ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 1991, p. 14-19
Vol. 35, No. 1
0066-4804/91/010014-06$02.00/0 Copyright © 1991, American Society for Microbiology
Antibacterial Activities In Vitro and In Vivo and Pharmacokinetics of Cefquinome (HR llyV), a New Broad-Spectrum Cephalosporin MICHAEL LIMBERT,* DIETER ISERT, NORBERT KLESEL, ASTRID MARKUS, KARL SEEGER, GERHARD SEIBERT, AND ELMAR SCHRINNER
Hoechst AG, Pharma Research, Postfach 80 03 20, 6230 Frankfurt/Main, Federal Republic of Germany Received 19 June 1990/Accepted 16 October 1990
Cefquinome is a new injectable aminothiazolyl cephalosporin derivative. It is stable against chromosomally and plasmid-encoded I8-lactamases and has a broad antibacterial spectrum. Staphylococcus aureus, streptococci, Pseudomonas aeruginosa, and members of the family Enterobacteriaceae (Escherichia coli, Salmonella spp., Klebsiella spp., Enterobacter spp., Citrobacter spp., and Serratia marcescens) are inhibited at low concentrations. Cefquinome is also active against many strains of methicillin-resistant staphylococci and enterococci. Its in vitro activity against gram-negative anaerobes is very limited. The high in vitro activity of cefquinome is reflected by its high in vivo efficacy against experimental septicemia due to different gram-positive and gram-negative bacteria. We studied the pharmacokinetic properties of cefquinome in mice, dogs, pigs, and calves. After single parenteral administrations, cefquinome displayed high peak levels, declining with half-lives of about 0.5, 0.9, 1.2, and 1.3 h, respectively. The areas under the concentration-time curve determined for dogs and mice showed linear correlations to the given doses. In dogs the urinary recovery was more than 70% within 24 h of dosing. At present cephalosporins are widely used for the treatment of infections in humans. The aminothiazolyl cephalosporins are particularly important because they possess favorable chemotherapeutic properties and are associated with low rates of adverse effects (1, 8). Cefotaxime was the first representative of this group of cephalosporins (11). During our efforts to find new derivatives with even broader antibacterial spectra or better pharmacokinetics, a series of polar cephalosporins was synthesized (3). From these compounds, cefpirome was selected to be developed for use in human medicine (6, 13). Cefquinome (HR 111V) (Fig. 1) is another representative of this group. Here we report the antibacterial activity of cefquinome in vitro, its therapeutic efficacy in experimental infections, and its pharmacokinetic properties in animals. (This work was presented in part at the 29th Interscience Conference on Antimicrobial Agents and Chemotherapy, Houston, Tex., 1989, abstr. no. 479 and 480.)
diluted cultures of the strains concerned. The MIC was taken as the lowest concentration at which no visible growth could be detected after 24 h at 37°C. The MICs for methicillin-resistant Staphylococcus aureus were read after 48 h at 30°C. The MICs for 50 and 90% of the isolates (MlC50 and MIC90, respectively) were calculated from the results. The susceptibility of obligate gram-positive and gramnegative anaerobes was tested by using the agar dilution test on Wilkins-Chalgren agar (Oxoid, Wesel, Federal Republic of Germany). Overnight cultures of the appropriate test strains diluted 1:10 in fresh thioglycolate medium (Oxoid) were used as the inoculum. The MICs of the antibiotics were determined after the plates had been incubated in anaerobic jars (Oxoid) for 48 h at 37°C. f8-Lactamase stability. The enzymes used were released from the bacteria by ultrasonication and partially purified by chromatography on Sephacryl S-200 superfine (Pharmacia, Freiburg, Federal Republic of Germany). For the spectrophotometric UV test (11), 2 ml of a 10' M solution of cefquinome and 50 ,ul of enzyme solution were mixed. The A255 was monitored for 10 min at room temperature. The relative rates of hydrolysis were compared with the decay of cephaloridine. Affinity to PBPs. The binding of cefquinome to penicillinbinding proteins (PBPs) of Escherichia coli K-12 was determined as previously described (12) by using 125I-ampicillin. Influence of nutrient medium. The effect of the composition of the culture medium was examined in the serial macrodilution test. Geometrical dilution series of the compound were prepared in different fluid nutrient media and inoculated with suspensions of the test organisms. The following commercial bacterial culture media were used: Mueller-Hinton medium (Difco), ABM III (Difco), standard I (Merck), heart infusion medium (Difco), brain heart infusion medium (Difco), standard bouillon (Oxoid), 1% Tryptone (Difco), Iso-Sensitest broth (Oxoid), and nutrient broth (Oxoid). The effect of pH on the activity of cefquinome was determined in the serial macrodilution test with Mueller-
MATERIALS AND METHODS Antimicrobial agents. Cefquinome, cefotaxime, and cefpirome were synthesized by Hoechst AG, Frankfurt, Federal Republic of Germany. Cefoperazone and cefuroxime are commercially available. Bacterial strains. The bacterial strains used were clinical isolates obtained from various hospitals in Europe between 1978 and 1988 and strains maintained in our own laboratories. All bacterial strains were stored on agar slants at room temperature or as suspensions in liquid nitrogen. Antimicrobial susceptibility testing. The susceptibility of aerobic bacteria was tested by means of the agar dilution test on Mueller-Hinton agar (Difco, Detroit, Mich.). When streptococci were tested, the agar was supplemented with 10% horse blood. Plates were inoculated with a multipoint inoculator (Greiner, Nuirtingen, Federal Republic of Germany) which delivered 5 x 104 CFU per spot of stationary, freshly *
Corresponding author. 14
IN VITRO AND IN VIVO ACTIVITIES OF CEFQUINOME
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N I OCH3
FIG. 1. Chemical structure of cefquinome (HR 111V).
Hinton medium (Difco) which had been adjusted to pH values between 5.5 and 9. Mouse protection test. NMRI mice of both sexes, weighing 18 to 22 g, were used in the experiments. The mice were kept in groups of 10 in Makrolon cages and were given grain feed (Nestlen KG, Mannheim, Federal Republic of Germany) and tap water ad libitum. The animals were infected intraperitoneally with 0.3 ml of a bacterial suspension in 5% hog gastric mucin adjusted to a multiple lethal dose. Untreated animals died within 6 to 48 h after infection, depending on the bacterial species. The mice were treated subcutaneously (s.c.) immediately and 4 h after infection. They were observed for 10 days, and a daily record of deaths was kept. The median effective doses (ED50s), i.e., the doses required to protect 50% of the animals from death, were calculated by probit analysis (7) from the number of animals surviving on day 10 after the infection. Pharmacokinetic studies. Groups of NMRI albino mice, weighing 19 1 g, were dosed s.c. with 10 and 40 mg of cefquinome per kg. At defined intervals after dosing, blood samples were obtained from a cut at the tip of the tail by using a 10-,ul capillary (wetted with sodium citrate) and kept at 4°C. Urine was collected in metabolism cages. Groups of three male beagle dogs, weighing about 22 kg each, were dosed with 5, 10, and 20 mg/kg intravenously (i.v.) via the cephalic vein. Blood samples were withdrawn from the cephalic vein in the opposite leg. Urine was collected by catheterization. Groups of five or six male and female pigs (Deutsches veredeltes Landschwein), weighing approximately 18 kg each, were injected i.v. with 10 mg of cefquinome via the venajugularis or s.c. in the base of the left ear. Blood samples were withdrawn from the contralateral jugu-
lar vein. Male and female calves weighing between 110 and 140 kg were dosed with 10 mg of cefquinome per kg i.v. via the verajugularis. Preliminary pharmacokinetic results were obtained after s.c., intramuscular, and intratracheal application. Bioassay. Blood, serum, plasma, and urine samples were bioassayed by the agar diffusion test with Mueller-Hinton agar supplemented with 10% sheep blood and seeded with Streptococcus pyogenes A77 as the assay strain. Standard solutions were prepared from pooled murine blood and urine and from autologous body fluids taken from untreated dogs, pigs, and calves. Cefquinome concentrations were calculated by regression analysis, using the standard curves in which the logarithms of the concentrations were proportional to the areas of the inhibition zones. Pharmacokinetic calculations. Curve fitting was carried out by nonlinear regression with the computer program PHAKOK. Pharmacokinetic analysis of the concentrationtime data after i.v. administration indicated that the best curve fits were usually achieved by using an open twocompartment model. Mean values and standard deviations of the pharmacokinetic parameters were calculated from individual fitted curves. RESULTS Antibacterial spectrum. The results of the susceptibility testing of numerous clinical isolates of gram-positive and gram-negative bacteria are summarized in Tables 1 and 2. The data indicate that cefquinome has high antibacterial activity in vitro against nearly all strains tested. The MICs of cefquinome (Table 1) for methicillin-susceptible and methicillin-resistant staphylococci are nearly identical to those of cefpirome, i.e., in the range of 0.195 to 50 pug/ml. Thus, the MIC50s and MIC90s of cefquinome (1.563 and 25 ,ug/ml, respectively) are slightly lower than those of cefotaxime. The MIC90 of cefquinome against streptococci of serogroups A, B, and C is 0.024 ,ug/ml and is therefore slightly lower than that of cefpirome and cefotaxime. Against enterococci the MIC90 of cefquinome was about 32 ,ug/ml, indicating that against this group of bacteria cefquinome is slightly more active than cefpirome but significantly more active than cefotaxime. All the species of the Enterobacteriaceae tested are usu-
TABLE 1. In vitro activities of cefquinome, cefpirome, and cefotaxime against gram-positive bacteria Organism (no. tested)
Staphylococcus aureus Methicillin susceptible (40)
HR 111V CPO CTX HR 111V CPO CTX
0.195-25 0.391-25 0.781-50 1.563-50 0.781-50 3.125->100
0.781 0.781 1.563 12.5 12.5 25
1.563 1.563 6.25 25 50 100
Streptococcus spp., serogroups A, B, and C (36)
HR 111V CPO CTX
'0.006-0.781 0.006-0.781 s0.006-3.125
Methicillin resistant (30)
HR 111V, Cefquinone; CPO, cefpirome; CTX, cefotaxime.
4.0 4.0 128
32 64 >128
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TABLE 2. In vitro activities of cefquinome, cefpirome, and cefotaxime against gram-negative bacteria MIC (I.g/ml) 50%0
Escherichia coli (40)
HR 111V CPO CTX
Tm) (h) Tnax
AUCO_ (mg. h/liter)
7.5 + 2.9 25.9 ± 5.1
0.38 + 0.16 0.45 ± 0.13
7.8 + 1.9 26.8 ± 7.7
a p1/2, Elimination half-life; Cmax, peak concentration of drug in serum; Tmax, time to Cmax; UR, urinary recovery.
AUCO, area under the concentration-time curve from 0 h to infinity;
ANTIMICROB. AGENTS CHEMOTHER.
LIMBERT ET AL.
TABLE 5. Pharmacokinetic parameters of cefquinome in dogs after i.v. administrationa Body wt
5 10 20
3 3 3
(kg) 21.8 21.5 21.2
+ + +
0.8 2.2 2.6
0.85 ± 0.10
26.6 ± 4.7 50.4 ± 13.7 104.6 + 5.9
0.20 + 0.06 0.24 + 0.09 0.22 ± 0.04
70.1 ± 13.5 79.3 ± 27.9 67.6 + 5.4
76.8 ± 8.7 73.8 ± 9.7 61.5 ± 6.9
1.5 + 0.2 2.6 + 0.2 1.4 ± 0.5
0.98 ± 0.28 0.96 ± 0.08
AUCO_x, area under the concenration-time curve from 0 h to infinity; V, volume of distribution at steady state; CL,0t, total body
a tj/2, Elimination half-life; clearance; UR, urinary recovery.
susceptible to cefquinome and the other cephalosporins tested. Binding to the PBPs. Figure 2 shows an autoradiogram exhibiting PBP patterns obtained by various dilutions of cefquinome. This compound, as all other aminothiazolyl cephalosporins, binds preferentially to PBP 3 of E. coli K-12. Significant binding was found at concentrations as low as 0.1 ,ug/ml. A concentration of 1 p,g/ml results in a further binding to PBP lA/B. 1-Lactamase stability. The susceptibilities of cefquinome, cefpirome, and cefotaxime to a number of different types of 1-lactamases from gram-negative bacteria were tested. By using the susceptibility of cephaloridine as the standard, relative rates of hydrolysis (RRH) were calculated. The data clearly indicate that cefquinome, with one exception (OXA-1, RRH = 100), is highly resistant to hydrolysis (RRH < 2) by plasmid-encoded (TEM-1, TEM-2, OXA-2, OXA-3, SHV-1, PSE-1, PSE-2, PSE-3, PSE-4) 3-lactamases from E. coli, Klebsiella pneumoniae, or P. aeruginosa as well as chromosomally encoded 13-lactamases from Citrobacter spp., Enterobacter cloacae and K. oxytoca. The RRHs of cefpirome are greater than 2 with the plasmid-encoded P-lactamases of the types OXA-1 (RRH, 99), PSE-2 (RRH, 20), and PSE-3 (RRH, 3) and from the chromosomally encoded enzyme from K. oxytoca (RRH, 3). Cefotaxime was hydrolyzed by 3-lactamases OXA-1 (RRH, 32), OXA-2 (RRH, 7), PSE-2 (RRH, 7), and PSE-3 (RRH, 6) and the enzymes from Citrobacter spp. (RRH, 8) and K. oxytoca (RRH, 6). These results show that cefquinome is more stable than cefpirome or cefotaxime against P-lactam-
Effect of medium composition and pH on in vitro activity. The MICs of cefquinome for S. aureus SG 511 and E. coli 1507E in nine commercial culture media (Mueller-Hinton medium, ABM III, standard I, heart infusion medium, brain heart infusion medium, standard bouillon, 1% Tryptone, Iso-Sensitest, and nutrient broth 2) were determined. The MICs of the compound for S. aureus SG 511 and E. coli 1507E varied only by one dilution step (0.313 to 0.625 and 0.062 to 0.125 ,ug/ml, respectively). Thus, the antibacterial activity of cefquinome does not depend upon the composition of the culture medium. The antibacterial activity of cefquinome for the same test organisms does not vary
Dose (mg/kg) and route
10 s.c. 10 i.v.
markedly in Mueller-Hinton medium adjusted to pH values ranging from 5.5 to 9.0. In vivo efficacy in mouse septicemia. The high in vitro activity of cefquinome (Table 3) against the pathogens involved was reflected by excellent in vivo activity against the septicemias induced in mice by these strains. Cefquinome has good in vivo activity against S. aureus, which is not typical for most other broad-spectrum cephalosporins including cefotaxime. Table 3 clearly demonstrates that cefquinome is superior to cefotaxime, cefoperazone, and cefuroxime as regards protection against the experimental S. aureus Giorgio and SG 511 infections in mice. The ED50s of cefquinome for these strains are 0.96 and 1.6 mg/kg, respectively, which is 6 to 12 times more active than the reference compounds. The activity demonstrated by cefquinome against infections due to Streptococcus pyogenes A77 was also higher than that of cefotaxime, cefoperazone, and cefuroxime. The Streptococcus pneumoniae infection also responded well to cefquinome. The ED50 (0.17 mg/kg) of cefquinome was 2 to 15 times lower than those of the reference compounds. In addition, Table 3 shows the chemotherapeutic efficacies of cefquinome and the three reference compounds in the treatment of infections caused by gram-negative bacteria. Cefquinome showed similar activity to cefotaxime in E. coli 078 and Salmonella typhimurium MZ II infections, with ED50s of 0.04 and 0.07 mg/kg, respectively. Cefoperazone and, in particular, cefuroxime, however, were distinctly less active against the infections caused by these strains. Similar results were obtained for septicemias induced by K. pneumoniae DT-S or Proteus mirabilis ATCC 14273. The ED50s of cefquinome for these bacteria were calculated to be 0.39 and 0.88 mg/kg, respectively. Only in the infection due to Pasteurella multocida was cefquinome somewhat less active than cefotaxime and cefoperazone. Pharmacokinetics. The binding of cefquinome to mouse, dog, horse, and calf serum proteins, as measured by a dialysis method, was less than 10% (data not shown). Pharmacokinetic parameters in mice, dogs, pigs, and calves after single parenteral doses of cefquinome are presented in Tables 4 to 7, respectively. Cefquinome was rapidly distributed after parenteral administration and showed high and sustained levels in blood in the species tested. The mean
TABLE 6. Pharmacokinetic parameters of cefquinome in pigs after s.c. and i.v. administration' Vss AUC>:c Cmax Body wt T (h) t (h) max 1/2 (liters/kg) (mg h/liter) (jj.g/ml) (kg)
17.9 + 0.7 18.2 ± 0.5
1.23 + 0.28 1.32 ± 0.18
26.1 ± 6.4
0.80 ± 0.16
62.3 ± 17.4 66.7 ± 27.2
0.24 ± 0.11
(ml/min) 48.5 ± 16.6
tl/2, Elimination half-life; C,.,,,, peak concentration of drug in serum; Tmax, time to Cmax; AUC-,,_, area under the concentration-time curve from 0 h to infinity;
V.S volume of distribution at steady state; CL,01, total body clearance.
IN VITRO AND IN VIVO ACTIVITIES OF CEFQUINOME
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TABLE 7. Pharmacokinetic parameters of cefquinome in calvesa Body wt (kg)
Dose (mg/kg) and route
10 i.v. 10 s.c.
10 i.m. 10 i.t.
118.5 ± 8.9 138.5 137.0 133.0
h T max (h)
AUC>x (mg. h/liter)
3.6 4.5 4.2
1.8 2.0 1.8
84.7 + 25.3 19.4 24.0 20.8
1.33 ± 0.41
a i.m., Intramuscular, i.t., intratracheal; t1/2, elimination half-life; Cmax, peak concentration of drug in serum Tmax, time to concentration-time curve from 0 h to x; V, volume of distribution at steady state; CL,0,, total body clearance.
elimination half-lives in dogs (Table 5), pigs (Table 6), and calves (Table 7) after i.v. administration of 10 mg/kg were 0.98, 1.32, and 1.33 h, respectively. The correlation between the doses and the total areas under the concentration-time curves in dogs (Table 5) proved to be linear. Preliminary results in calves indicate little difference between the serum profiles after s.c., intramuscular, or intratracheal administration of cefquinome (Table 7). The calculated volume of distribution at steady state was very similar in all species tested, corresponding to about 25% of the body volume. Cefquinome was excreted mainly via the kidneys. In mice (Table 4) and in dogs (Table 5) about 80% and 63 to 78%, respectively, of the dose was recovered in the urine as antibacterially active substance within 24 h of administration. DISCUSSION
The introduction of a methoxyimino-aminothiazolyl moiety in the acyl side chain of cephalosporins brought about significant enhancement of activity, extension of the antibacterial spectrum-especially against gram-negative bacteria-and high resistance to inactivation by P-lactamases (3, 9). The introduction of polar C-3'-substituents into a cephem nucleus improves the activity of the aminothiazolyl cephalosporins, especially against staphylococci and P. aeruginosa, without loss of activity against members of the Enterobacteriaceae. Cefquinome is a representative of this group of cephalosporins, as are cefpirome (5) and cefepime (2). The results of our investigations proved that cefquinome has a broad spectrum of activity. The spectrum includes staphylococci, streptococci, and many strains of methicillin-resistant staphylococci and enterococci. The most important members of the Enterobacteriaceae (E. coli, Klebsiella spp., Salmonella spp., Citrobacter spp., Enterobacter spp., and Proteus spp.) are inhibited at low concentrations. Most strains of P. aeruginosa are also susceptible to cefquinome. Owing to its resistance to various plasmid-encoded or chromosomally encoded P-lactamases, cefquinome is also active against bacteria that are resistant to earlier cephalosporins. Its action against gramnegative strict anaerobic bacteria (e.g., Bacteroidesfragilis) is limited, as is that of cefpirome and cefepime (2, 4). The in vitro activity of cefquinome, similarly to those of other cephalosporins, does not depend on the composition or pH of the test medium. The broad antibacterial spectrum and the high in vitro activity of cefquinome are reflected by high in vivo efficacy in experimental infections. In mouse models of septicemia induced by both gram-positive and gram-negative bacteria, cefquinome possessed high therapeutic efficacy. All infections were cured by the administration of cefquinome. The ED50s were usually below 1 mg/kg. The pharmacokinetic properties of cefquinome make a
261 ± 128
area under the
major contribution to its high in vivo activity. After parenteral administration to mice, dogs, pigs, or calves, high and sustained levels were measured in blood. The elimination half-lives are comparable to those of other polar cephalosporins. Cefquinome is excreted mainly by the kidneys: within 24 h of administration, 60 and 80% of the administered dose was recovered in the urine of mice and dogs, respectively. In conclusion, cefquinome is a new representative of the polar aminothiazolyl cephalosporins. With its broad antibacterial spectrum and favorable pharmacokinetics, it merits further investigation into its chemotherapeutic properties. REFERENCES 1. Caprile, K. A. 1988. The cephalosporin antimicrobial agents: a comprehensive review. J. Vet. Pharmacol. Ther. 11:1-32. 2. Clarke, A. M., S. J. V. Zemcov, and I. M. Wright. 1985. HR 810 and BMY-28142, two new cephalosporins with broad-spectrum activities: in vitro comparison with other P-lactam antibiotics. J. Antimicrob. Chemother. 15:305-310. 3. Durckheimer, W., F. Adam, G. Fischer, and R. Kirrstetter. 1988. Recent developments in the field of cephem antibiotics. Adv. Drug Res. 17:61-234. 4. Jones, R. N., and E. H. Gerlach. 1985. Antimicrobial activity of HR 810 against 419 strict anaerobic bacteria. Antimicrob. Agents Chemother. 27:413-415. 5. Jones, R. N., C. Thornsberry, and A. L. Barry. 1984. In vitro evaluation of HR 810, a new wide-spectrum aminothiazolyl a-methoximino cephalosporin. Antimicrob. Agents Chemother.
25:710-718. 6. Klesel, N., M. Limbert, E. Schrinner, K. Seeger, G. Seibert, and I. Winkler. 1984. Chemotherapeutic properties of the new cephalosporin antibiotic HR 810 in laboratory animals. Infection 12:286-292. 7. Litchfield, J. T., and F. Wilcoxon. 1949. A simple method of evaluating dose effect experiments. J. Pharmacol. Exp. Ther. 96:99-113. 8. Neu, H. C. 1982. Clinical use of cephalosporins. Lancet ii:252255. 9. Neu, H. C. 1983. Structure-activity relation of new beta-lactam compounds and in vitro activity against common bacteria. Rev.
Infect. Dis. 5(Suppl. 2):319-336. 10. Ross, G. W., and C. H. O'Callaghan. 1975. ,-Lactamase assay. Methods Enzymol. 43:69-85. 11. Schrinner, E., M. Limbert, L. Penasse, and A. Lutz. 1980. Antibacterial activity of cefotaxime and other newer cephalosporins (in vitro and in vivo). J. Antimicrob. Chemother.
6(Suppl. A):13-17. 12. Schwarz, U., K. Seeger, F. Wengenmayer, and H. Strecker. 1981. Penicillin-binding proteins of Escherichia coli identified with a 1241-derivative of ampicillin. FEMS Microbiol. Lett. 10:107-109. 13. Seibert, G., N. Klesel, M. Limbert, E. Schrinner, K. Seeger, I. Winkler, R. Lattrell, J. Blumbach, W. Durckheimer, K. Fleischmann, R. Kirrstetter, B. Mencke, B. C. Ross, K.-H. Scheunemann, W. Schwab, and M. Wieduwilt. 1983. HR 810, a new parenteral cephalosporin with broad antibacterial spectrum. Arzneim. Forsch. 33:1084-1086.