Single- and multiple-dose pharmacokinetics of linezolid and co-amoxiclav in healthy human volunteers

Olaf Burkhardt1, Klaus Borner2, Nicolas von der Höh1, Peter Köppe3, Mathias Wilhelm Pletz1, Carl Erik Nord4 and Hartmut Lode1,*

1 Department of Chest and Infectious Diseases, Chest Hospital Heckeshorn, affil. Freie Universität Berlin, Zum Heckeshorn 33, D-14109 Berlin; 2 Institute of Clinical Chemistry and Pathobiochemistry and 3 Department of Medical Physics, University Hospital Benjamin Franklin, Free University of Berlin, Berlin, Germany; 4 Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden

Received 16 January 2002; returned 15 April 2002; revised 30 May 2002; accepted 24 June 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In an open, randomized, two-period crossover study the pharmacokinetics of linezolid and co-amoxiclav were investigated after single- and multiple-dose administration in 12 healthy volunteers (six females and six males). Linezolid was given in tablets of 600 mg twice a day for 7 days and co-amoxiclav in tablets of 1000 mg (875 + 125 mg) once a day for 7 days. The wash-out period was 4 weeks between the administration of the two antibacterial agents. Blood and urine samples were collected on days 1 and 7 before and at different time points up to 24 h after medication. The concentrations of the antibiotics in serum and urine were measured by validated high-performance liquid chromatography methods. Linezolid exhibited a mean Cmax of 14.5 ± 4.6 mg/L after Tmax of 47.5 ± 20.1 min on day 1, with a significant increase to 24.0 ± 6.9 mg/L on day 7 (P < 0.01). The AUDtot (total area under the data) revealed a significant increase from 140.5 ± 28.3 mg·h/L on day 1 to 220.2 ± 42.6 mg·h/L on day 7 (P < 0.01). There were no significant differences in terminal elimination half-life between days 1 and 7 (9.53 ± 2.87 versus 7.97 ± 3.08 h) or in total clearance (71.6 ± 17.6 versus 81.5 ± 14.7 ml/min·1.73 m2). Results are in agreement with the assumption of a limited accumulation of linezolid under the dosage regimen given. Serum linezolid concentrations in females were always higher than those in males. The volume of distribution Vss/f differed significantly between females and males (41.6 ± 4.2 versus 52.2 ± 3.3 L/70 kg; P < 0.01). Pharmacokinetic parameters of amoxicillin and clavulanic acid found in this study were similar to previously published data. No accumulation was found with co-amoxiclav. No serious adverse event was observed with the study drugs.

Keywords: linezolid, co-amoxiclav, pharmacokinetics, multiple dosing


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
With a worldwide increasing incidence of infections due to Gram-positive organisms, which are resistant to standard therapies, there is a need for new antimicrobial agents with a new and different mode of action compared with currently available agents. Linezolid belongs to a new class of antibacterial agents, the oxazolidinones,1 which are active against a variety of Gram-positive pathogenic bacteria, including methicillin-resistant strains of Staphylococcus aureus and Staphylococcus epidermidis, vancomycin-resistant strains of Enterococcus spp. and penicillin-resistant pneumococci.2 Linezolid acts as a protein synthesis inhibitor.3 It binds to the 23S ribosomal RNA of the 50S ribosomal subunit on the bacterial ribosome and prevents formation of an initiation complex in protein synthesis in a fashion similar to macrolides, lincosamides, chloramphenicol and streptogramins. Although linezolid has been the subject of several review studies,4,5 only two reports on multiple-dose kinetics, in six male subjects, have been published so far.6,7 In our investigations on the ecological effects of linezolid versus co-amoxiclav on the normal intestinal microflora,8 we also evaluated the pharmacokinetics of linezolid and co-amoxiclav after single and multiple oral doses in six male and six female volunteers.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Volunteers

Six male and six female healthy Caucasians, between 23 and 39 years old (mean 32.2 ± 4.3 years for females and 31.3 ± 6.1 for males), average body height 166.7 ± 7.1 cm for females and 182.3 ± 4.3 cm for males, average body weight 65.2 ± 4.5 kg for females and 82.2 ± 8.1 kg for males and average body surface 1.73 ± 0.06 m2 for females and 2.03 ± 0.11 m2 for males, participated in the study. All had normal renal and hepatic function, the mean creatinine clearance was 101.6 ± 21.1 ml/min·1.73 m2. All volunteers included in the study had normal findings from physical examination, electrocardiogram and laboratory tests (including haematological and biochemical parameters, hepatitis and human immunodeficiency virus serological tests, tests for drug abuse, urinalysis and negative pregnancy test). Further exclusion criteria were regular use of medications, abuse of alcoholic beverages, symptoms of significant illness within 3 months before the study period, history of gastrointestinal, liver or kidney disease potentially interfering with absorption, metabolism or excretion of drugs, history of central nervous system disorders, allergy or hypersensitivity to the study drugs, blood donation of more than 500 mL during the previous 3 months, participation in a clinical trial within 3 months before the study period, and pregnancy. Written informed consent was obtained from all volunteers prior to the study. The study was approved by the Ethics Committee of University Medical Centre Benjamin Franklin, Free University of Berlin, Berlin, Germany.

Study design and protocol

The study was performed in an open, randomized, two-period crossover design and was divided into two periods of 35 days. The treatment regimens were (i) one 600 mg linezolid tablet (Pharmacia & Upjohn, Kalamazoo, MI, USA) in the morning and one 600 mg linezolid tablet in the evening for 6 days; and (ii) one 1000 mg co-amoxiclav tablet in a new formulation (875 mg amoxicillin and 125 mg clavulanic acid; SmithKline Beecham, Harlow, UK) each morning for 7 days. Each volunteer received first one treatment regimen and then the crossover regimen. The wash-out period was 4 weeks between the administration of the two antibiotic regimens. Strenuous physical activity, smoking, intake of alcohol and of stimulating beverages containing xanthine derivatives (tea, coffee and soft drinks containing caffeine) were prohibited from 24 h before until 48 h after drug administration, the latter in order to avoid analytical interference.

Oral drug administration was done at 8:00 a.m. and at 8:00 p.m., in a sitting position with 100 mL of carbon dioxide-free water at ambient temperature. The test drug was taken after fasting for 12 h and in fasting conditions on days 1–7 in the morning and 2 h after dinner for the evening administration. Breakfast was served 2 h after drug administration on days 1 and 7. Drug intake was observed directly in each volunteer on day 1 and on the morning of days 2 and 7; all other medications had to be confirmed directly by telephone calls from the volunteer to the responsible physician and each volunteer recorded a diary protocol with the exact time of each medication, possible adverse event and stools, including the quality of faeces. All diaries were checked for possible differences with the protocol made when receiving the phone calls. In the case of adverse events, the investigating physician performed a precise interview and, if necessary, initiated consultations or additional diagnostic or therapeutic procedures.

Sampling

Blood samples (10 mL) were taken from a peripheral vein on days 1 and 7 before and 30, 60, 90, 120, 180, 240, 360, 480, 720 and 1440 min (day 7) after medication through an indwelling venous cannula. The samples were allowed to clot at room temperature for ~30 min. The samples were subsequently centrifuged at 1300g for 10 min at 4°C. The serum samples were stored at –80°C until analysis.

Urine samples were collected on days 1 and 7. On both days, urine fractions were collected pre-dose and over the following intervals: 0–3, 3–6, 6–12 and 12–24 h after administration. The urine volumes were measured after each collection interval and two 5 mL aliquots were saved. The samples were stored without preservatives in closed sterile tubes at –80°C.

Specimens were protected against light and heat during collection, storage and analysis.

High-performance liquid chromatography (HPLC)

Concentrations of linezolid, amoxicillin and clavulanic acid in serum and urine were determined by validated HPLC methods.9,10,11 Validation of the method for linezolid gave the following results for serum (and urine): detection limit 0.07 mg/L (2.4 mg/L), lower limit of quantification 0.14 mg/L (4.7 mg/L), linear range 20 mg/L (500 mg/L), intra-assay variability (CV) 1.8–2.5% (0.8–1.0%), inter-assay variability (CV) 1.8–2.3% (0.4–2.3%), recovery 99–102% (93–103%). Validation of the method for amoxicillin yielded the following results for serum (and urine): detection limit 0.15 mg/L (1.6 mg/L), lower limit of quantification 0.5 mg/L (15.6 mg/L), linear range 20 mg/L (1500 mg/L), intra-assay variability (CV) 1.1–3.7% (0.7–1.1%), inter-assay variability (CV) 1.7–11.2% (1.7–2.4%), recovery 96.2–101.5% (100.7–101.3%). Validation of the method for clavulanic acid yielded the following results for serum (and urine): detection limit 0.06 mg/L (3.9 mg/L), lower limit of quantification 0.12 mg/L (7.8 mg/L), linear range 5 mg/L (200 mg/L), intra-assay variability (CV) 1.5–5.9% (1.2–6.4%), inter-assay variability (CV) 3.6–4.8% (4.5–19.6%), recovery 98.8–101% (89.5–105.3%).

Pharmacokinetic calculations and statistical evaluations

The serum concentrations of linezolid were analysed assuming an open two-compartment model and the data of amoxicillin and clavulanic acid with a one-compartment model. Models were selected using the Schwarz criterion.12 These models were used to calculate time between drug administration and start of absorption (Tlag), terminal half-life and total area under the curve (AUCtot) by integration of the regression curve. The highest observed serum concentration was taken for peak concentration (Cmax) and time to peak concentration (Tmax). All other parameters [total area under the data (AUDtot), volume of distribution at steady state (Vss/f), mean residence time (MRT) and urinary recovery] were analysed non-compartmentally. The AUD was calculated with the trapezoidal rule. AUCtot and AUDtot of linezolid on each day were calculated up to 12 h after administration and on day 7 up to 24 h. Residual areas were calculated by integration of the compartmental regression line from the last data point to infinity and added to the areas from zero to 12 or 24 h (day 7). The dose-dependent parameters (AUCtot, AUDtot, Vss/f and Cmax) were adjusted to a body weight of 70 kg. Clearance values were normalized to a body surface of 1.73 m2. Recoveries in urine were extrapolated to infinity. The pharmacokinetic determinations were made using REVOL software as previously described.13 Student’s t-test and Wilcoxon’s rank test were used for statistical analysis and P values <0.05 were considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pharmacokinetics

Concentration versus time curves of linezolid, amoxicillin and clavulanic acid are shown in Figures 13. The pharmacokinetic data for the three drugs obtained on days 1 and 7 are listed in Tables 1 and 2.



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Figure 1. Mean (S.D.) serum concentrations of linezolid on days 1 and 7.

 


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Figure 3. Mean (S.D.) serum concentrations of amoxicillin and clavulanic acid on days 1 and 7.

 

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Table 1.  Pharmacokinetic parameters (mean ± S.D.) of linezolid on days 1 and 7
 

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Table 2.  Pharmacokinetic parameters (mean ± S.D.) of amoxicillin and clavulanic acid on days 1 and 7
 
Single-dose kinetics of linezolid

On day 1 the linezolid absorption started after a mean Tlag of 0.16 ± 0.19 h and reached a Cmax of 14.5 ± 4.6 mg/L after Tmax of 0.79 ± 0.34 h (Table 1). The first dose of 600 mg linezolid resulted in peak levels of 13.2 ± 3.8 mg/L after 1 h, with a decrease to 11.6 ± 3.6 mg/L after 2 h, 8.84 ± 3.0 mg/L after 4 h and 3.62 ± 1.7 mg/L after 12 h (Figure 1). The area under the serum concentration–time curve (AUCtot) was 138 ± 27.6 mg·h/L, the AUDtot was 140.5 ± 28.3 mg·h/L. The volume of distribution at steady state (Vss/f) was 46.9 ± 6.61 L. The pharmacokinetics of linezolid could be described by an open two-compartment model with a terminal elimination half-life of 9.53 ± 2.87 h. The MRT was 9.90 ± 2.28 h. The mean renal clearance (CLren) of linezolid was 36.7 ± 9.7 ml/min·1.73 m2, the total clearance (CLtot) 71.6 ml/min·1.73 m2. Within 12 h after the first dose of linezolid, 29.2 ± 6.7% of the dose was recovered in urine as unmodified parent drug. After intake of linezolid, two additional peaks were observed in chromatograms of the urine samples. These potential metabolites could not be identified because of the lack of pure reference materials.

Multiple-dose kinetics of linezolid

On day 7 serum peak and trough levels showed a clear accumulation, with a linezolid concentration of 9.01 ± 4.4 mg/L before medication and a maximum concentration of 21.07 ± 7.3 mg/L after 1 h, with a decrease to 19.38 ± 6.6 mg/L after 2 h, 15.39 ± 5.5 mg/L after 4 h, 6.45 ± 2.6 mg/L after 12 h and 1.98 ± 1.1 mg/L after 24 h (Figure 1). Calculations of Cmax, AUCtot, AUDtot, CLren and urinary recovery revealed significant differences (P < 0.05) between days 1 and 7. All other relevant pharmacokinetic parameters, Tmax, Tlag, CLtot and terminal half-life, revealed no significant differences between days 1 and 7 (see Table 1).

Sex-related differences in linezolid pharmacokinetics

Serum concentrations, adjusted to 70 kg body weight, were always higher in females than in males (Figure 2). The distribution volume was accordingly significantly lower (P < 0.01) in females than in males (Table 1).



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Figure 2. Mean (S.D.) serum concentrations of linezolid in male and female volunteers.

 
Pharmacokinetics of amoxicillin and clavulanic acid

Calculations of relevant pharmacokinetic parameters of amoxicillin and clavulanic acid revealed no significant differences between days 1 and 7 (Table 2). The serum concentration–time curves of days 1 and 7 showed an excellent agreement (Figure 3). On day 7, amoxicillin had a mean Cmax of 9.38 ± 3.41 mg/L, a terminal elimination half-life of 1.29 ± 0.77 h and an AUCtot of 27.9 ± 6.22 mg·h/L. The mean Cmax of clavulanic acid was 2.79 ± 1.32 mg/L, the terminal half-life 1.01 ± 0.15 h and the AUCtot 7.21 ± 2.00 mg·h/L. No accumulation was observed for either drug.

Safety and tolerance

The overall tolerance of linezolid and co-amoxiclav was good. No severe adverse events occurred. No significant differences in the values of clinical parameters and safety laboratory test results were found before and after the study. No volunteer was excluded during the study. The main adverse events after linezolid administration were headaches (two subjects) and gastrointestinal disturbances such as flatulence and diarrhoea (two subjects). One male volunteer complained of Candida mycosis of the glans penis. Under therapy with co-amoxiclav three volunteers complained of headaches. Two female subjects complained of candidiasis vulvovaginalis; one complained of mycosis of the body and the other of cystitis. All adverse events were classified as mild to moderate.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Linezolid is the first of a new class of antibacterial agents, the oxazolidinones, with good activity against Gram-positive bacteria, including multidrug-resistant strains. Mean trough levels of linezolid 12 h after a single oral administration of 600 mg were 3.6 ± 1.7 mg/L and after multiple administration 9.0 ± 4.4 mg/L. In vitro studies demonstrated that linezolid inhibited 90% (MIC90) of methicillin-susceptible and -resistant S. aureus strains at 2–4 mg/L.1416 Against both penicillin-sensitive and -resistant Streptococcus pneumoniae, linezolid had a MIC of 1–4 mg/L17,18 and against Enterococcus faecium and Enterococcus faecalis, either vancomycin-sensitive or -resistant, linezolid yielded an MIC90 of 4 mg/L.15,19,20 This study demonstrates that a dose of 600 mg twice daily results in drug serum concentrations higher than MICs for most clinically significant Gram-positive pathogens during the entire dosing interval.

Pharmacokinetic modelling showed that the serum concentrations of linezolid best fitted an open two-compartment model. The single- and multiple-dose pharmacokinetics of linezolid in our study are similar to those previously reported by Gee et al.6 and Slatter et al.7 After a single dose, linezolid was absorbed with a Tmax of <1 h to give a Cmax of 14.5 ± 4.6 mg/L, and an MRT of 9.90 ± 2.28 h. The AUDtot on day 1 was 140.5 ± 28.3 mg·h/L. Linezolid and the two main metabolites have been reported to be predominantly eliminated by the kidneys, accounting for 83.9 ± 3.3% of the radioactive label.7 In this study 29.2 ± 6.7% of the dose was recovered in urine as parent compound within the first 12 h. Two potential metabolites could be detected in chromatograms from urine samples, as has been similarly reported by Slatter et al.7

After multiple drug administration of linezolid, Cmax, AUCtot, AUDtot, CLren and urinary recovery revealed significant differences between days 1 and 7. The significant increase of maximum serum concentration on day 7 could be considered as dose-dependent accumulation. When adjusted to 70 kg body weight, serum concentrations were higher in females than in males and resulted from the fact that the volume of distribution Vss/f in female volunteers was significantly lower than in male volunteers (41.6 ± 4.2 versus 52.2 ± 3.3 L/70 kg; P < 0.01).

Pharmacokinetic parameters of amoxicillin and clavulanic acid in the modified formulation used in this study were similar to previously published data.10,21

The overall tolerance of linezolid and the other two drugs administered in our study was acceptable. The main adverse events were headaches, diarrhoea and candidiasis, and no volunteer had to be withdrawn from the study because of them. Furthermore, no drug-related changes in vital signs, clinical chemistry, haematology or urinalysis were observed in the study.


    Acknowledgements
 
The technical assistance of E. Borner, H. Hartwig, M. Rau and G. Schreiber is gratefully acknowledged.


    Footnotes
 
* Corresponding author. Tel: +49-30-8002-2223; Fax: +49-30-8002-2623; E-mail: haloheck{at}zedat.fu-berlin.de Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Dressler, L. D. & Rybak, M. J. (1998). The pharmacologic and bacteriologic properties of oxazolidinones, a new class of synthetic antimicrobials. Pharmacotherapy 18, 456–62.[ISI][Medline]

2 . Moellering, R. C., Jr (1999). A novel antimicrobial agent joins the battle against resistant bacteria. Annals of Internal Medicine 130, 155–7.[Free Full Text]

3 . Swaney, S. M., Aoki, H., Clelia Ganoza, M. & Shinabarger, D. L. (1998). The oxazolidinone linezolid inhibits initiation of protein synthesis in bacteria. Antimicrobial Agents and Chemotherapy 42, 3251–5.[Abstract/Free Full Text]

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14 . Zurenko, G. E., Yagi, B. H., Schaadt, R. D., Allison, J. W., Kilburn, J. O., Glickman, S. E. et al. (1996). In vitro activities of U-100592 and U-100766, novel oxazolidinone antibacterial agents. Antimicrobial Agents and Chemotherapy 40, 839–45.[Abstract]

15 . Rybak, M. J., Hershberger, E., Moldovan, T. & Grucz, R. G. (2000). In vitro activities of daptomycin, vancomycin, linezolid, and quinupristin–dalfopristin against staphylococci and enterococci, including vancomycin-intermediate and -resistant strains. Antimicrobial Agents and Chemotherapy 44, 1062–6.[Abstract/Free Full Text]

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17 . Mason, E. O. J., Lamberth, L. B. & Kaplan, S. L. (1996). In vitro activities of oxazolidinones U-100592 and U-100766 against penicillin-resistant and cephalosporin-resistant strains of Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 40, 1039–40.[Abstract]

18 . Spangler, S. K., Jacobs, M. R. & Appelbaum, P. C. (1996). Activities of RPR 106972 (a new oral streptogramin), cefditoren (a new oral cephalosporin), two new oxazolidinones (U-100592 and U-100766), and other oral and parenteral agents against 203 penicillin-susceptible and -resistant pneumococci. Antimicrobial Agents and Chemotherapy 40, 481–4.[Abstract]

19 . Eliopoulos, G. M., Wennersten, C. B., Gold, H. S., Moellering, R. C., Jr (1996). In vitro activities of new oxazolidinone antimicrobial agents against enterococci. Antimicrobial Agents and Chemotherapy 40, 1745–7.[Abstract]

20 . Bostic, G. D., Perri, M. B., Thal, L. A. & Zervos, M. J. (1998). Comparative in vitro and bactericidal activity of oxazolidinone antibiotics against multidrug-resistant enterococci. Diagnostic Microbiology and Infectious Disease 30, 109–12.[ISI][Medline]

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