Free and total cefazolin plasma and interstitial fluid concentrations at steady state during continuous infusion

Grant W. Howard1, Evan J. Begg1, Stephen T. Chambers2, Jane Vella Brincat1,*, Mei Zhang1 and Carl M. J. Kirkpatrick1

Departments of 1 Clinical Pharmacology and 2 Infectious Diseases, Christchurch Hospital, PO Box 4710, Christchurch, New Zealand

Received 7 December 2001; returned 13 March 2002; revised 15 May 2002; accepted 25 May 2002


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Free and total concentrations of cefazolin were compared in plasma and interstitial fluid during continuous intravenous infusion therapy. Seven patients, median age 53 (25–74) years, were administered a constant infusion of cefazolin at a mean (±S.D.) dose of 3.5 g (±1.1) per 24 h for >=5 days. Four blisters were induced on the forearm of each patient for sampling of interstitial fluid. Free concentrations in plasma and interstitial fluid were similar, and correlated better than total concentrations (r2 = 0.82, P = 0.005 versus r2 = 0.54, P = 0.056). In all patients, the free concentrations in the interstitial fluid were at least two-fold the MIC90 for Staphylococcus aureus.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cefazolin is widely used for the treatment of Gram-positive infections including cellulitis.1 This cephalosporin has been used as part of home-based intravenous antibiotic programmes because it is well tolerated, stable and has a relatively narrow spectrum.2 The use of a first generation cephalosporin is considered desirable in this setting to reduce antibiotic pressure and help restrict the emergence of resistance.

Cefazolin has a volume of distribution of ~10 L and is 75–85% plasma bound.3 The total body clearance is ~3.6 L/h in patients with normal renal function with ~90% of the dose eliminated unchanged via the kidneys. The reported mean elimination half-life is ~2 h (range 1.5–2.5 h).3 Cefazolin, therefore, needs to be administered at least twice daily by intravenous bolus or by a constant infusion over 24 h to achieve therapeutic concentrations.

A constant infusion may be the most appropriate method of delivering ß-lactam antibiotics, as the major determinant of ß-lactam antibiotic efficacy is widely considered to be the time above the MIC at the site of infection.4 With constant infusions at steady state, and assuming the principles of diffusion, it would be expected that concentrations of free drug (not protein bound) should be similar in the plasma and the interstitial fluid.4 If this can be demonstrated, then free drug concentrations in plasma can easily be measured to check the adequacy of dosing regimens.

The aim of this study was to measure free and total cefazolin concentrations in both plasma and interstitial fluid under steady state conditions using a blister model during continuous infusion.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient selection and drug administration

Seven consecutive patients were recruited from those who had been enrolled in the home antibiotic programme for the treatment of uncomplicated cellulitis. All patients received cefazolin (Eli Lilly, Auckland, New Zealand) at a fixed dose by continuous intravenous infusion for at least 5 days before recruitment to ensure steady state conditions. The usual starting dose was 3 g per 24 h adjusted at the discretion of the attending physician for body size, severity of infection and renal function. Cefazolin was administered via a Homepump ECLIPSE C-Series (5 mL/h) (I-flow Corporation, Lakefront, CA, USA) through a peripherally inserted catheter (PIC line) (Arrow International Inc., Reading, PA, USA). Patients were excluded if their calculated creatinine clearance by the Cockcroft & Gault equation5 was outside the normal range for their age, weight and gender.

Interstitial fluid and blood/plasma sampling

Interstitial fluid (0.2 mL per patient) was harvested from four skin blisters induced on the forearm of each patient using the technique first described by Schreiner et al.4 Blood (5 mL) was also taken at the beginning and end of the period of blister formation to provide comparative protein free and total drug concentrations in plasma. Samples were stored at –30°C prior to analysis.

Processing of samples

Total and free cefazolin concentrations in plasma and interstitial fluid were analysed by the HPLC method of Kamani et al.,6 with minor modifications. Free drug concentrations and degree of protein binding were determined by ultrafiltration (2600g for 30 min at 37°C) using a Diaflo ultrafiltration membrane, YMT DISCS, 30K NMWL, 14 mm (Amicon Inc., Beverly, MA, USA), for 30 min. The filtrate (50 µL) was injected into the Kontron HPLC system and chromatography performed using an AQUA C18 5 µm 75 x 4.6 mm, ID column (Phenomenex, Torrance, CA, USA). The mobile phase was a mixture of 0.01 M phosphate buffer pH 6.5 and acetonitrile [90:10 (v/v)] at a flow rate of 1 mL/min. This provided a retention time for cefazolin of ~8 min.

Statistical analysis

Descriptive statistics and linear regression analysis were undertaken using GraphPad Prism, Version 3.0 (GraphPad Software, San Diego, CA, USA).

Ethics committee approval

The study was reviewed and approved by the regional ethics committee. Patients were informed about all aspects of the study, including possible de-pigmentation of the skin areas used for blister formation, and gave written consent.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Demographics

Five males and two females were recruited into the study: median age 53 years (range 25–74), median weight 90 kg (range 71–170), mean creatinine clearance (±S.D.) 1.9 mL/s (±0.9), mean daily dose (±S.D.) 3.5 g (±1.1), and mean dose per kg per day (±S.D.) 36 mg/kg/day (±6.1). The patients received no medications that were known to interfere with the clearance or protein binding of cefazolin.

Assay

Standard curves for total and free cefazolin were linear (r2 > 0.99) over the concentration ranges 1.5–200 and 0.04–20 mg/L, respectively. The limits of quantification for total and free cefazolin were 1.5 and 0.04 mg/L, respectively. The absolute recoveries of total cefazolin concentrations from plasma and interstitial fluid were >95%, whereas recoveries during ultrafiltration at concentrations of 0.4, 4.0 and 16 mg/L were ~90%. Intra- and inter-day coefficients of variation (CV%) of assay precision for total cefazolin were <7 and <5.5%, respectively (concentration range 4.0–160 mg/L). For free cefazolin the intra- and inter-day CV% were <1 and <2%, respectively (concentration range 0.4–16 mg/L). Cefazolin was found to be stable in plasma and interstitial fluid at –30°C for at least 4 weeks.

Cefazolin concentrations

The free and total plasma and interstitial fluid concentrations of cefazolin are presented in Table 1 and Figure 1. The total and free plasma concentrations from samples taken at the beginning and end of blister formation were not significantly different (paired t-test, P = 0.88 and 0.38, respectively). Total concentrations (mean ± S.D.) in plasma were higher than those in interstitial fluid in six of seven patients and lower in one (32 ± 17 versus 17.4 ± 8.3 mg/L). Linear regression analysis of the total concentrations in the plasma and interstitial fluid revealed a squared correlation coefficient (r2) of 0.55 (P = 0.056), with similar results observed for the free concentrations. In five of the seven patients free interstitial concentrations were slightly lower than in plasma, and in the other two patients these were equal (mean ratio = 0.84, 95% CI = 0.696–0.998). Linear regression analysis showed a strong correlation between the free concentrations in the plasma and the interstitial fluid [r2 of 0.82 (P = 0.005)].


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Table 1.  Free and total cefazolin concentrations in plasma and interstitial fluid
 


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Figure 1. Free and total cefazolin concentrations in plasma and interstitial fluid.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have shown that at steady state during constant infusion of cefazolin the free drug concentration in interstitial fluid approximates to that of the free drug concentration in plasma. This supports the postulate of Barza & Cuchural,7 based on pharmacokinetic modelling, that penetration into interstitial fluid results from diffusion. Several other methods have been used to measure drug concentrations of antibiotics in peripheral sites.8,9. These have produced disparate results, possibly because they did not measure free concentrations directly, but made estimates of these.

Unlike other cephalosporins, cefazolin demonstrates saturable protein binding in the range of concentrations encountered in clinical practice,10 creating problems in studies where antibiotic concentrations vary over the period of investigation. In contrast, saturable protein binding should not influence the free drug concentrations in either compartment at steady state during continuous infusion, as the only determinants of these are the drug dose administered and free drug clearance.

Our studies have confirmed the results of Waterman et al.,11 who showed similar results using bolus dosing in an animal model. Furthermore, we have confirmed that with a constant infusion, at steady state, free concentrations in plasma reflect interstitial fluid free concentrations, and therefore can be compared with the MICs of infecting organisms. For ß-lactam antibiotics clinical efficacy is improved when concentrations exceed the minimum required to inhibit bacterial proliferation (MIC) for the majority (>50%) of the dosing interval.12 It was, therefore, reassuring that the lowest free concentration in the interstitial fluid was 2 mg/L, as this exceeded the MIC90 for the two most likely organisms, Staphylococcus aureus (1 mg/L) and Streptococcus pyogenes (0.1 mg/L),13 suggesting that the dose regimen used was appropriate.

Further studies need to be undertaken with regard to the time taken to achieve steady state conditions, with or without loading doses, to elucidate the distribution kinetics of cefazolin given by constant infusion. Nevertheless, this study provides an important step in validating home intravenous antibiotic programmes, particularly those based on constant infusion techniques.


    Footnotes
 
* Corresponding author. Tel: +64-3-3640640; Fax: +64-3-3641003; E-mail: jane.vella-brincat{at}cdhb.govt.nz Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Steckelberg, J. M., Rouse, M. S., Tallan, B. M., Osmon, D. R., Henry, N. K. & Wilson, W. R. (1993). Relative efficacies of broad-spectrum cephalosporins for treatment of methicillin-susceptible Staphylococcus aureus experimental infective endocarditis. Antimicrobial Agents and Chemotherapy 37, 554–8.[Abstract]

2 . Grayson, M. L., Silvers, J. & Turnidge, J. (1995). Home intravenous antibiotic therapy. A safe and effective alternative to inpatient care. Medical Journal of Australia 162, 249–53.[ISI][Medline]

3 . Dollery, C. (1999) Therapeutic Drugs. Churchill Livingstone, London, UK.

4 . Schreiner, A., Bergan, T., Hellum, K. B. & Digranes, A. (1981). Pharmacokinetics of ampicillin in serum and in dermal suction blisters after oral administration of bacampicillin. Reviews of Infectious Diseases 3, 125–31.[ISI][Medline]

5 . Cockcroft, D. W. & Gault, H. M. H. (1976). Prediction of creatinine clearance from serum creatinine. Nephron 16, 31–41.[ISI][Medline]

6 . Kamani, C., Low, C. L., Valerie, T. T. H. & Chui, W. K. (1998). HPLC determination of cefazolin in plasma, urine and dialysis fluid. Journal of Pharmacy and Pharmacology 50, 118.

7 . Barza, M. & Cuchural, G. (1985). General principles of antibiotic tissue penetration. Journal of Antimicrobial Chemotherapy 15, 59–75.[ISI][Medline]

8 . Vondracek, T. G. (1995). Beta-lactam antibiotics: is continuous infusion the preferred method of administration? Annals of Pharmacotherapy 29, 415–24.[Abstract]

9 . Peterson, L. R., Gerding, D. N. & Fasching, C. E. (1981). Effects of method of antibiotic administration on extravascular penetration: cross-over study of cefazolin given by intermittent injection or constant infusion. Journal of Antimicrobial Chemotherapy 7, 71–9.[ISI][Medline]

10 . Ryan, D. M. (1993). Pharmacokinetics of antibiotics in natural and experimental superficial compartments in animals and humans. Journal of Antimicrobial Chemotherapy 31, 1–16.[ISI][Medline]

11 . Waterman, N. G., Raff, M. J., Scharfenberger, L. & Barnwell, P. A. (1976). Protein binding and concentrations of cephaloridine and cefazolin in serum and interstitial fluid of dogs. Journal of Infectious Diseases 133, 642–7.[ISI][Medline]

12 . Craig, W. A. (2001). Does the dose matter? Clinical Infectious Diseases 33, S233–7.[ISI][Medline]

13 . Mandell, R. R., Douglas, R. G. & Bennett, J. E. (1990) Principles and Practice of Infectious Diseases. Churchill Livingstone, New York, NY, USA.





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