Derivative of Carbenicillin

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 197, p Copyright 197 American Society for Microbiology Vol. 1, No. 3 Printed in U.S.A. Carbenicillin Indanyl Sodium, an ly Active Derivative of Carbenicillin ARTHUR R. ENGLISH, JAMES A. RETSEMA, VERNE A. RAY, AND JOHN E. LYNCH Medical Research Laboratories, Pfizer Inc., Groton, Connecticut Received for publication 13 December 1971 Carbenicillin indanyl, an orally active derivative of carbenicillin, is active against a broad spectrum of bacterial species. Although the ester has in vitro antimicrobial activity per se when evaluated in Brain Heart Infusion broth, the in vivo antibacterial activity seen in mice and rats reflects primarily the efficient hydrolysis of the ester to carbenicillin. With an acute systemic infection in mice as a test system, orally administered carbenicillin indanyl protected mice against lethal infections produced by Escherichia coli, Salmonella choleraesuis, Pasteurella multocida, Proteus vulgaris, Staphylococcus aureus, and Streptococcus pyogenes. The dose that protected % of the animals against each of these infections was comparable to that of parenteral carbenicillin. Against experimental urinary-tract disease in rats produced by E. coli, P. vulgaris, and Pseudomonas aeruginosa, it was again observed that carbenicillin indanyl provided activity comparable to that of parenterallv administered carbenicillin. Because of its unique activity against Pseudomonas aeruginosa and indole-positive Proteus species usually resistant to ampicillin, carbenicillin has assumed an important role as a parenteral chemotherapeutic agent for the serious medical problems caused by these microorganisms. Carbenicillin is administered parenterally as di carbenicillin. This formulation is, however, poorly absorbed or acid-labile, or both, when given orally and is not therapeutically effective. The -indanyl ester of carbenicillin (Fig. 1) is an acid-stable form of carbenicillin. It is well absorbed from the gastrointestinal tract and is rapidly hydrolyzed in vivo to carbenicillin. Laboratory studies presented in this report demonstrate that the ester provides bacteriological activity comparable to that of parenterally administered carbenicillin. MATERIALS AND METHODS Antibiotics. Carbenicillin indanyl was used as the crystalline mono salt; carbenicillin, as the amorphous di salt. When appropriate, ampicillin and cephalexin were used in comparative studies. Ampicillin was used as the trihydrate and cephalexin as the monohydrate. The latter two compounds were provided by, respectively, the Biological Division of Bristol Laboratories, Syracuse, N.Y., and Eli Lilly & Co., Indianapolis, Ind. The hydrolysis of carbenicillin indanyl to carbenicillin after oral administration to animals was readily demonstrated through the use of a descending paper chromatographic system. The solvent system contained n-butanol, water, ether, and acetone in a ratio of 3:1:1:1. Serum and urine samples collected at appropriate times after carbenicillin indanyl dosage were spotted on chromatographic paper strips (Whatman no. 4) which were then developed in the solvent system for 1 hr at room temperature. After drying, the strips were placed onto the surface of bioplates, with Escherichia coli 1A66, Staphylococcus aureus 01A00, or Pseudomonas aeruginosa as indicator organisms. In each study, controls included carbenicillin indanyl and carbenicillin di prepared in normal animal sera and urine. Antibacterial activity in vitro. Minimal inhibitory concentrations (MIC) of carbenicillin indanyl and the other antibiotics were determined by a serial dilution technique for a variety of bacterial isolates that were predominantly of clinical origin. Final concentrations of antibiotic ranged from 00 to,ug/ml. Assays were usually accomplished in sterile DisPoso trays (Lindbro Chemical Co., New Haven, Conn.) with a final volume of 1 ml per cup. Brain Heart Infusion (BHI) broth (Pfizer Diagnostics) was used for all organisms with the exception of Haemophilus influenzae. For this organism, BHI medium was enriched with 1% Supplement C (Difco). Test inocula consisted of 0. ml of an overnight culture grown at 37 C and diluted to Inocula for Streptococcus pyogenes and H. influenzae were used at 10-'. After incubation at 37 C for approximately 0 hr, the tests were read visually, with the MIC being designated as the least amount of drug preventing visual growth as evidenced by the absence of gross turbidity (). Penicillin G competitive uptake studies. S. aureus cells from overnight growth were used to inoculate 18

2 186 ENGLISH ET AL. ANTIMICROB. Ao. CHEMOTHER. CH-C-HH3 / 6'cH 0 CONa 6 -/-phenyl -- (-,ndony/oxycarbony/)j aceta'mido penici//anic ocid FIG. 1. Carbenzicillini inidanyl. BHI cultures. After a 4-hr incubation, cells were harvested by centrifugation, and the pellets were suspended in cold buffer at a concentration of 1 to mg (dry weight) per ml. The buffer was composed of 1.0% glucose (w/v) and 0.0 M KHPO4 neutralized to ph 7.0 with NaOH. Amounts of ml of the cell suspension were added to tubes containing 14C-penicillin G (Amersham- Searle) alone or in combination with the 3-lactam antibiotic under test. Tube contents were immediately mixed with a vortex mixer and incubated at 0 C for 0 min. All binding reactions were terminated by the addition of.0 ml of ice cold buffer (B) containing 0.1 M NaHPO4 and.0 mg of 'C-penicillin G per ml. After mixing, cells were collected on prewashed nitrocellulose membrane filters (4 um pore size; Millipore Corp.) and were washed three times with 3.0-ml portions of buffer B and once with 10 ml of cold distilled water containing 1.0 mg of 'C-penicillin G per ml. The above methodology was adapted from that of Rogers (10) and Edwards and Park (4). Radioactivity was counted as described previously (9) İn vitro acid stability studies. Carbenicillin indanyl was added to synthetic gastric juice (ph.0) at 400 or 800 Ag/ml, and the solution was maintained at 37 C for 1 hr. A 0.-ml portion was serially diluted in BHI broth through 10 cups which were inoculated with 0. ml of a suitably diluted culture of S. auireus. MIC values were read after incubation at 37 C for approximately 0 hr, and were compared with standard MIC values against the same test organism. The percentage loss of biological activity was calculated from the differences of MIC values. Comparative data were obtained by use of penicillin G and carbenicillin di. In vivo studies. Acute systemic infections in mice were produced by intraperitoneal inoculation of standardized cultures suspended in % hog gastric mucin. The severity of infection was consistently at 1 to 10 LDloo, i.e., 1 to 10 times the number of organisms needed to kill % of the mice. Treatment with test antibiotics was initiated 0. hr after challenge, and a second dose was administered 4 hr later. After a holding period of 4 days, living mice were counted, and the per cent survivors was calculated. These values were then converted to probits, and a PDss value in milligrams per kilogram was calculated by means of a probit method (1). The PD is defined as the dose of antibiotic in milligrams per kilogram required to protect % of the treated mice against the otherwise lethal infection. Experimental infections were produced in rats to assess the therapeutic potential of carbenicillin indanyl against urinary-tract disease. The details of the technique as used in this laboratory for Proteus vulgaris and Pselidomontas aeruginosa have been published previously (7). Experimental infections with E. coli 08 utilized the urether ligation technique followed by intravenous administration of the organism (8). In all instances, rats were dosed once a day for 8 days, with the first dose being administered the day after infection. Rats were killed 4 hr after the last dose, kidneys were removed and macerated, and viable counts of organisms were made on appropriate media. Such counts are expressed as log averages, and efficacy is measured on the basis of a reduction in log counts compared with infected controls. RESULTS AND DISCUSSIONS The indanyl ester of carbenicillin provides chemical stability which prevents degradation by gastric juice and, in addition, contributes the lipophilic properties necessary for good absorption from the gastrointestinal tract. The indanyl moiety plays a temporary role only, as it is rapidly removed in vivo to regenerate the active parent compound, i.e., carbenicillin. Biological activity in vivo is due to a preponderance of carbenicillin, as only trace amounts (0.1 to 1.0,ug/ml) of carbenicillin indanyl have been detected in serum (3). Indanyl carbenicillin TABLE 1. Comnparison of carbenicillin indanyl with carbenicillin clisodiuim antd ampicillin Microorganism Pseuidomiionzas aerugiiiosa Proteus vulgaris. P. mirabilis. Escherichia coli. Enterobacter aerogenies Haemnophilus inluflenzzae. Pasteurella niuiltocido. Serratia marcescens... Klebsiella pnzeumoniiae.. Salnmoniella species Staphlvlococcus aur-eus Non-penicillinase... Penicillinase. Staphylococcus epidernzidis Streptococcus pyogenes. S. faecalis... Diplococcus pneunmoniiae No. of strains tested 14 Minimal inhibitory concna (pg/ml) Carbenicillin indanyl Carbeni- di > Ampi ciin > a Median based on three to five replicates. b Median values encompass 14 difterent species, each species usually represented by a single strain.

3 VOL. I) 197 TABLE. Competitive uptake studies CARBENICILLIN INDANYL SODIUM Mole fractions 1C penicilfin causing %0 inhibition" 1C-benzylpenicillin... Carbenicillin Carbenicillin indanyl Ampicillin Phenoxymethylpenicillin Mole fraction of 1C-penicillin producing %0 inhibition of "4C-benzylpenicillin binding at ph 7.0. TABaE 3. Cwnulative percentage of37 Pseudomonas aeruginosa clinical isolates susceptible to carbenicillin indanyl and carbenicillin di Antibiotic concn (pg/mi) Antibiotic _ >00 Carbenicillin indanyl Carbenicillin di per se has not been detected (<0.1,ug/ml) in urine from any species; however, the regenerated parent compound, carbenicillin, is found in large amounts. Within 1 hr postadmninistration of carbenicillin indanyl to dogs, rats, and mice, only carbenicillin was detected in serum and in urine samples based on chromatographic evidence. Carbenicillin indanyl also can be converted to carbenicillin in vitro in appropriate laboratory media and reagents, although at a rate considerably slower than the occurrence in vivo. In BHI medium, the ester is converted to carbenicillin within a period of to 16 hr; conversion occurs after 4 hr when added to a glucose-salts synthetic medium. The antibacterial spectrum and MIC values obtained with carbenicillin indanyl were expected to approximate closely those of the parent penicillin. As presented in Table 1, carbenicillin indanyl, like carbenicillin di, is active against a broad spectrum of bacterial species in vitro (6). Carbenicillin indanyl and carbenicilhin generally exhibit comparable activity against the important gramnegative organisms. Those differences in activity that do occur are usually represented by a single dilution in the series. It should be pointed out TABLE 4. Cumulative percentage of Proteus clinical isolates susceptible to carbenicillin indanyl and other g-lactam antibiotics 187 Isolate Antibiotic Antibiotic concn (pg/mi) P. mirabilis (34 Carbenicillin in isolates) danyl Carbenicillin di Ampicillin P. vulgaris (13 Carbenicillin in isolates) danyl Carbenicillin di Ampicillin Other indole- Carbenicillin in positive Pro- danyl teus strains (1 isolates) Carbenicillin di Ampicillin Escherichia coli Carbenicillin in ( isolates) danyl Carbenicillin di Ampicillin

4 188 ENGLISH ET AL. that the MIC values for carbenicillin indanyl have not been corrected for its increased molecular weight resulting from the indanyl moiety. Thus, the indanyl ester contains only 76% by weight of carbenicillin as the biologically active compound. Unlike ampicillin and other penicillins, carbenicillin indanyl and carbenicillin di are active against most strains of indole-positive Proteus species, Pseudomonas, and Serratia marcescens. Carbenicillin indanyl is as active against E. coli, P. mirabilis, and various Salmonella species as are carbenicillin and ampicillin. Carbenicillin indanyl is considerably more active in vitro against susceptible grampositive microorganisms than is carbenicillin di. As shown in Table 1, carbenicillin indanyl is from 8 to 60 times more active than is carbenicillin di against the grampositive microorganisms presented. This enhanced in vitro potency is the result of the decreased hydrophilic properties and the increased lipophilic character of the ester. These physical-chemical properties of penicillins have a profound effect upon the uptake and binding of the antibiotics to the gram-positive bacterial cells (J. A. Retsema and V. A. Ray, in preparation). Similar effects were observed in the gramnegative bacteria only with Klebsiella pneumoniae. It should be noted that the improved activity of carbenicillin indanyl against such organisms is not manifested in vivo; the drug is rapidly metabolized after oral absorption so that it produces the same biological response as carbenicillin di. Additional experiments were designed to measure the increased activity of carbenicillin indanyl against S. aureus. Data from penicillin G competitive uptake studies are presented in Table. Carbenicillin indanyl competes for 4C-penicillin G binding sites on S. aureus (Oxford strain) better than either carbenicillin or ANTIMICROB. Ao. CHEMOTHER. ampicillin but not benzylpenicillin or phenoxymethylpenicillin. It appears that the ester moiety of carbenicillin indanyl facilitates binding in comparison with either carbenicillin or ampicillin. Since the indanyl ester function changes carbenicillin from a very polar compound to a more lipophilic penicillin, the hydrophobic character of indanyl penicillin may assist in its transport through the gram-positive cell wall to the penicillin-sensitive site, the transpeptidase enzyme (4, 10). Such facilitated transport is reflected in the MIC values of 0.0 to 0.08 for carbenicillin indanyl compared to 0.3 to 0.8 for carbenicillin with S. aureus Oxford. This interpretation is in complete agreement with results obtained by Biagi and co-workers using chromatographic techniques (). They found that the more lipophilic penicillins and cephalosporins have better activity against gram-positive bacteria. Both carbenicillin indanyl and carbenicillin were considerably more active in vitro against penicillinase-producing isolates of S. aureus than was ampicillin. As presented in Table 1, the median MIC of ampicillin for these isolates was 00 Ag/ml. The general equivalency of carbenicillin in- TABLE. Stability of carbenicillin indanyl in synthetic gastric juice at 37 C for I hr Antibiotic MIC (,g/ml) against S. aureusa BHI (ph Gastic activity 7.) (ph.0)at ri ph.0(% Carbenicillin indanyl Carbenicillin di Benzylpenicillin a Median values. TABLE 6. Efficacy of carbenicillin indanyl and carbenicillin in vivo against selected pathogens Infecting organism PDss (mg/kg with 9% confidence limits)0 Carbenicillin indanyl Carbenicillin, subcutaneous Escherichia coli 1A ± ± ±.3 Salmonella choleraesuis 8B ± ± ± 3.7 Pasteurella multocida 9A ± ± ±. Proteus vulgaris 7A9.43. ± ± t 61.8 Staphylococcus aureus 01A ± ± ±.8 Streptococcus pyogenes 0C ± ± ±.4 a Expressed as carbenicillin free acid.

5 VOL. 1, 197 CARBENICILLIN INDANYL SODIUM 189 TABLE 7. Efficacy of carbenicillin indanyl and selected atlibiotics againist strainis of Pseudomonas, Proteus, and Escherichia coli in micea PDso (mg/kg with 9%O confidence limits) Infecting organism Carbenicillin indanyl b Carbenicillinb Ampicillin (oral) (subcutaneous) (oral) Pseudomonas aeruginiosa JH i ± 87.8 >400 A >400 IF ± i >400 R ± 94.1 >400 Proteus rettgeri IC ± ± 33. >400 P. morganii IF >400 P. vulgaris 1K >400 P. mirabilis KI i 80 Escherichia coli JH ± i 36.7 (est.)c JH (est.) 3.8 (est.) > (est.) a Cephaloridine was inactive at 400 mg/kg subcutaneously against all Proteus. It was not evaluated against the E. coli infections. I Expressed as carbenicillin free acid. c Estimated. strains of Pseudomonas and danyl to carbenicillin di against gram-negative bacteria is further attested by comparative studies against several recent clinical isolates (Tables 3 and 4). As presented in Table 3, carbenicillin indanyl and carbenicillin appear to have equivalent activity against clinical Pseudomonas isolates. Carbenicillin indanyl, carbenicillin di, and ampicillin had very similar activity against recent P. mirabilis and E. coli isolates (Table 4). However, both carbenicillin indanyl and carbenicillin were superior to ampicillin against strains of P. vulgaris and other indole-positive Proteus species (Table 4). Carbenicillin indanyl is markedly more stable to an acidic environment than is carbenicillin di. Exposure of carbenicillin indanyl to ph.0 (in a synthetic gastric juice) for 1 hr at 37 C resulted in no detectable loss of biological activity (Table ). In contrast, carbenicillin di and benzyl penicillin were almost totally destroyed under these experimental conditions. Based on chromatographic evidence, carbenicillin indanyl remained stable under the conditions of these experiments for as long as 3 hr. In vivo studies. The activity of carbenicillin indanyl, administered either orally or subcutaneously, against experimental infections in mice produced by E. coli, Salmonella choleraesuis, Pasteurella multocida, Staphylococcus au- TABLE 8. Comparative activity of carbenicillin indanyl against experimental urinary-tract infection in rats produced by Escherichia coli Test compound Carbenicillin indanyl Carbenicillin Cephaloridine Cephalexin Ampicillin Cephaloglycin Route of administration a Median value of four experiments. b Average value of two experiments. Dosage (mg/ kg) t Log of viable count/g of kidney Treated Tetd Infected controls a 7.4a.83b 7.87b a a.b. 4b 7. 70b 8.0b reus, Streptococcus pyogenes, the four major species of Proteus, and several Pseudomonas strains, is presented in Tables 6 and 7. As mentioned previously, carbenicillin indanyl

6 190 ENGLISH ET AL. ANTIMICROB. AG. CHEMOTHER. TABLE 9. Comparative activity of carbenicillin indanyl against urinary-tract infections in rats produced by Proteus vulgaris and Pseudomonas aeruginosa Infecting organism Test compound Route (mg/kg) Dosage Log of viable count/g of ~kidney Treated Infected controls P. vulgaris Carbenicillin indanyl a 6.8a Carbenicillin a 6.8a Nitrofurantoin P. aeruginosa Carbenicillin indanyl Carbenicillin Ampicillin.30.7 Nitrofurantoin.76.7 a Average of two experiments. per se has not been detected in urine from any species; however, the regenerated compound, carbenicillin, is found in large amounts. Therefore, the various PD values obtained in the experimental infection studies with carbenicillin indanyl dosage have been corrected to represent milligrams of carbenicillin free acid per kilogram. In addition, the PD values obtained after administration of carbenicillin di also have been converted to represent milligrams of carbenicillin free acid per kilogram. The PD values of carbenicillin indanyl administered orally or subcutaneously were nearly identical against the various experimental infections presented in Table 6. In addition, the oral PD values of carbenicillin indanyl against the experimental infections presented in Tables 6 and 7 were comparable to those of parenteral carbenicillin and further demonstrate that the ester form of carbenicillin is an orally effective form of carbenicillin. The activity of carbenicilhin indanyl against experimental urinary-tract infections in rats is presented in Tables 8 and 9. Table 8 shows the activity of the six drugs used in our studies against E. coli as the infecting organism. The counts of viable organisms per gram of kidney tissue for the infected controls are consistently high, ranging from an organism count of log 6.76 to log 8.0. Carbenicillin indanyl was as active as the other antibioticd studied, when tested at the same levels. Cephaloglycin, unexpectedly, did not perform well when administered orally at relatively high doses. Several additional trials with oral cephaloglycin at these levels have been consistent with the values presented. Parenterally administered cephaloglycin is, however, significantly active against E. coli urinary-tract infections in the rat. As shown in Table 9, carbenicillin indanyl, carbenicillin, and nitrofurantoin are generally able to reduce significantly the count of viable P. vulgaris cells in the rat kidney at the dosages evaluated. Again, the activity of carbenicillin indanyl is quite similar to that of carbenicillin. Neither ampicillin nor nitrofurantoin at mg/kg was able to reduce the count of viable P. aeruginosa cells in the kidney tissue. Carbenicillin indanyl and carbenicilhin at the two higher levels effected a significant response. At mg/kg, both carbenicillin and its ester exhibit marginal activity. LITERATURE CITED 1. Baston, H. C An introduction to statistics in the medical sciences, p Burgess Publishing Co., Minneapolis, Minn.. Biagi, G. L., M. C. Guerra, A. M. Barbaro, and M. F. Gamba, Influence of lipophilic character on the antibacterial activity of cephalosporins and penicihins. J. Med. Chem. 13: Butler, K., A. R. English, A. K. Knirsch, and J. J. Korst Carbenicillin indanyl (CP 1,464-): metabolism and laboratory studies with an oral dosage form of carbenicillin. Del. Med. J. 43: Edwards, J. R., and J. T. Park Correlation between growth inhibition and binding of various penicillin and cephalosporins to Staphylococcus aureus. J. Bacteriol. 99:

7 VOL. 1, 197 CARBENICILLIN INDANYL SODIUM 191. English, A. R d-6-deoxyocytetracycline. I. Some biological properties. Proc. Soc. Exp. Biol. Med. 1: English, A. R Laboratory studies with carbenicillini. Antimicrob. Ag. Chemother. 1968, p English, A. R., T. J. McBride, L. H. Conover, and P. N. Gordon Substituted nitrofurantoins as urinary tract anti-infectives. Antimicrob. Ag. Chemother. 1966, p Guze, L. B., and P. B. Beeson Experimental pyelonephritis. 1. Effect of ureteral ligation of the rat. J. Exp. Med. 104: Retsema, J. A., and T. W. Conway, Inactivation of Escherichia coli ribosomes by 1-fluoro-, 4-dinitrobenzene. J. Mol. Biol. : Rogers, H. J The inhibition of mucopeptide synthesis by benzylpenicillin in relation to irreversible fixation of antibiotic by staphylocci. Biochem. J. 103:90-10.