Penetration of linezolid into bone, fat, muscle and haematoma of patients undergoing routine hip replacement

A. M. Lovering1,*, J. Zhang2, G. C. Bannister3, B. J. A. Lankester3, J. H. M. Brown3, G. Narendra3 and A. P. MacGowan1

1 Bristol Centre for Antimicrobial Research & Evaluation and 3 Department of Orthopaedics, Southmead Hospital, Bristol BS10 5NB, UK; 2 Institute of Antibiotics, Huashan Hospital, Fu Dan University, Shanghai 200040, Peoples Republic of China

Received 22 October 2001; returned 22 February 2002; revised 4 March 2002; accepted 21 March 2002


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Twelve patients undergoing total hip replacement were given 600 mg of linezolid as a 20 min iv infusion along with conventional prophylaxis of 1 g of cefamandole immediately before surgery. Routine total hip arthroplasty was carried out, and at timed intervals during surgery samples of bone, fat, muscle and blood were collected for assay by high-performance liquid chromatography analysis. Samples of the haematoma fluid that formed around the operation site and further blood samples for assay were also collected at timed intervals following the operation. The penetration of linezolid into bone was rapid, with mean concentrations of 9.1 mg/L (95% CI 7.7–10.6 mg/L) achieved at 10 min after the infusion, decreasing to 6.3 mg/L (95% CI 3.9–8.6 mg/L) at 30 min. Correction for the simultaneous blood concentrations gave mean values for bone penetration of 51% at 10 min, 60% at 20 min and 47% at 30 min. Although the penetration of linezolid into fat was also rapid, mean concentrations and degree of penetration were c. 60% of those in bone; at 10 min they were 4.5 mg/L (95% CI 3.0–6.1 mg/L; penetration 27%); at 20 min they were 5.2 mg/L (95% CI 4.0–6.4 mg/L; penetration 37%); and at 30 min, 4.1 mg/L (95% CI 3.3–4.8 mg/L; penetration 31%). For muscle the corresponding values were 10.4 mg/L (95% CI 8.1–12.7 mg/L; penetration 58%) at 10 min, 13.4 mg/L (95% CI 10.2–16.5 mg/L; penetration 94%) at 20 min and 12.0 mg/L (95% CI 9.2–14.8 mg/L; penetration 93%) at 30 min. Mean concentrations of linezolid in the haematoma fluid drained from around the operation site were 8.2 mg/L at 6–8 h and 5.6 mg/L at 10–12 h after the infusion, and 7.0 mg/L at 2–4 h following a second 600 mg infusion given 12 h post-operatively. We conclude that linezolid exhibits rapid penetration into bone, fat and muscle of patients undergoing hip arthroplasty, to achieve levels in excess of its MIC for susceptible organisms (<=4 mg/L); therapeutic concentrations were maintained in the haematoma fluid that surrounds the operation site for >16 h.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Linezolid is the first member of a new synthetic class of antimicrobials, oxazolidinones, to enter clinical practice. Linezolid is characterized by a broad spectrum of activity against Gram-positive organisms and acts by inhibition of an early stage in the protein synthesis pathway.13 This mode of action, although not fully characterized, is not common to other classes of antibacterial and cross-resistance associated with the known mechanisms responsible for acquired resistance does not occur with linezolid.1 As a result, linezolid retains activity against the clinically important strains of methicillin-resistant Staphylococcus aureus (MRSA), glycopeptide-intermediate S. aureus (GISA) and glycopeptide-resistant enterococci (GRE).

Linezolid is rapidly absorbed following an oral dose to give concentrations similar to those following parenteral administration, with bioavailability c. 100%.47 Linezolid has a relatively high volume of distribution (c. 50 L in adults), and achieves levels in inflammatory fluid similar to those in blood, suggesting good penetration into extra-vascular sites and a possible role in the treatment of infections involving a wide range of soft tissue sites.8 Linezolid is cleared primarily through the renal route with a half-life of c. 5 h, which permits a twice daily dosing regimen.46

MRSA is an established problem in many hospitals across the world, causing problems of both colonization and nosocomial infection.9 When these infections involve either bone or joint prostheses they present a particularly difficult problem in patient management, frequently resulting in prolonged periods of hospitalization for parenteral therapy, and often only modest therapeutic success. As linezolid has both a microbiological and pharmacokinetic profile, which suggests a potential use in the treatment of such infections, we have evaluated the penetration of linezolid, after a single 600 mg iv dose, into the bone and associated fat and muscle of 12 patients undergoing routine total hip replacement and receiving standard prophylaxis with cefamandole.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study protocol

This was approved by the local medical research ethics committee and all patients gave written informed consent. Twelve patients who had not received antimicrobials in the preceding 72 h and were to undergo routine hip replacement were enrolled into the study. Immediately before the induction of anaesthesia a 20 min infusion of 600 mg of linezolid followed by a single bolus of 1 g of cefamandole were administered through a forearm vein. The operation began immediately at the end of the infusion and all timings are expressed relative to this time point. Further doses of cefamandole were administered 8 and 16 h after the start of the operation and a further dose of linezolid was given 12 h after the start of the operation. Routine total hip replacement was carried out on all patients, and samples of bone, fat, muscle and blood were collected 10, 20 and 30 min after the start of the operation. Samples of the haematoma fluid draining from the operation site were collected over the periods 6–8, 10–12 and 14–16 h after the start of the operation, and samples of blood were collected at 8, 12 and 16 h immediately before the administration of further doses of cefamandole or linezolid. Blood and haematoma samples were centrifuged, and the supernatant removed and stored at –70°C along with the bone, fat and muscle samples until assayed.

HPLC assay procedures

Samples were assayed for the presence of cefamandole and linezolid by a high-performance liquid chromatography (HPLC) method that permitted the simultaneous assay of both agents, adapted from Tobin et al.10 Chromatography was performed on a Hypersil 5ODS column (HPLC Technology Ltd, Macclesfield, UK) using a mobile phase of methanol/water/phosphoric acid (30:69:1) with the addition of 2 g/L heptane sulphonic acid (Sigma Chemical Co.) and the pH adjusted to 4.5. Detection of both compounds was by UV absorbance at 254 nm with quantification by the external standard method. Serum and haematoma samples were diluted with an equal volume of acetonitrile, centrifuged at 5000g and 10 µL of the supernatant was injected into the chromatograph. Bone, fat and muscle samples were processed as previously described and the two antibiotics were extracted in phosphate-buffered saline (PBS) at 4°C for 5 h.11,12 In brief, bone samples (c. 0.5 g) were crushed, a volume of PBS equal to twice the weight of bone added (1 g {cong} 1 mL) and the antimicrobial agents extracted at 4°C for 5 h. Any samples with visible evidence of blood contamination were discarded and, where possible, the total sample collected was processed. After extraction the samples were centrifuged at 5000g and an aliquot of the aqueous layer was treated with acetonitrile, as described for serum samples. Samples of fat and muscle were treated as described for bone, but were finely sliced rather than crushed.

The assay response was linear over the concentration range 0.5–100 mg/L, with a lower limit of detection of 0.1 mg/L for linezolid and 0.5 mg/L for cefamandole. Recovery of the two agents from spiked bone and fat samples was in the range 95–110%, depending on drug, concentration and tissue, and for the purposes of this study was taken as 100%. Intra- and inter-day accuracy and precision were assessed by the use of quality control standards with limits for accuracy of 10% and coefficient of variability for precision of 5% used.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Following a 600 mg infusion of linezolid, mean serum concentrations for the 12 patients were 19.2 mg/L (median 16.9 mg/L; range 10.7–38.2 mg/L) at 10 min after the infusion, dropping to 14.3 mg/L (median 13.2; range 5.4–21.3 mg/L) at 30 min after the infusion (Table 1). By 8 h after the infusion mean levels had fallen to 5.1 mg/L (median 4.1 mg/L; range 3.4–12.0 mg/L), further decreasing to 2.8 mg/L (median 2.2 mg/L; range 0.5–9.2 mg/L) at 12 h (Table 1). Although too few samples were taken to make a conventional assessment of the serum half-life of linezolid, a review of the data indicates that these results are consistent with a half-life of c. 4–5 h.


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Table 1.  Concentrations of linezolid and cefamandole in bone, fat, muscle and haematoma in 12 patients undergoing total hip replacement
 
The penetration of linezolid into bone was rapid, with mean concentrations of 9.1 mg/L (range 4.3–13.0 mg/L), 8.6 mg/L (range 5.2–23.7 mg/L) and 6.3 mg/L (2.6–17.3 mg/L) found at 10, 20 and 30 min, respectively, after the end of the infusion (Table 1). Although there was considerable variation in concentrations (range 2.6–23.7 mg/L), in only one of the 36 samples assayed was the concentration of linezolid <4 mg/L; a concentration of 2.6 mg/L was measured in the 30 min sample from patient six, compared with 7.4 and 5.2 mg/L at 10 and 20 min, respectively. The lowest bone concentration of cefamandole (5.3 mg/L) was also measured in this sample. When compared with simultaneous blood concentrations, the mean bone penetration of linezolid was 51.0% at 10 min, 60.0% at 20 min and 47% at 30 min (Table 2); overall penetration was 51% (95% CI 43–75%).


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Table 2.  Degree of penetration of linezolid and cefamandole into bone, fat and muscle in 12 patients undergoing total hip replacement
 
Penetration of linezolid into the muscle surrounding the operation site was also rapid, achieving mean concentrations of 10.4 mg/L at 10 min after the infusion and a peak concentration of 13.4 mg/L by 20 min after the end of the infusion (Table 1). When compared with simultaneous blood concentrations these values represented penetration of 58.3% at 10 min, 94.3% at 20 min and 93.0% at 30 min (Table 2). As with bone and muscle, penetration of linezolid into fat was also rapid, with mean peak concentrations of 5.2 mg/L achieved 20 min after the infusion. However, both the concentrations and degree of penetration into fat relative to serum levels were approximately half the values obtained for bone (Tables 1 and 2). The good penetration and levels of linezolid observed in the soft tissues surrounding the operation site at the time of surgery were also seen in the drainage from the wound site. Mean concentrations of linezolid in drainage fluid were 8.2 mg/L 6–8 h and 5.6 mg/L 10–12 h after the first dose (Table 1) and 7.0 mg/L 2–4 h after the second dose (Table 2).

For all tissue samples the concentrations of cefamandole were significantly higher (Welch’s t-test, P < 0.01) than those of linezolid, but when corrected for the simultaneous blood concentrations the penetration of linezolid into these tissues was significantly higher (P < 0.05).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study we have attempted to characterize the tissue distribution and penetration profile of linezolid during routine orthopaedic surgery. Although the role of antibiotic administration in this procedure has been for prophylaxis, the study objective was to gain data to help support decisions on the role of linezolid in the treatment of bone and associated soft tissue infections. In designing the study we have used a two-drug administration protocol to ensure adequate prophylaxis whilst at the same time providing comparative data for one of the major classes of antimicrobial used in the treatment and prophylaxis of such infections, namely the ß-lactams. Although the use of such a protocol avoids much of the variability seen in conventional two-group study designs, a central assumption is the lack of a pharmacodynamic interaction between the two agents being co-administered.11,12 The decision to use a ß-lactam was based on the apparent lack of interaction between the pharmacokinetics of ß-lactams and linezolid, and from similarities in both PK/PD parameters (time above MIC) and the MICs of the two agents for susceptible pathogens.7,13 However, the limitations of comparison between groups of agents as dissimilar as the ß-lactams and the oxazolidinones must be recognized.

Following a 600 mg infusion in patients undergoing routine hip arthroplasty, linezolid penetrated rapidly into bone, fat and muscle at the operation site, reaching peak concentrations at between 10 and 20 min after the end of the infusion. For bone, these concentrations were c. 50% of the simultaneous blood levels, whereas for muscle and fat they were c. 90% and 30%, respectively. In all but one bone sample the concentration of linezolid exceeded the MIC for susceptible pathogens (<=4 mg/L);1,3 concentrations in the other two samples from the same patient exceeded the MIC, but this sample also had a significantly lower cefamandole concentration than other samples. Although we are unable to offer an explanation for the apparently lower levels of both agents in this sample, it does highlight the heterogeneous nature and variable penetration of antimicrobials into bone, and further illustrates the complexity of such studies.11,12

With the soft tissue samples, fat concentrations of linezolid at the time of operation were similar to its MIC for susceptible pathogens, whereas muscle concentrations exceeded the MIC by a factor of three or four, suggesting good penetration into the tissues surrounding the bone. This is supported by the concentrations of linezolid found in the drainage from these tissues, which exceeded simultaneous blood levels for up to 12 h after dosing and were above the MIC for susceptible pathogens throughout the dosing interval.

The concentrations of cefamandole found in this study are in agreement with the findings of our earlier studies,11,12 and we found broad similarities in the bone and soft tissue disposition of linezolid and cefamandole. Both agents exhibit rapid penetration into the bone and soft tissues, with concentrations similar to or exceeding their MICs for susceptible pathogens at the time of operation. Inhibitory levels of both agents were also maintained in the haematoma around the operation site for the entire dosing interval. However, in making such comparisons it must be recognized that the two agents differ in their mode of action and other aspects, and that similar pharmacokinetic disposition in bone may not necessarily predict similarities in efficacy. Although these data have been obtained for healthy, well perfused bone the values for linezolid are similar to the single reported level of 9.0 mg/L for necrotic bone from a patient with osteomyelitis,14 suggesting that our findings may be relevant to treatment of infected tissue.

In conclusion, we find that linezolid has a similar distribution and penetration profile in bone and associated soft tissues to cefamandole, an agent that may be used in the treatment of infections in these tissues. This would indicate a potential role for linezolid in the management of such patients, in line with an encouraging clinical report,14 but further clinical evaluation is needed.


    Acknowledgements
 
This study was supported by a grant from Pharmacia Ltd, Milton Keynes, UK.


    Footnotes
 
* Corresponding author. Tel: +44-117-9595653; Fax: +44-117-9593217; E-mail: Lovering_a{at}southmead.swest.nhs.uk Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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2 . Xiong, L., Kloss, P., Douthwaite, S., Andersen, N. M., Swaney, S., Shinabarger, D. L. et al. (2000). Oxazolidinone resistance mutations in 23S rRNA of Escherichia coli confirms the central region of domain V as the primary site of drug action. Journal of Bacteriology 182, 5325–31.[Abstract/Free Full Text]

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5 . Hendershot, P. E., Antal, E. J., Welshman, I. R., Batts, D. H. & Hopkins, N. K. (2001). Linezolid: pharmacokinetic and pharmacodynamic evaluation of coadministration with pseudoephedrine HCl, phenylpropanolamine HCl, and dextromethorpan HBr. Journal of Clinical Pharmacology 41, 563–72.[Abstract/Free Full Text]

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8 . Gee, T., Ellis, R., Marshall, G., Andrews, J., Ashby, J. & Wise, R. (2001). Pharmacokinetics and tissue penetration of linezolid following multiple oral doses. Antimicrobial Agents and Chemotherapy 45, 1843–6.[Abstract/Free Full Text]

9 . World Health Organisation. (2001). WHO Global Strategy for Containment of Antimicrobial Resistance. WHO, Geneva, Switzerland.

10 . Tobin, C. M., Sunderland, J., White, L. O. & MacGowan, A. P. (2001). A simple, isocratic high-performance liquid chromatographic assay for linezolid in human serum. Journal of Antimicrobial Chemotherapy 48, 605–8.[Abstract/Free Full Text]

11 . Lovering, A. M., Perez, J., Bowker, K. E., Reeves, D. S., MacGowan, A. P. & Bannister, G. (1997). Comparison of the penetration of cefuroxime and cefamandole into bone, fat and haematoma in patients undergoing total hip replacement. Journal of Antimicrobial Chemotherapy 40, 99–104.[Abstract]

12 . Lovering, A. M., Walsh, T. R., Bannister, G. C. & MacGowan, A. P. (2001). The penetration of ceftriaxone and cefamandole into bone, fat and haematoma and relevance of serum protein binding to their penetration into bone. Journal of Antimicrobial Chemotherapy 47, 483–6.[Abstract/Free Full Text]

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14 . Melzer, M., Goldsmith, D. & Gransden, W. (2000). Successful treatment of vertebral osteomyelitis with linezolid in a patient receiving hemodialysis and with persistent methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus bacteremias. Clinical Infectious Diseases 31, 208–9.[ISI][Medline]