a Department of Clinical Pharmacology, Alfred Hospital, Prahran, 3181 b Department of Pharmacy, Alfred Hospital, Prahran, 3181 c Department of Microbiology and Infectious Diseases, Alfred Hospital, Prahran, 3181 d Victorian Centre of Ambulatory Care Innovation, Alfred Hospital, Prahran, 3181 e Department of Biochemistry, St Vincent's Hospital, Melbourne , 3065 f University of Sydney Department of Medicine and Department of Geriatric Medicine, The Canberra Hospital, Garran, ACT 2605, Australia
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Abstract |
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Introduction |
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The aim of this study was to model clinical cephazolin concentrationtime profiles in vitro as a benchmark of exposure, then to systematically re-examine the individual influence of AUC, peak and maintained antibiotic concentration, ratio of concentration(s) to MIC, and duration of exposure on the action of cephalosporins.
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Materials and methods |
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Escherichia coli NCTC 10418, with an MIC of cephazolin of 1 mg/L, were cultured under standard conditions in the presence and absence of standardized concentrationtime profiles of cephazolin (Eli Lilly, West Ryde, NSW, Australia). An overnight culture of E. coli in Brain-Heart Infusion Broth (BHIB) (Oxoid, Basingstoke, UK) was diluted to 107 cfu/mL in 0.1% peptone water (Difco Laboratories, Detroit, MI, USA) and a 1 mL sample of the 107 cfu/mL culture was added to the experimental culture broth resulting in an initial density of 106 cfu/mL.19 Viable counts were determined from colony formation on the surface of nutrient plates (Oxoid). The lower limit of detection was 20 cfu/mL.
Modelling of 1 g and 2 g im cephazolin post-injection profiles
In-vitro modelling of clinical exposures following 1 g im dosing20 involved determination of the AUC from published data using the trapezoidal rule,21 followed by design of an equal AUC profile approximating the clinical profile (Figure 1 (i)a). Cephazolin in BHIB was added in four increments over 30 min to produce a peak of 60 mg/L (Figure 1 (i)a). After 2 h, a 1:1.5 dilution was made to reduce the concentration to 40 mg/L, then cephazolin concentrations were halved every 2 h until 10.5 h. The concentrations were halved again at 24 h (Figure 1 (i)a). Samples for the determination of viable cell counts were taken at 0 and 0.5 h then two hourly for 10.5 h and at 24 and 48 h. For the 2 g im dose profile, all drug concentrations were doubled assuming linear extrapolation of data (Figure 1 (i)b).
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In studies of time and concentration dependence, the cephazolin AUC was kept constant while time and concentration were varied. Maintaining a constant AUC of 288 mg/L (the AUC associated with a 1g im injection) cephazolin concentrations of 288, 48, 24, 12 and 6 mg/L were kept for 1, 6, 12, 24 and 48 h, respectively, after which time the drug was washed out by centrifuging the culture twice for 12 min at 3.2 x 103 rpm. The E. coli pellet was resuspended in drug-free BHIB after each centrifugation and incubated at 37°C for the remainder of the experiment. To determine viable cell counts, samples were taken at 0, 1, 2, 4, 6, 8, 10, 12, 24 and 48 h post-dose.
The AUC studies related to the 2 g im profile (576 mg·h/L) involved double the concentrations of the above profiles.
Time and concentration dependence of maintained concentration exposures
To investigate time dependence and concentration dependence of cephazolin activity, bacteria were exposed to maintained cephazolin concentrations of 6, 12 and 24 mg/L for 48 h on the basis of results from the AUC experiments. Samples for determination of viable cell counts were taken at 0, 1, 2, 4, 6, 8, 10, 24 and 48 h post-dose.
Cephazolin stability studies
The stability of cephazolin in the culture medium was studied by addition of cephazolin as cefazolin sodium powder (Lot No. A7080A; Eli Lilly, Indianapolis, IN, USA) to broth at concentrations across the range 624 mg/L with incubation at 37°C for 72 h. Samples were taken at 0, 0.5, 1.0, 1.5, 2.0, 4.0 6.0, 8.0, 10.0 24.0, 48.0 and 72.0 h. Samples were immediately frozen at 20°C until assay, and assays were performed within 20 min of thawing each sample.
Cephazolin determinations were performed using a HPLC system (Waters Associates, Milford, MA, USA), consisting of a Model 501 Pump and a Model 440 UV absorbance detector at 254 nm with an Omniscribe chart recorder (Houston Instruments, TX, USA). Cephazolin samples (20 µL) were injected directly on to the column and chromatographed on a Waters C-18 10 µm radial-pak column with a mobile phase of 18% methanol, 82% 0.03 M phosphate buffer at a flow rate of 2.0 mL/min. The assay was linear over the range 225 mg/L, with an intra-assay CV of 10.78 % at 2 mg/L and 2.1% at 24 mg/L.
The stability of the cephalosporin was high with no significant between-sample change in concentration detectable over a 72 h period at concentrations of 6 and 12 mg/L. Among the 24 mg/L replicates there was a small (<13%) but significant difference between the 24 h and the 48 h and 72 h samples; however, the 48 and 72 h samples did not differ from each other.
Determination of pharmacokinetic parameters and statistical analysis
The AUC was calculated using the trapezoidal rule21 and a one-way Tukey ANOVA (Minitab Release 8, Minitab Inc, PA, USA) was used for statistical analysis using a significance level of 0.05.
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Results |
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Control studies of E. coli growth from seeding concentrations of approximately 106 cfu/mL showed a plateau at or near 109 cfu/mL (Figure 1 (ii)a,b). Exposure to concentration modelling 1 g and 2 g im dosing with cephazolin caused initial bactericidal effects from 0.5 h. Live colony counts fell from 106 to 103 within 30 min, and continued to decline until 10.5 h (Figure 1). Early (2.5 h) bactericidal action was concentration-dependent (Table). There were no detectable live colonies from plated subcultures at 8.5 h for the 2 g profile and at 10.5 h with both 1 g and 2 g exposure modelling. This was followed by a variable recovery process measured at 24 h and 48 h (Figure 1 (ii)a,b). There were individual between-dose differences in subculture colony count means at 24 and 48 h. However, there was considerable variation in response and no systematic differences between the sustained effects of 1 g and 2 g profiles (Table).
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The dependence of cephalosporin effect on concentration and time was investigated by exposures designed to reflect the opposite extremes of maximum concentration exposure and maximum time exposure. Exposure to cephazolin for 1 h caused a concentration-dependent effect (P < 0.05; Figure 2a,b). With maintained concentrations of cephazolin, bacterial populations continued to decline steadily to levels at or near 102 cfu by 8 h. After 12 h variable regrowth resumed (Figure 2). Continuing 24 h exposure to 6, 12 and 24 mg/L cephazolin failed to prevent resumption of growth (Figure 2c).
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E. coli growth after maintained concentration exposure
Exposure to cephazolin, maintained at concentrations of 6, 12 and 24 mg/L, caused a sustained decrease in cfu over the first 6 h (Figure 2c). Cultures exposed to 12 and 24 mg/L declined until 8 and 10 h, respectively, with variable recovery to 48 h (Figure 2c).
There was clear-cut concentration dependence of initial (1 h) exposure over these exposure ranges (P < 0.05). However, the trend to systematic continuing differences did not reach statistical significance.
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Discussion |
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Upon exposure to constant AUC profiles, an initial (1 h) concentration-dependent bactericidal effect was observed with all cephazolin profiles. Such concentration dependence of the initial antibacterial activity of cephalosporins is in agreement with the conclusions of several studies.3,4,11,22 Maintained concentrations were most efficacious for continued suppression of bacterial growth over 24 h in these AUC profile studies.
Where cephazolin concentrations of 6, 12 and 24 mg/L were maintained for 48 h, there were initial (1 h) concentration-dependent differences between the results of these three concentrations. While there was a trend to earlier recovery of bacterial growth in cultures exposed to lower concentrations, these differences were not statistically significant.
The most effective cephazolin exposure profile was that based on the im injection profile, hence, further analysis of the exposure parameters of the im injection profile is needed in order to identify the major determinants of bactericidal efficacy of cephalosporins beyond concentration, AUC and time of exposure.
Our results showing regrowth of bacteria despite concentrations maintained well above MIC indicate that the design of cephalosporin dosage schedules based principally upon the maintenance of concentrations above the MIC15,23 may not be adequate. The current general clinical practice of intermittent dosing should be maintained in the interim. If the responses to the im dose profiles presented here represent empirical evidence of an additive effect between bactericidal responses to dynamically variable initial concentrations and maintained concentrations, then this principle may represent a basis for improving the efficacy of cephalosporins. Equally the results from the im profiles could provide a basis for allowing the extension of interdose intervals with cephazolin and other cephalosporins beyond 8 h to 12 h.24
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Acknowledgments |
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Notes |
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References |
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2 . Hyatt, J. M., McKinnon, P. S., Zimmer, G. S. & Schentag, J. J. (1995). The importance of pharmacokinetic/pharmacodynamic surrogate markers to outcome. Focus on antibacterial agents. Clinical Pharmacokinetics 28, 14360.[ISI][Medline]
3 . Shah, P. M., Junghanns, W. & Stille, W. (1976). Dosis-Wirkungs-Beziehung der bacterizidie bei E. coli, K. pneumoniae and Staphylococcus aureus. Deutsche Medizinische Wochenschrift 101, 3258.[ISI][Medline]
4 . Nishino, T. & Nakazawa, S. (1976). Bacteriological study on effects of ß-lactam group antibiotics in high concentrations. Antimicrobial Agents and Chemotherapy 9, 103342.[ISI][Medline]
5 . Nishi, T., Nakao, M. & Tsuchiya, K. (1981). Relevance of in vitro antibacterial activities of cefotiam and cefazolin to their therapeutic effects on experimental pneumonia caused by Klebsiella pneumoniae DT-S in mice. Journal of Antibiotics 34, 2319.[ISI][Medline]
6 . Hanberger, H., Nilsson, L. E., Kihlstrom, E. & Maller, R. (1990). Postantibiotic effect of ß-lactam antibiotics on Escherichia coli evaluated by bioluminescence assay of bacterial ATP. Antimicrobial Agents and Chemotherapy 34, 1026.[ISI][Medline]
7 . van Ogtrop, M. L., Mattie, H., Guiot, H. F., van Strijen, E., Hazekamp-van Dokkum, A.-M. & van Furth, R. (1990). Comparative study of the effects of four cephalosporins against Escherichia coli in vitro and in vivo. Antimicrobial Agents and Chemotherapy 34, 19327.[ISI][Medline]
8 . Tauber, M. G., Doroshow, C. A., Hackbarth, C. J., Rusnak, M. G., Drake, T. A. & Sande, M. A. (1984). Antibacterial activity of ß-lactam antibiotics in experimental meningitis due to Streptococcus pneumoniae. Journal of Infectious Diseases 149, 56874[ISI][Medline]
9 . Grasso, S., Meinardi, G., De Carneri, I. & Tamassia, V. (1978). New in vitro model to study the effect of antibiotic concentration and rate of elimination on antibacterial activity. Antimicrobial Agents and Chemotherapy 13, 5706.[ISI][Medline]
10 . Shah, P. M., Troche, G. & Stille, W. (1979). Effect of concentration on bactericidal activity of cefotaxime. Journal of Antimicrobial Chemotherapy 5, 41922.[ISI][Medline]
11 . Craig, W. A. & Ebert, S. C. (1992). Continuous infusion of ß-lactam antibiotics. Antimicrobial Agents and Chemotherapy 36, 257783[ISI][Medline]
12 . Vogelman, B., Gudmundsson, S., Leggett, J., Turnidge, J., Ebert S. & Craig. W. A. (1988). Correction of antimicrobial pharmacokinetic parameters with therapeutic efficacy in an animal model. Journal of Infectious Diseases 158, 83147.[ISI][Medline]
13 . Roosendaal, R., Bakker-Woudenberg, I. A., van den Berg, J. C. & Michel, M. F. (1985). Therapeutic efficacy of continuous versus intermittent administration of ceftazidime in an experimental Klebsiella pneumoniae pneumonia in rats. Journal of Infectious Diseases 152, 3738.[ISI][Medline]
14 . Lavoie, G. Y. & Bergeron, M. G. (1985). Influence of four modes of administration on penetration of aztreonam, cefuroxime and ampicillin into interstitial fluid and fibrin clots and on in vivo efficacy against Haemophilus influenzae. Antimicrobial Agents and Chemotherapy 28, 40412.[ISI][Medline]
15 . Drusano, G. L. (1988). Role of pharmacokinetics in the outcome of infections. Antimicrobial Agents and Chemotherapy 32, 28997.[ISI][Medline]
16 . Leggett, J. E., Ebert, S., Fantin, B. & Craig, W. A. (1990). Comparative dose-effect relations at several dosing intervals for ß-lactam, aminoglycoside and quinolone antibiotics against Gram-negative bacilli in murine thigh-infection and pneumonitis models. Scandinavian Journal of Infectious Diseases, Supplementum 74, 17984.
17 . Craig, W. (1993). Pharmacodynamics of antimicrobial agents as a basis for determining dosage regimens. European Journal of Clinical Microbiology and Infectious Diseases 12, Suppl. 1, 68.[ISI]
18 . Pangon, B., Joly, V., Vallois, J., Abel, L., Bure, A., Brion, N. et al. (1987). Comparative efficacy of cefotiam, cefmenoxime, and ceftriaxone in experimental endocarditis and correlation with pharmacokinetics and in vitro efficacy. Antimicrobial Agents and Chemotherapy 31, 51822.[ISI][Medline]
19 . Bastone, E. B., Li, S. C., Ioannides-Demos, L. L., Spicer, W. J. & McLean, A. J. (1993). Kill kinetics and regrowth patterns of Escherichia coli exposed to gentamicin concentrationtime profiles stimulating in vivo bolus or infusion dosing. Antimicrobial Agents and Chemotherapy 37 , 9147.[Abstract]
20 . Cahn, M. M., Levy, E. J., Actor, P. & Pauls, J. F. (1974). Comparative serum levels and urinary recovery of cefazolin, cephaloridine, and cephalothin in man. Journal of Clinical Pharmacology 14, 616.[ISI][Medline]
21 . Gibaldi, M. & Perrier, D. (1982). Pharmacokinetics. 2nd Edn, Marcel Dekker Inc., New York., 4459.
22 . Goto, S., Sakamoto, H., Ogawa, M., Tsuji, A. & Kuwahara, S. (1982). Bacericidal activity of cefazolin, cefoxitin, and cefmetazole against Escherichia coli and Klebsiella pneumoniae. Chemotherapy 28, 1825.[ISI][Medline]
23 . Drusano, G. L. (1990). Human pharmacodynamics of ß-lactams, aminoglycosides and their combination. Scandinavian Journal of Infectious Diseases, Supplementum 74, 23548.
24 . Leder, K., Turnidge, J. D. & Grayson, M. L. (1998). Home-based treatment of cellulitis with twice-daily cefazolin. The Medical Journal of Australia 169, 51922.[ISI][Medline]
Received 22 December 1998; returned 23 February 1999; revised 13 April 1999; accepted 11 May 1999