Pirbenicillin, a New Semisynthetic Penicillin with Broad- Spectrum Activity

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1 ANTmCROBAL AGaNTs AND CHDMoTHzRA&Y, Apr. 1976, p Copyright ) 1976 American Society for Microbiology Vol. 9, No. 4' Printed in U.SA. Pirbenicillin, a New Semisynthetic Penicillin with Broad- Spectrum Activity GERALD P. BODEY,* VCTORO RODRGUEZ, AnD SUZANNE WEAVER Department ofdevelopmental Therapeutics, The University oftexas System Cancer Center, M. D. Anderson Hospital and Tumor nstitute, Houston, Texas 7725 Received for publication 24 November 1975 Pirbenicillin is a new semisynthetic penicillin which inhibited 67% of isolates of Proteus aeruginosa tested in our laboratory, 93% of P. mirabilis, 31% of Enterobacter spp., 41% of Serratia spp., and 58% of Escherichia coli at a concentration of 6.25 Mg/ml. ts activity appeared to be inoculum dependent and it was virtually inactive against 1O7 inocula ofp. aeruginosa. t was more active than carbenicillin or ticarcillin, but less active than BL-P1654 againstp. aeruginosa. Carbenicillin and ticarcillin appeared to be more active than pirbenicillin against Proteus spp., but pirbenicillin was active against some isolates of Klebsiella spp. The synthesis of ampicillin represented an important advance in antibiotic research because this was the first truly broad-spectrum penicillin. The subsequent discovery of carbenicillin extended the spectrum of penicillins to include indole positive Proteus spp. and Pseudomonas aeruginosa (1). Clinical studies demonstrated that this antibiotic was efficacious for the treatment ofpseudomonas infections, even in patients with severely impaired host defenses (3). Because of its rather marginal activity, carbenicillin must be administered in high doses which are associated with undesirable side effects such as electrolyte imbalance and coagulation abnormalities (4). Consequently, research has been directed toward synthesizing new penicillin derivatives with greater antipseudomonal activity. One of these derivatives, ticarcillin, is more active than carbenicillin in vitro and has been used successfully at lower doses for the treatment of Pseudomonas infections (6). Pirbenicillin, 6-[D-2-phenyl-2-(N- 4-pyridyl-formimidyl aminoacetamido)-acetamidol-penicillanic acid (CP-33, 994) is a newly synthesized penicillin derivative which appears to have substantially greater activity than carbenicillin against P. aeruginosa. This report presents the results ofin vitro studies ofpirbenicillin which indicate that it may be a potentially useful antibiotic. MATERALS AND METHODS Susceptibility tests were conducted on 525 clinical isolates of gram-negative bacilli and 139 clinical isolates of gram-positive cocci, using the dilution technique with an automatic microtiter system (Canalco: Autotiter nstruction Manual). All orga- 668 nisms were inoculated into Mueller-Hinton broth (Difco) and incubated at 37 C for 18 h. For gramnegative bacilli, a.5-ml sample of a 1-3 dilution of this broth culture (approximately 15 colonyforming units [CFU]/ml) was used as the inoculum. For gram-positive cocci, a.5-ml sample of a 1-2 dilution of this broth culture (approximately 16 CFU/ml) was used as the inoculum for susceptibility testing. All gram-negative bacilli used in this study were cultured from blood specimens obtained from patients between 1967 and The patients were hospitalized at this institution and had underlying malignant diseases. A total of 1 isolates each ofp. aeruginosa and Escherichia coli, 119 isolates ofproteus spp., 7 isolates of Serratia spp., 86 isolates of Enterobacter spp., and 5 isolates ofklebsiella spp. was used. All gram-positive cocci used in this study were cultured from specimens obtained from hospitalized patients, most of whom did not have cancer. A total of 25 isolates of Streptococcus pyogenes, 14 isolates of Streptococcus pneumoniae and 1 isolates of Staphylococcus aureus was used. The susceptibility of isolates ofs. aureus to penicillin G was determined by the broth dilution method. solates inhibited by less than.1,ug/ml were selected as penicillin G susceptible, and those isolates resistant to more than 25,ug/ml were selected as penicillin G resistant. Pirbenicillin was supplied as a powder by Pfizer Central Research, Groton, Conn. BL-P [D-a- (3-guanylureido)-phenylacetamido]penicillanic acid was supplied by Bristol Laboratories, Syracuse, N. Y. Carbenicillin and ticarcillin were supplied by Beecham Pharmaceuticals, Bristol, Tenn. Twofold serial dilutions of the antibiotics were made with Mueller-Hinton broth, to which phosphate buffer was added to a ph of 7.2. The minimal inhibitory concentration (MC) was determined after incubation at 37 C for 18 h. All wells containing trace growth or no discernible growth were subcultured Downloaded from on November 9, 218 by guest

2 VOL. 9, 1976 on sheep blood agar. A calibrated pipette was used to transfer.1 ml of the inoculum. The minimum bactericidal concentration (MBC) was determined after incubation at 37 C for 18 h. The MBC was defined as the lowest concentration of drug which yielded less than 5 colonies on subculture (less than 5 colonies per.1 ml of inoculum). Comparison studies were conducted simultaneously. The concentration of pirbenicillin in media was determined by an agar well method with Sarcina lutea ATCC9341 as the test organism. This organism was prepared so that a 1:1 dilution had an absorbance of.6 at a wavelength of 58 nm, and 4 ml of this preparation was added to 1 liter of antibiotic medium no. 1 (Difco). After mixing thoroughly, 14 ml of broth was poured in plates (1 by 15 mm). Wells (.75-mm diameter and.75-mm depth) were cut into the agar and filled with.5 ml of each specimen. The plates were incubated at 37 C for 18 h. Zones of inhibition were measured with a caliper, and the concentration was determined from a standard curve. A standard curve was prepared every time samples were assayed. All studies were performed in triplicate. The effect of pirbenicillin on the growth of 1 isolates ofp. aeruginosa was determined in the following fashion. All 1 isolates were inoculated into Mueller-Hinton broth and incubated at 37 C for 18 h. A.1-ml aliquot of broth containing each Pseudomonas isolate was inoculated into each of five tubes containing 9.9 ml of unbuffered Mueller-Hinton broth. Two of the five tubes contained pirbenicillin at a concentration equal to the MC for that Pseudomonas isolate. Two other tubes contained pirbenicillin at a concentration equal to four times the MC for that Pseudomonas isolate. The fifth tube served as a control. All of the tubes were incubated at 37 C for 24 h. After vigorous agitation, a.1-ml aliquot was removed from each tube at, 2, 4, 6, 8, 1, 12, 15, 18, 21, and 24 h and then subcultured on Mueller-Hinton agar. The number ofviable organisms present was determined from these subcultures. At 8 h, additional pirbenicillin was added to two tubes (one containing the MC and one containing four times the MC) so that the concentration was the same as at the onset of the experiment, assuming that all of the original pirbenicillin had been destroyed. The concentration of pirbenicillin in each tube was assayed at 8 h and at 24 h. The MC was determined for all organisms still growing at 24 h. RESULTS The in vitro activity of pirbenicillin against gram-positive cocci and gram-negative bacilli is shown in Fig. 1. Nearly all isolates of S. pyogenes and S. pneumoniae were inhibited by.1,ug/ml or less. All isolates of penicillin G- sensitive S. aureus were inhibited by 1.56,g/ ml, whereas only 4% of penicillin G-resistant S. aureus was inhibited by 3.12,ug ofpirbenicillin per ml. At a concentration of 6.25 pg/ml, pirbenicillin inhibited 31% of isolates ofentero- 1 8 % 6t - - E 4 C- PRBENCLLN Minimum nhibitory Concentrotion FG. 1. n vitro activity of pirbenicillin against gram-negative bacilli and gram-positive cocci. Numbers in parentheses indicate number ofisolates tested. bacter spp., 41% ofserratia spp., 58% ofe. coli, 67% of P. aeruginosa, and 93% of P. mirabilis. At 5,ug/ml, the antibiotic inhibited 34% of isolates of Klebsiella spp. and 28% of indolepositive Proteus spp. Generally, using the buffered Mueller-Hinton broth, the MC was also the MBC. However, the MBC for isolates of P. aeruginosa was usually two to four times higher than MC. For example, 6.25,ug/ml was the MC and MBC for 58% of isolates ofe. coli, the MC for 41%, and MBC for 34% of isolates of Serratia spp., and the MC for 67% of isolates of P. aeruginosa, but the MBC for only 4% of these latter isolates. The effect of inoculum size on the MC and MBC was determined for 1 isolates each ofe. coli and P. aeruginosa (Fig. 2). For most isolates, at both inoculum sizes, the MC and MBC were similar. However, the MC and MBC for isolates of E. coli increased fourfold when the inoculum was increased from 15 CFU/ml to 17 CFU/ml. noculum size had a major effect on the inhibitory activity of pirbenicillin against isolates of P. aeruginosa. Only one isolate was inhibited by 4,ug of pirbenicillin per ml when an inoculum of 17 CFU/ml was used. The effect of ph on susceptibility of 28 isolates of P. aeruginosa to pirbenicillin, BL- P1654, carbenicillin, and ticarcillin was investigated (Fig. 3). Phosphate buffer was added to Mueller-Hinton broth to a final ph of 6.4 and Downloaded from on November 9, 218 by guest

3 67 BODEY, RODRGUEZ, AND WEAVER 7.2. BL-P1654 was the most active antibiotic at both H+ concentrations and carbenicillin was the least active. Pirbenicillin, carbenicillin, and ticarcillin were more active at ph 7.2, whereas BL-P1654 was more active 'at ph 6.4. The difference between the MC and MBC was similar at both H+ concentrations for carbenicillin, BL-P1654, and pirbenicillin. The difference between MC and MBC for ticarcillin was greater at ph 6.4 than at ph 7.2. z.4 Z E Minimum nhibitory Concentration (yig/mi) FG. 2. Effect of inoculum size on MC and MBC of pirbenicillin. Ten isolates each of P. aeruginosa and E. coli were tested. ANTMCROB. AGENTS CHZMOTHER. The activity of the antibiotics in different media was studied, using these same isolates of P. aeruginosa (Fig. 4). BL-P1654 was the most active antibiotic in every medium and was most affected by the composition of the media. All of the antibiotics were most active in M9 broth which does not contain preformed amino acids. They were least active in Mueller-Hinton broth buffered at ph 7.2. Except for BL-P1654, the greatest differences of MC and MBC were observed with M9 broth. nterestingly, the MCs for carbenicillin and ticarcillin were the lowest with M9 broth, but the MBCs were the highest in these medium. The activity of pirbenicillin against 25 isolates each of various gram-negative bacilli was compared with ticarcillin and carbenicillin, using Mueller-Hinton broth buffered at ph 7.2 (Fig. 5-8). Carbenicillin was least active against E. coli, whereas ticarcillin and pirbenicillin had similar activity. Pirbenicillin was substantially less active than ticarcillin and carbenicillin against indole-positive and -negative Proteus spp. The activity of all three antibiotics was similar against isolates of Enterobacter spp. and Serratia spp. Pirbenicillin was substantially more active than carbenicillin against isolates of P. aeruginosa. Pirbenicillin was the only antibiotic with activity against isolates of Klebsiella spp. Downloaded from (1) a) 4- (,). L o ph MC * ph 6.4-MBC 2 a ph 7. 2-MC - ph72-mbc O '-k Minimum nhibitory Concentration (plg/ml) Effect ofph on activity ofpenicillins against P. aeruginosa. Twenty-eight isolates were tested. FG. 3. on November 9, 218 by guest

4 VOL. 9, 1976 PRBENCLLN O 4. 2 %+.- cn o 4 / 2,,'. MBC M-9 Broth MC * MBC Broin Heort nfusion o13 MBC mueller Hinton Brothi Minimum nhibitory Concentration (jig/ml) FG. 4. Effect of media on activity of antipseudomonal penicillins against 28 isolates ofp. aeruginosa. The effect of pirbenicillin on the proliferation of 1 isolates of P. aeruginosa was studied. Figure 9 shows the median results with six isolates which were initially susceptible to 12.5,ug of pirbenicillin per ml. At 8 h, the concentration had decreased from 16/ml to 13/ml when exposed to 12.5,ug/ml (MC) or 5,ug/ml (four times MC) of pirbenicillin. About 5% of the antibiotic had been inactivated at this time. Hence, during a portion ofthis 8-h period, organisms incubated in media containing the MC of pirbenicillin were exposed to a subinhibitory concentration. f no additional antibiotic was added at this time, the organisms proliferated so that the concentration was the same as the controls at 24 h. The MC for these persistent organisms had increased from 12.5,ug/ml to 1,ug/ml. Even if additional drug was added at 8 h, the organisms were able to proliferate subsequently, but only after a 4-h delay. These persistent organisms also had developed increased resistance. Organisms exposed to 5,ug of pirbenicillin per ml were all killed eventually because the concentration of pirbenicillin remained above the MC. Complete killing occurred sooner if additional drug was added at 8 h. Similar results were observed with four isolates of P. aeruginosa which had MCs of 6.25, 6.25, 25, and 5,ug/ml. When pirbenicillin was incubated alone, there was no appreciable destruction of drug at 8 h. However, at 24 h, 7 to 22% was inactivated. DSCUSSON Pirbenicillin is a broad-spectrum penicillin with activity against both gram-positive cocci and gram-negative bacilli. t is more active than carbenicillin or ticarcillin against P. aeruginosa, but less active than BL-P1654. Clinical studies of the latter antibiotic have been abandoned because of its potential nephrotoxicity. Pirbenicillin is less active than either carbenicillin or ticarcillin against Proteus spp. t is as active as carbenicillin against Serratia spp. and Enterobacter spp. These results are in agreement with those of Retsema et al. who also demonstrated that pirbenicillin was more active than carbenicillin against experimental Pseudomonas infections in mice (J. A. Retsema, A. R. English, and J. E. Lynch, Prog. Abstr. ntersci. Conf. Antimicrob. Agents Chemother., 15th, Washington, D.C., Abstr. 252, 1975; Retsema and English, Prog. Abstr. ntersci. Conf. Antimicrob. Agents Chemother., 15th, Washington, D.C., Abstr. 253, 1975). Downloaded from on November 9, 218 by guest

5 672 BODEY, RODRGUEZ, AND WEAVER 1 ANnimacRoB. AGZWM CHEMOTHZR. * Pirbenicillin A Ticorcillin o Corbenicllin 8 U, c -.' 4 a E L-, l t Minimum nhibitory Concentration (ag/ml) FG. 5. Comparative activity ofsemisynthetic penicillins against E. coli. Twenty-five isolates were tested. There has been some controversy over the bactericidal activity of pirbenicillin. Lopez et al. found considerable differences between the MC and MBC against isolates ofp. aeruginosa (C. Lopez, H. Standiford, B. Tatum, F. Calia, S. Shimpif, M. Synder, and R. Hornick, Prog. Abstr. ntersci. Conf. Antimicrob. Agents Chemother., 15th, Washington, D.C., Abstr. 254, 1975). These differences appear to be due to the chemical instability of pirbenicillin in alkaline media (Retsema et al., Prog. ntersci. Conf. Antimicrob. Agents Chemother., Abstr. 252, 1975). The ph of Mueller-Hinton broth increases to over 8 during growth of P. aeruginosa causing degradation of pirbenicillin. This problem can be circumvented by buffering the media. Consequently, we used only Mueller- Hinton broth buffered to ph 7.2 for our studies, whereas Lopez et al. used unbuffered media. The difference between MC and MBC tended to be minimal in buffered media. The efficacy of pirbenicillin was profoundly influenced by inoculum size. This was especially true for isolates of P. aeruginosa which became resistant to pirbenicillin when the inoculum was increased from 15 to 17 cells. The activity of other penicillins is also affected by 4 a* t * Pirbenicillin E "-, # ii *~~~~ Ticarcillin O lil o~~~~~ Carbenicillin 2 - o Minimum nhibitory Concentration (jig/ml) FG. 6. Comparative activity ofsemisynthetic penicillins against P. mirabilis. va 1 - a C-) 2 r 8[ 6 F 4 [ 2. f.i4 /1 A,- / 1 * Pirbenicillin A Ticarcillin o Carbenicillin Minimum nhibitory Concentration (,.g/ml) FG. 7. Comparative activity ofsemisynthetic penicillins against indole-positive Proteus spp. t Downloaded from on November 9, 218 by guest

6 VOL. 9, 1976 (A - 7 V 4- C._ cj C-) ~ / * Pirbenicillin * ricorcillin O Carbenicillin OW. A Minimum nhibitory Concentration (pg/ml) FG. 8. Comparative activity ofsemisynthetic penicillins against P. aeruginosa. 1 E U, a 8 a the size of inoculum (5, 7). Pirbenicillin was most effective in a basic medium such as M9 which contains no preformed amino acids. The effect of media on the activity of BL-P1654, a related semisynthetic penicillin, has been described previously (2). When isolates ofp. aeruginosa with an inoculum size of 16 organisms/ml are exposed to the MC for pirbenicillin in unbuffered Mueller-Hinton broth, killing occurs during the first 8 h. However, sufficient drug is destroyed, so that the concentration falls below the MC. At this suboptimal concentration, persisting organisms develop resistance and begin to proliferate. This can be avoided by exposing the organisms to a higher concentration initially. ncreasing the concentration to above the MC at 8 h has little effect, indicating that resistance probably has already developed. Unfortunately, we only repeated MC determinations on organisms that survived at 24 h. We have observed similar results with a few isolates of E. coli, and Lopez et al. reported similar results with P. aeruginosa (Prog. ntersci. Conf. Antimicrob. Agents Chemother., Abstr. 254, 1975). These observations suggest that it may be important to maintain serum concentrations continuously above the MC of infecting organisms when treating patients with this drug. Pirbenicillin is an interesting new semisyn- %- 6 a._ -4 O4 u 2 c Drug Concentrotion (#g/ml) PRBENCLLN ,12.5,ag/ml / zooig/ml MC -OOg/ml * Control O Tube A TubeB A Tube C O TubeD Downloaded from on November 9, 218 by guest 1 1-., 'w Hours FG. 9. Effect ofpirbenicillin on growth ofsix isolates ofp. aeruginosa, all ofwhich were initially inhibited by 12.5 pg/ml. Tube A initially contained 12.5 pg ofpirbenicillin per ml and tube C initially contained 5 pg ofpirbenicillin per ml. No additional drug was added to these tubes at 8 h. Tube B initially contained 12.5 pgl ml and an additional 12.5 pg/ml was added at 8 h. Tube D initially contained 5 pgml and an additional 5 pgml was added at 8 h. For details of experiment, see text.

7 674 BODEY, RODRGUEZ, AND WEAVER thetic penicillin with broad-spectrum activity. Although it is not as active against P. aeruginosa as BL-P1654 in vitro, it does not cause nephrotoxicity in animals. This drug deserves clinical investigation since it may be more effective than carbenicillin or ticarcillin. t may also prove efficacious against K. pneumoniae since it inhibits 7% of these isolates in vitro at 2 jug/ml. Although this represents only marginal activity, it may be possible to maintain adequate serum concentrations with high doses of drug. ACKNOWLEDGMENTS This investigation was supported by Public Health service grant CA 142 from the National Cancer nstitute and by a grant-in-aid from Pfizer Central Research, Groton, Conn. LTERATURE CTED 1. Acred, P., D. M. Brown, E. J. Knudsen, G. N. Robinson, and R. Sutherland New semisynthetic ANTMaCROB. AGENTS CHEMOTHER. penicillin active against Pseudomonas pyocyanea. Nature (London) 215: Bodey, G. P., and D. Stewart. n vitro studies on semisynthetic -(substituted-ureido) penicillins. Appl. Microbiol. 21: Bodey, G. P., J. P. Whitecar. Jr., E. Middleman, and V. Rodriguez Carbenicillin therapy of Pseudomonas infections. J. Am. Med. Assoc. 218: Brown, C. H.,, E. A. Natelson, M. W. Bradhaw, T. W. Williams, Jr., and C. P. Alfrey, Jr The hemostatic defect produced by carbenicillin. New Engl. J. Med. 219: Neu, H. C., and E. B. Winshell n vitro studies of a semisynthetic penicillin, 6-[D(-)a-carboxy-3- thienylacetamido] penicillanic acid (BRL 2288) active against Pseudomonas, p Antimicrob. Agents Chemother Rodriguez, V., G. P. Bodey, N. Horikoshi, J. nagaki, and K. B. McCredie Ticarcidin therapy of infections. Antimicrob. Agents Chemother. 4: StandUford, H. C., A. C. Kind, and W. M. M. Kirby Laboratory and clinical studies of carbenicillin against gram-negative bacilli, p Antimicrob. Agents Chemother Downloaded from on November 9, 218 by guest