18 h at 37 C. Single colonies were disregarded in MIC determinations. The procedure for MIC determinations in broth, which utilized an inoculum of about 106 cells/ml, has been described previously (2). Bactericidal studies. Bactericidal properties of CP-35,587 and the other antibiotics were evaluated in killing-curve experiments. These were initiated by diluting cultures, which had been grown overnight on a rotary shaker, 100-fold in brain heart infusion broth containing the appropriate concentration of antibiotic. During incubation at 37 C on a rotary shaker, samples were removed at various times and diluted at 2-log intervals, and 0.1 ml was plated out in triplicate on brain heart infusion agar plates. After overnight incubation, colonies were counted and recorded as viable colony-forming

At one time it was believed that the antibacterial activity of penicillin required the presence of a carboxyl group at C3. However, it was later found that the carboxamido group at the C3 locus possesses interesting antibacterial activity per se (4), and ca-methoxy-n-propyl penicillinamide was effective against experimental infections in mice produced by penicillin G-resistant and penicillin-susceptible staphylococci (5). This paper describes the microbiological properties of 3-(5-tetrazolyl)penam, hereafter referred to as CP-35,587, in which the C3 carboxyl of penicillin has been replaced with a 5tetrazolyl group (W. C. Barth, West German patent 2449863, 1975) and which exhibited broad-spectrum activity with particularly good activity against Klebsiella species. MATERIALS AND METHODS Microorganisms. The microorganisms used to assess the spectrum and initial activity of CP-35,587 were primarily pathogenic strains well adapted to growth under laboratory conditions. To supplement these microorganisms, fresh clinical isolates were obtained from hospitals in several geographical areas in the eastern United States. Antibiotics. CP-35,587, a white, water-soluble crystalline solid, was used either as the sodium salt or in the zwitterion form (Fig. 1). Ampicillin trihydrate, amoxicillin, cephaloridine, and cephalexin used in various phases of these studies were commercial products of, respectively, Bristol Laboratories, Beecham-Massengill, and Eli Lilly & Co. Ap-

units. ,B-Lactamase studies. The hydrolysis of CP35,587, penicillins, and cephalosporins was determined, respectively, by the microiodometric method of Novick and by decreases in ultraviolet absorbance 132

VOL. 10, 1976

BROAD-SPECTRUM ACTIVITY OF CP-35,587

133

at 37 C for 1 h and then ultrafiltrated with conical ultrafiltration cones (Centriflo, CF 50A, Amicon Corp., Lexington, Mass.; cutoff level molecular weight, 50,000) for 30 min at 4 C. Initial antibiotic concentration in serum (zero-time sample) and the residual concentration in ultrafiltrate were determined by bioassay procedures using standard curves prepared in, respectively, serum or ultrafiltrate.

FIG. 1. Structure of CP-35,587, 3-(5-tetrazolyl)penam.

(7, 8). Conditions were identical for both assays, i.e., 0.5 M potassium phosphate, pH 6.5, at 37 C. The penicilloic acid of CP-35,587 was prepared by incubation with a purified Bacillus cereus penicillinase. The resulting product was found to obey the Novick equation (7) for determination of penicillin hydrolysis. Cell-free extracts containing /8-lactamases were prepared by sonication of harvested and washed cultures, which had been grown in brain heart infusion broth on a rotary shaker incubator. When indicated, the de novo synthesis of 3-lactamases was induced by growing a log-phase culture in the presence of a sublethal concentration of ampicillin for 1.5 h before harvesting. Induced cell-free extracts were dialyzed overnight at 4 C against a 1,000-fold greater volume of buffer (0.005 M sodium cacodylate, pH 6.5, and 0.001 M 2-mercaptoethanol). Protein was determined by the biuret method (6). Experimental infections in mice. Acute systemic infections in mice were produced by intraperitoneal inoculation of standardized bacterial cultures suspended in 5% hog gastric mucin. The challenge dose was generally 1 to 10 lethal doses, i.e., 1 to 10 times the number of organisms needed to kill 100% of the mice within a 4-day period. The dosage regimen for all experimental infections was initiated 0.5 h postchallenge, with subsequent dosing at 4 and 24 h. After 4 days, a 50% protective dose value, expressed in milligrams per kilogram, was calculated by a probit method (1). The 50% protective dose is defined as the dose of antibiotic, in milligrams per kilogram, that will protect 50% of the treated mice from the otherwise lethal infection. Pharmacokinetic studies in mice and dogs. CP35,587 and the other antibiotics were administered in a water-carboxymethyl-cellulose-Tween diluent. Mouse whole blood samples were collected via the intra-orbital sinus bleeding technique (9). Serum from dogs was removed from clotted blood samples obtained via jugular vein puncture. Biological assays were performed using a conventional largeplate disk assay, with Micrococcus lutea (ATCC 9341) as the assay organism. Standard curves for the assay of mouse whole blood and dog sera were prepared in the respective fluid from normal animals. Serum-binding studies. The binding of CP-35,587 and other antibiotics to human and dog serum proteins was determined in an ultrafiltration procedure. The various antibiotics were added to serum samples to approximate 50 gg/ml. After zero-time portions were removed, the samples were incubated

RESULTS MIC values of CP-35,587 against large numbers of clinically isolated microorganisms of diverse genera are summarized in Table 1. Amoxicillin and cephalexin are included for comparison. CP-35,587 was active against nearly all of the microorganisms studied, the most striking feature being the degree of activity against Klebsiella pneumoniae isolates. MIC values for CP-35,587 compared favorably (50 and 75% end points) with those of cephalexin and clearly distinguish the novel spectrum and degree of activity of CP-35,587 from that of any known penicillin. The further extension of the CP35,587 spectrum over that of amoxicillin is indicated by activity against Enterobacter, Proteus morgani, and Serratia species. The spectrum advantage of CP-35,587 over cephalexin included the three genera mentioned above for amoxicillin and, in addition, Streptococcus faecalis, Citrobacter, Providencia, and Haemophilus influenzae. Based on MIC values, which included those for 50 and 75% of the gram-negative isolates

studied, CP-35,587 generally was more active than amoxicillin and cephalexin. CP-35,587 was more active against gram-positive organisms than was cephalexin but was not as active against gram-positive bacteria as ampicillin. MIC values of all antibiotics against 90% of the gram-negative isolates were routinely higher than those at the 50 and 75% end points. This increase in MICs probably resulted from a few multiply resistant isolates carrying R-factor present in each genus. A 100-fold increase in inoculum size (from 200 to 20,000 cells) in a series of gram-negative microorganisms was without significant effect on agar dilution MIC values of CP-35,587. A further increase of 10 times (200,000 cells) generally produced a rise in MIC from the original, which represented a single twofold dilution in concentration. In general, similar data were obtained with ampicillin and cephalexin. The bactericidal activity of CP-35,587 with high inocula (>3 x 107 cells/ml) is clearly evident in the killing-curve experiments described by Fig. 2. CP-35,587 was bactericidal againstK. pneumoniae, Escherichia coli, and Serratia marcescens. At equal concentrations amoxicil-

134

ANTimicROB. AGENTS CHEMOTHZR.

ENGLISH, RETSEMA, AND LYNCH

TABLE 1. Activity of CP-35,587 against fresh clinical cultures MIC value (,ug/ml) to inhibit 50, 75, or 90% of strains tested

Organism

Kkebsiella pneumoniae Escherichia coli Enterobacter Serratia marcescens Citrobacter Providencia SalmoneUa typhimurium Proteus mirabiisb Proteus vulgarisb Pseudomonas aerugi-

No. of isolates tested 57 32 31 23 16 10 16

15 16 29

CP-35,587 75% 90% 12.5 100 12.5 >200 50 >200 25 100 25 >200 200 200 12.5 12.5

50% 6.25 6.25 25 25 12.5 50 6.25

12.5 100 200

Amoxicillin

50% 200 6.25 200 100 100 100 1.56

12.5 12.5 0.78 >200 >200 >200 >200 >200

75% 200 100 >200 >200 >200 >200 3.12

Cephalexin 90%

>200 >200 >200 >200

200'

1.56

3.12

50a. d

50%

75% 90% 6.25 6.25 6.25 6.25 12.5 12.5 100 >200 >200 25 25 100 50 >200 3.12 12.5 25 25 >200 >200

25

100

nosa

Hemophilus influenzaeb Staphylococcus aureus Staphylococcus epidermidis Streptococcus faecalis Streptococcus pyogenesb

Streptococcus pneumo-

10 31 9

1.56 12.5 1.56

3.12 25 3.12

3.12 50 3.12

0.39 12.5 0.1

3.12 50 0.39

29

1.56

1.56

1.56 0.05

0.78

1.56

4'

3r

0.05

niagebI a Ampicillin tese instead of amoxicillin. b

3.12 1.56'

1.56a 0.0078 0.0003=9

6.25 6.25 1.56 100

12.5 12.5 1.56 100

25' 25 3.12 200

0.39 0.78

IIII

Mean value of three determinations. Medium for swarming Proteus strain was brain heart infusion (BHI) broth, instead of BHI agar. Medium for H. influenzae was BHI broth and agar enriched with 5% (vol/vol) Fildes and 2% (vol/vol) IsoVitaleX. Medium for S. pyogenes and S. pneumoniae was BHI agar plus 5% blood. H. influenzae, S. pyogenes, and S. pneumoniae cultures grown overnight were diluted 10-fold for use as inoculum. Incubations were for 18 h at 37 C except for H. influenzae, which was incubated for 40 h at 37 C. e Cefazolin tested instead of cephalexin. d Note that approximately 80% of the strains were resistant to ampicillin. ' In the case of eight or less strains, the MIC value given is the amount required to inhibit 90% of the strains.

lin was bactericidal against only E. coli and cephalexin was bactericidal against only K. pneumoniae. The lack of bactericidal activity of cephalexin (5 ug/ml) against E. coli was confirmed with a second strain. The resistance of CP-35,587 to hydrolysis by crude ,B-lactamase preparations from diverse gram-negative bacteria and S. aureus is presented in Table 2. Ampicillin and cephalexin data are included for comparison. CP-35,587 was highly refractory to the (8-lactamase preparations from most strains of K. pneumoniae tested, one of two strains of E. coli, and representative strains of S. rmarcescens and Enterobacter sp. Hydrolysis of CP-35,587 did occur with single strains of K. pneumoniae (53A077) and E. coli (51A129). Both of these cultures are multiply antibiotic-resistant clinical isolates and probably contain (3-lactamases mediated by transferable R-factors. Both cultures had MIC values of 200 pg/ml (Table 2). The E. coli strain, 51A002, which does not hydrolyze CP-35,587, is not a multiply antibioticresistant isolate. It is apparent, therefore, that the (8-lactamase present in this monoresistant (ampicillin) isolate and the typical K. pneumonia strains is quite different than the 38lactamase(s) present in the multiply resistant

strains 51A129 and 53A077. Although some hydrolysis of CP-35,587 occurred with Proteus, Pseudomonas, and S. aureus, the hydrolysis rates were significantly less than that of one or both of the other antibiotics. CP-35,587 proved to be a very effective therapeutic agent against experimental infections produced by a variety of gram-negative pathogens and S. aureus in mice. These data, accompanied by comparative values for amoxicillin and cephalexin, are presented in Table 3. In agreement with in vitro data, CP-35,587 showed good activity against experimental infections produced by five K. pneumoniae isolates. Neither amoxicillin nor ampicillin protected against these infections. Its efficacy against ampicillin-resistant Klebsiella clearly distinguishes CP-35,587 from any known penicillin. The activity of CP-35,587 was also noteworthy against infections produced by Proteus, S. marcescens, and Enterobacter sp. Dependent upon the infections under study, either amoxicillin or cephalexin, or both, were inactive. Therefore, the spectrum of CP-35,587 is an extension of the combined spectra of amoxicillin plus cephalexin. Oral and subcutaneous activities were quite similar, although the parenteral

VOL. 10, 1976

BROAD-SPECTRUM ACTVIVTY OF CP-35,587 Serratia

Escherichia coli

Klebsiella pneumoniae

135

marcescens

1 x 1010.

1

x

z

lx

2 zO U.

x

o106

1 x

104

1x

103

1

J

0 LU

-.1

1

HOURS

2

3

45 6

7 :

HOURS

FIG. 2. Bactericidal activity of CP 35,587 compared with ampicillin

or

HOURS

amoxicillin and cephalexin.

TABLE 2. Comparative (&lactamase resistance of CP-35,587 and other semisynthetic (&lactam antibioticsa Source of 9-lactamase (cell-free extracts)

Hydrolysis rate (gmolIh/mg of protein) Ampicillin

CP-35,587

Cephalexin

53A079b

326 156 578

Laboratory evaluation of 3-(5-tetrazolyl) penam, a new semisynthetic beta-lactam antibacterial agent with extended broad-spectrum activity.

ANTImcboBL AozNrs AND CHMoTHzURPY, July 1976, p. 132-138 Vol. 10, No. 1 Printed in U.S.A. Copyright 0 1976 American Society for Microbiology Labora...
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