Epicillin: Experimental Chemotherapy, Pharmacodynamics, and Susceptibility Testing

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INFECrION AND IMMUNITY, July 1971, p. 50-53 Vol. 4, No. I Copyright @ 1971 American Society for Microbiology Printed in U.S.A. Epicillin: Experimental Chemotherapy, Pharmacodynamics, and Susceptibility Testing

H. GADEBUSCH, G. MIRAGLIA, F. PANSY, AND K. RENZ Squibb Institute for Medical Research, New Brunswick, New Jersey 08903 Received for publication 10 December 1970

Epicillin exhibits excellent therapeutic activity when administered orally or subcutaneously to mice infected with a variety of pathogenic bacteria. These included single strains of Streptococcus pyogenes, non-penicillinase-producing Staphylococcus aureus, Proteus mirabilis, Escherichia coli, Salmonella schottmuelleri, and Pseudo- monas aeruginosa, strains of microorganisms selected as typical of those that fre- quently cause serious clinical infections. In oral chemotherapeutic comparisons of tetracycline, chloramphenicol, cephalexin, ampicillin, and epicillin, the latter, with but minor exceptions, exhibited activity comparable to that of most of the other antibiotics studied. Oral administration to mice of a loading dose of epicillin or ampicillin resulted in similar peak blood levels and decay curves. The concentration in urine and the excretion rates were significantly higher in epicillin than in ampicillin-treated animals. Presumptive evidence suggests that the interpretation of zone diameters obtained with 10-,ug epicillin discs should be the same as is used for 10-,ug ampicillin discs.

As part of broad studies in the area of new Animals. Female white Swiss mice of the CF1-S semisynthetic anti-infective agents, we became strain (Carworth Farms, average weight 18 to 20 g) interested in structures derived from 1,4-cyclo- were used in all experiments. Bacterial cultures. All test organisms were clinical hexadienyl-substituted-a-amino acids. Whereas isolates maintained under liquid nitrogen as part of the 1 ,4-cyclohexadienyl ring, much like the ben- the Squibb stock culture collection. Streptococcus zene ring, was expected to be planar, differences pyogenes SC 3862 is generally known as C-203. For all in affinities for active sites on various enzymes other organisms, the sole designation is that of the might be anticipated. Thus, in addition to modi- Squibb collection. Staphylococci and all gram-nega- fied potency, alteration in patterns of absorption, tive organisms were prepared by harvesting cells tissue penetration, and distribution as well as regenerated from liquid nitrogen on Brain Heart metabolism and excretion could distinguish the Infusion( BHI; Difco) agar for 18 to 20 hr. Appropri- aromatic from the dihydro forms. In the peni- ate concentrations of cells were suspended in 5% hog cillin family, the physical, chemical, and biologi- gastric mucin (Wilson Laboratories, Chicago, Ill., type 1701-W) and stored in vials held at -60 C (dry- cal properties in vitro of epicillin have been de- ice chest) until needed. Streptococci and pneumococci scribed (1). The present report will deal with (i) were freshly prepared by cultivation in Trypticase Soy an evaluation of this drug in experimental bac- broth (BBL) for 18 to 20 hr at 37 C. Dilutions con- terial infections in mice and a comparison of its taining the desired number of cells to comprise the in- activity with that of other antibacterial agents, fecting dose(s) were made in primary growth media (ii) its blood levels achieved and its urinary excre- prior to injection into the test animals. The number tion in mice, as well as (iii) preliminary correla- of viable units per dose was confirmed by plate tion of susceptibility data with clinical response. count for each experiment. Efficacy (in vivo) tests. Experimental infections were MATERIALS AND METHODS produced by the intraperitoneal injection of suitably diluted cultures containing 1,000 LD5o (streptococci, Antibiotics. Antibiotics were supplied by Eli Lilly pneumococci) or 100 LD5o (staphylococci and all and Co. (cephalexin monohydrate, lot 413-214-145); gram-negative bacteria). Under these conditions, all Parke Davis and Co. (chloramphenicol, lot 581024, untreated control animals died within 72 hr. Mice 890 jug/ml); and Squibb (epicillin, crystalline, lots infected with streptococci and pneumococci were NNO29NA, 948 ,ug/mg; epicillin-sodium salt, lots given medication in divided doses at 1 and 5 hr after NNO01NA, 890 ug/ml; ampicillin trihydrate, lot infection: those infected with E. coli, at 1 hr after 25B, 874 ,g/mg; ampicillin-sodium salt, lot 2A, 954 infection; and all others, in divided doses at 1 hr be- ug/mg; tetracycline HCl, lot 70817, 972 ,ug/ml). fore and 4 hr after infection. All medication was 50 VOL. 4, 1971 EPICILLIN ON EXPERIMENTAL INFECTIONS 51 administered in 5% gum acacia or water by gavage, Disc susceptibiilty testing. All strains of bacteria by subcutaneous injection, or by a combination of were tested for susceptibility to epicillin by the disc- methods. At least three different doses of the test diffusion procedure, essentially as described by Bauer antibiotic were used in twofold increments; each dose et al. (2). Mueller-Hinton agar, with and without 2% group consisted of 10 mice. All animals were observed rabbit blood, poured to a depth of ca. 4 mm was used for a period of 6 days, and the EDro was determined as the test medium. After incubation for 18 to 24 hr by the method of Reed and Muench (4). Tests were at 37 C, arithmetic means were computed from the repeated at least twice. The statistical formula for measurements of three zones. estimation of the 95% confidence limits of the geo- Minimal inhibitory concentration determinations. metric mean ED5, was adapted from J. Ciminera as Twofold broth and agar dilution tests were used to reported by Miller et al. (3). determine the susceptibility to epicillin of clinical Blood and urine samples. Groups of five mice were isolates as described in the previous paper (1). given a single 400 mg/kg dose of the sodium salt of epicillin or ampicillin by gavage, with water or 5% RESULTS AND DISCUSSION gum acacia as a vehicle. The groups of animals were Efficacy studies. Model infections establshed housed in metabolism cages to permit the collection with the aid of three gram-positive (S. pyogefes output the 6-hr after of the total urine during period SC 3862, S. aureus SC 2399, Diplococcus pneu- drug administration. At specified intervals, groups of moniae SC 5479) and three gram-negativee (Esch- mice were bled from the orbital sinus; the serum was then separated by centrifugation and stored at 4 C. erichia coli SC 8294, Proteus mirabilis SC 3873, Both sera and urines were assayed on the day of col- Salmonella schottmuelleri SC 3850) organisms lection by a disc-diffusion assay with Staphylococcus have responded to therapy with either subcutane- aureus P-209 as the test organism (1). ous or oral, or a combination of these routes,

TABLE 1. Chemotherapeutic activity of epicillin in experimental bacterial infections in mice

Geometric mean of ED,, (mg/kg)a Model infection Oral Subcutaneous

Streptococcus pyogenes SC 3862 ...... 1.7 (1.4-2.3) 0.2 (0.15-0.25) Staphylococcus aureus SC 2399 ...... 3.2 (2.6-3.9) 0.15 (0.1-0.23) (non-penicillinase producer) S. aureus SC 2400 ...... 1,250 (740-2,200) (penicillinase producer) Diplococcus pneumoniae SC 5479...... 3.4 (2.6-4.5) Escherichia coli SC 8294...... 55 (37-106) 12.9 (10-19.9) Proteus mirabilis SC 3873 ...... 15 (13-20) 13.2 (11-18) Salmonella schottmuelleri SC 3850 ...... 16 (13-42) 14.8 (10-20) Pseudomonas aeruginosa SC 8327 >800 413 (402-459) a Confidence limits (95%) in parentheses.

TABLE 2. Comparison of the oral chemotherapeutic activity of epicillin with that offour broad/medium spectrum antibiotics in experimental murine infections

Chemotherapeutic activity expressed as geometric mean of oral EDso (mg/kg)" Model infection Epicillin Ampicillin Tetracycline Chloramphenicol Cephalexin

Streptococcus pyogenes SC 3862...... 2 (1.4-2.3) 1 (0.6-1.2) 237 (185-267) 201 (182-216) 9 (8.2-10.6) Diplococcus pneumoniae SC 5479 ...... 3 (2.6-4.3) 5 (2.5-10) >400 >400 92.3 Escherichia coli SC 8294..... 55 (37-106) 50 (43-60) 265 (232-300) 53 (49-62) 20 (19-21) Proteus mirabilis SC 3873 ...... 15 (13-20) 17 (10-20) NTb 625 48 (43-53) Salmonella schottmuelleri SC 3850...... 16 (13-42) 10 (6.3-13.0) 800 858 130 (100-171) a Confidence limits (95%) in parentheses. b NT = not tested. .52 GADEBUSCH ET AL. INFEC. IMMUN. with low doses of epicillin (Table 1). The MIC Drug Concentration in Urine for all of these organisms was < 1.5 ,ug/ml (1). 60 (0 6hours) Subcutaneous: oral ED50 ratios from 1:1 to 1 :4 50 for gram-negative bacteria indicated that the new 40' Epic illin Ampicillin penicillin was well absorbed. Although epicillin 30 1030 g/mI 650ug/ml is sensitive to beta lactamase, it has been shown in E this case with Staphylococcus aureus SC 2400 i 20 that massive doses of the antibiotic can protect C mice even against this penicillinase-producing It /2 = 60minutes organism. The strain of P. aeruginosa used in these studies 0 10.0 c is sensitive to epicillin in vitro (MIC 12.5 ug/ml) 0 o-o EPICILLIN and showed an ED50 of 413 mg/kg when the anti- - - AMPICILLIN -a biotic was injected subcutaneously. The results a of six separate experiments with carbenicillin w (MIC 25 ,g/ml) in this model infection yielded a c: geometric mean ED50 of 306 mg/kg, with esti- mated 95 % confidence limits of 304 to 360 mg/kg. The oral chemotherapeutic activity of epicillin 0. in mice was compared with that of other broad/ medium spectrum antibiotics (Table 2). In most cases, epicillin showed a degree of activity in vivo against six representative bacteria which was 3 greater than that exhibited by tetracycline, chlor- Hours Post Addministrat ion amphenicol, and cephalexin but similar to that FIG. 1. Comparison of the serum and urine concent- of ampicillin. tration of epicillin and ampicillin after oral adminis- Preclinical pharmacology. After the oral ad- tration of the drugs as their sodium salts (400 mg/kg) ministration to mice of a single loading dose of to CFI-S female mice. These data represent the results 400 mg/kg of the sodium salt of either epicillin offour separate experiments. or ampicillin, peak blood levels of ca. 20 ,ug/ml were reached at approximately 30 min (Fig. 1) animals during the 6-hr test period showed a mean and fell off rapidly during the ensuing 5.5 hr. concentration (based on total bioactivity) of The. serum half-life of both antibiotics was ap- epicillin (1,030 jig/ml) that was significantly proximately 60 min. Urine excreted by these higher (P < 0.001) than that obtained with ampi- EPICILLIN MIC (10-pgdisc) Log 2rn (pg/mli 6 64 5 32 a 4 6 3 8.0 2 4.0 2.0 0 .0 .50 -2 .250 -3 .125 -4 .0 6 2 5 - 5 .0313 -6 .0156

- 7 .0078 - T 5 10 15 20 25 30 35 40 45 ZONE DIAMETER (mm) FIG. 2. Regression cuirve for zone diameters as derived by the Bauer-Kirby disc-diffusion procedure and minimal inhibitory concentrationis (MIC) of100 clinical bacterial isolates. VOL. 4, 1971 EPICILLIN ON EXPERIMENTAL INFECTIONS 53 cillin (650 ,g/ml). The excretion rate of the two to epicillin were differentiated from those that antibiotics during the same period, based on came from subjects that had responded less administered dose, was 37% for epicillin and 26% satisfactorily (Fig. 2). On the basis of these early for ampicillin. This difference was statistically clinical studies, it appeared that the well known significant (P < 0.05). The reason(s) for these interpretation of zone diameters obtained with differences are being investigated. 10-,ug ampicillin discs (gram-positive cocci, en- Laboratory-clinical correlation studies. With the terococci, and gram-negative organisms; refer- initiation of clinical efficacy studies, preliminary ence 2) could also be applied tentatively to 10-,ug correlation between laboratory (MIC or zone epicillin discs. Interpretive values for Haemophilus size) and clinical (therapeutic response) data would be expected to be similar but must await became possible. Participating investigators were the results of additional clinical trials. asked to submit to the Squibb Institute slants of about to ACKNOWLEDGMENTS pretreatment cultures from patients We are indebted to William Trejo and his staff for the identifi- enter the Preliminary Efficacy Study (U.S. Phase cation and cataloging of the clinical cultures. In addition, we thank II). These cultures were identified with respect to W. A. Kolman and U. Barai for the statistical evaluations and genus and species and were tested by broth or the Clinical Research Department of the Institute, particularly agar dilution (MIC) and by Bauer-Kirby disc- J. A. Hubsher, for permitting us to use certain clinical data. diffusion (zone size) techniques. The data were LIRATURE CITED subsequently plotted (Fig. 2) and subjected to 1. Basch, H., R. Erickson, and H. H. Gadebusch. 1971. Epicillin: The correlation in vitro studies. Infec. Immun. 4:44-49. regression analysis. coefficient 2. Bauer, A. W., W. M. Kirby, J. C. Sherris, and M. Turck. 1966. between log2 MIC and zone diameter was 0.91. Antibiotic susceptibility testing by a standardized single disc The t test on the regression coefficient showed it method. Amer. J. Clin. Pathol. 45:493-496. to be significantly different from zero with P < 3. Miller, A. K., B. M. Brost, M. E. Valiant, H. Kropp, and D. Hendlin. 1970. Phosphonomycin V. Evaluation in mice. 0.001. Antimicrob. Ag. Chemother. 1969, p. 310-315. In addition, cultures from patients that had 4. Reed, L. J., and H. Muench. 1958. A simple method of esti- demonstrated a good to excellent clinical response mating fifty per cent end points. Amer. J. Hyg. 27:493-499.