Abstract

In a randomised crossover study, 14 volunteers received a single oral dose of 500 mg levofloxacin or 500 mg ciprofloxacin in order to assess plasma concentrations by high-pressure liquid chromatography (up to 24 h), urinary excretion and urinary bactericidal titres (UBTs) at intervals up to 120 h. The median maximum concentration of levofloxacin in plasma was 6.1 mg/L and that of ciprofloxacin was 2.3 mg/L. The median cumulative level of renal excretion of the administered dose of the parent drug was 81.2% for levofloxacin and 36.2% for ciprofloxacin. UBTs were determined for a reference strain and nine clinical uropathogens. The median UBTs of both quinolones measured within the first 12 h were between 0 and 1:≥1024, correlating with the minimum inhibitory concentrations (MICs) of the strains. For Gram-negative strains, the UBTs of both quinolones were comparable despite the lower MICs of ciprofloxacin. During further time courses, however, the UBTs of levofloxacin were significantly higher than those of ciprofloxacin. For Gram-positive strains, for which the MICs of levofloxacin were equal to or lower than those of ciprofloxacin, the UBTs of levofloxacin were already significantly higher from the beginning. It can be concluded that overall the doses of the two tested fluoroquinolones may be considered equivalent with regard to treatment of complicated urinary tract infections, although the recommended dosing is twice daily for ciprofloxacin and once daily for levofloxacin.

Keywords: Levofloxacin; Ciprofloxacin; Serum and urine pharmacokinetics; Urinary bactericidal titres

Article Outline

1. Introduction

2. Patients and methods

2.1. Study design and subject population

2.2. Drug administration

2.3. Sample collection

2.4. Sample preparation

2.5. Assay conditions

2.6. Calibration row and spiked quality controls

2.7. Pharmacokinetic indices

2.8. Determination of MICs and MBCs

2.9. Determination of UBTs

2.10. Test organisms

2.11. Statistical analyses

3. Results

3.1. Volunteers

3.2. Safety and laboratory test results

3.3. Concentrations in plasma and pharmacokinetic parameters

3.4. Urinary pHs, volumes and drug concentrations in urine

3.5. MICs and MBCs

3.6. UBTs and AUBTs

4. Discussion

5. Conclusion

Acknowledgements

References

1. Introduction

Urinary tract infections (UTIs) are usually caused by Escherichia coli or other species of the Enterobacteriaceae such as Proteus, Klebsiella, Enterobacter and Citrobacter species, but also by non-fermenters such as Pseudomonas aeruginosa and Gram-positive cocci such as enterococci and staphylococci, especially Staphylococcus saprophyticus [1]. For empirical antimicrobial therapy, an antibiotic agent should be chosen that includes most of the uropathogens in its antibacterial spectrum. In this regard, the fluoroquinolones are currently considered to be amongst the drugs of choice. However, they differ in their pharmacokinetic properties [2] and antibacterial activity. In addition, their antibacterial activity in urine is significantly reduced depending on urine pH and contents [3]. Levofloxacin is the levo isomer of the d,l-racemate of ofloxacin. It has an extended spectrum of activity compared with ciprofloxacin, with improved activity against Gram-positive bacteria and atypical organisms and a comparable good activity against Gram-negative bacteria [4].

In this ex vivo clinical study, fluoroquinolones such as levofloxacin and ciprofloxacin with different pharmacokinetic properties were compared with regard to sufficient dosages in the treatment of UTIs. To combine pharmacokinetic and pharmacodynamic parameters, the concentrations in urine and the urinary bactericidal titres (UBTs) for common uropathogens were determined to approximate more closely their antibacterial activities at the site of infection [5], [6], [7] and [8]. According to animal and human studies [9] and [10], the efficacy of fluoroquinolones can be considered comparable if peak concentration/minimal inhibitory concentration (MIC) and area under the concentration–time curve (AUC)/MIC or minimal bactericidal concentration (MBC) ratios are in the same range. The AUC/MBC ratio relates to the area under the UBT–time curve (AUBT) minus 24/1 h.

The purpose of the present study was to evaluate the concentrations in plasma, urinary excretion and bactericidal activity of levofloxacin (500 mg) versus ciprofloxacin (500 mg) in healthy volunteers receiving a single oral dose. Such data have not been published previously.

2. Patients and methods

2.1. Study design and subject population

The study was approved by the Ethics Committee of the Landesärztekammer Bayern, Munich, Germany, and was performed as an ex vivo, open, randomised, crossover clinical trial with 14 healthy volunteers. The volunteers were considered healthy on the basis of medical history, physical examination, haematology parameters (haemoglobin concentration and erythrocyte, leukocyte and platelet counts), serum chemistry parameters (creatinine, uric acid, gamma-glutamyltransferase, alkaline phosphatase and total bilirubin levels) and urinalysis (glucose and protein levels, white and red blood cell counts and lack of antibacterial activity, i.e. inhibition of Bacillus subtilis).

2.2. Drug administration

After giving written informed consent to participate in the study, each volunteer successively received one oral dose of 500 mg levofloxacin (Aventis Pharma GmbH, Bad Soden, Germany) or 500 mg ciprofloxacin (Bayer AG, Wuppertal, Germany) in a crossover design at an interval of 7 days according to the randomisation schedule. Study drugs were administered following an overnight fast. Following drug administration, subjects fasted for 2 h. Alcohol- and xanthine-containing beverages and meals and acidic drinks such as grapefruit juice were not allowed 12 h before and 24 h after drug administration. The volunteers were asked to drink sufficient and comparable amounts of water through both collection periods to ensure adequate urine production. A physical examination, electrocardiography and laboratory tests were performed before and after each phase of the study. Adverse events were recorded continuously throughout the trial period. The volunteers were monitored and urine collection was controlled in the study centre for the first 48 h and thereafter at the end of each sampling period up to 120 h.

2.3. Sample collection

Serum samples were collected at the following time points: pre dose and at 1, 2, 3, 4, 6, 12 and 24 h. All urine voided was collected over a 12 h interval prior to drug administration (to ascertain that the urine was antibiotic free) and at the following time intervals thereafter: 0–6, 6–12, 12–24, 24–36, 36–48, 48–72, 72–96 and 96–120 h. All samples were stored at −20 °C.

2.4. Sample preparation

All sample handling was done under protection from daylight. Serum samples were thawed and vigorously mixed, then precipitated with acetonitrile/perchloric acid containing the internal standard (pipemidic acid). Following centrifugation, 10 μL of the supernatant was analysed. Urine samples were thawed and vigorously mixed then diluted 1:100 with 0.05 M NaH₂PO₄ buffer containing the internal standard. Then, 17 μL of the diluted sample was analysed.

2.5. Assay conditions

Levofloxacin and ciprofloxacin were analysed with the same chromatographic conditions. The assay methods are ‘in house’ procedures; the ciprofloxacin assay was published in 2003 [7]. Chromatographic separation was performed with a reversed phase column, isocratic solvent system (citric acid buffer containing ammonium perchlorate and acetonitrile containing ion pairing reagent (90/10, v/v)), and the effluent was monitored by fluorescence detection (excitation 285 nm; emission 480 nm). Turbochrom 3 software (version 3.2, 1991; PE Nelson, Cupertino, CA) was used for evaluation of chromatograms. The retention times were 8.0 min and 4.5 min for ciprofloxacin and levofloxacin, respectively.

2.6. Calibration row and spiked quality controls

The drug concentrations in serum and urine samples were measured by comparison with a serum and urine calibration row, respectively. Calibration standards were prepared by adding defined amounts of standard solution of levofloxacin and ciprofloxacin to tested drug-free serum or urine.

Spiked quality controls (SQCs) were prepared for determination of interassay variation by addition of defined amounts of stock solution or spiked control of higher concentration to defined amounts of tested drug-free serum or urine. No interference was observed for levofloxacin, ciprofloxacin or the internal standard in serum and urine. Weighted linear regression (1/peak height ratio) was performed for calibration.

The linearity of the calibration could be proven between concentrations of 0.00234 μg/mL and 7.93 μg/mL for levofloxacin and 0.00230 μg/mL and 7.82 μg/mL for ciprofloxacin in serum (r² for levofloxacin, ≥0.9998; and r² for ciprofloxacin, ≥0.9996). The quantification levels were identical to the lowest calibration levels. The interassay precisions of the SQCs in serum were 2.6% (6.41 μg/mL), 2.6% (0.626 μg/mL), 4.3% (0.0488 μg/mL) and 7.2% (0.0122 μg/mL) (range of accuracies of SQCs, 97.1–98.2%) for levofloxacin and 2.2% (6.40 μg/mL), 2.4% (0.625 μg/mL), 4.3% (0.0487 μg/mL) and 7.7% (0.0122 μg/mL) (range of accuracies of SQCs, 93.0–98.2%) for ciprofloxacin.

The linearity of the calibration could be proven between concentrations of 0.250 μg/mL and 809 μg/mL for levofloxacin and 0.248 μg/mL and 800 μg/mL for ciprofloxacin in urine (r² for levofloxacin, ≥ 0.9997; and r² for ciprofloxacin, ≥0.9997). The quantification levels were identical to the lowest calibration levels. The interassay precisions of the SQCs in urine were 2.1% (641 μg/mL), 3.6% (62.0 μg/mL), 2.3% (4.77 μg/mL) and 3.3% (1.19 μg/mL) (range of accuracies of SQCs, 98.3–100.8%) for levofloxacin and 2.4% (640 μg/mL), 4.0% (61.9 μg/mL), 3.4% (4.77 μg/mL) and 6.3% (1.18 μg/mL) (range of accuracies of SQCs, 99.9–101.8%) for ciprofloxacin.

2.7. Pharmacokinetic indices

The concentrations of drugs were estimated by standard non-compartmental methods. They were determined with data from each of the sample collection times and concentrations assayed at those times. Concentrations below the lower limit of quantification were set equal to zero. The pharmacokinetic calculations were performed on a 333 MHz computer with a Pentium II processor and the Microsoft Excel 97 program (1985–1998; Microsoft Co., Redmond, WA). The equations were entered into the program manually and were checked by recalculation of the urine concentrations for randomly selected data sets using the pharmacokinetic program WinNonlin Professional (version 2.0, 1994–1998; Pharsight Corp., Palo Alto, CA).

2.8. Determination of MICs and MBCs

MICs and MBCs were determined by a microdilution test with Mueller–Hinton broth (CM 405; Oxoid, Wesel, Germany) for MICs and Mueller–Hinton broth and Columbia agar supplemented with 5% blood (Merck, Darmstadt, Germany) for MBCs. The inoculum of Mueller–Hinton broth ranged from 1.3 × 10⁵ to 9.4 × 10⁵ colony-forming units (CFU)/mL. The MIC was defined as the lowest concentration inhibiting visible growth after incubation at 37 °C for 18 h in ambient air [11]. The MBC was determined in a second step according to the guidelines recommended by the National Committee for Clinical Laboratory Standards [8]. In brief, cultures were grown to mid-logarithmic phase for the final inoculum, which was confirmed by actual count and was added to the wells using a multipoint inoculator (0.0015 mL). The subcultured broth was spotted with a multipoint inoculator on Columbia agar and incubated for 24 h at 37 °C. The number of colonies subsequently grown was used to determine the lethal endpoint. Bactericidal activity was defined as a >99.9% (>3 log) reduction in the numbers of CFU.

2.9. Determination of UBTs

A logarithmic serial dilution (dilution range 1:2 to 1:1024) was prepared by combining a 1:1 mixture of the urine sample and the individual's antimicrobial agent-free urine collected prior to drug administration [5], [6] and [12]. UBTs were then determined by microdilution, with each well of the microplates containing 100 μL of the prepared dilution. A UBT of 0 was defined as no bactericidal activity, and a UBT of 1 was used when only undiluted urine displayed bactericidal activity.

2.10. Test organisms

Nine pathogens were cultured from the urine of patients with UTIs. The pathogens included one strain of E. coli (nalidixic acid resistant), one strain of Klebsiella pneumoniae, one strain of Proteus mirabilis, one strain of P. aeruginosa, two strains of S. aureus (one fluoroquinolone susceptible and one fluoroquinolone resistant), one strain of S. saprophyticus (methicillin susceptible) and two strains of Enterococcus faecalis (one fluoroquinolone susceptible and one fluoroquinolone resistant). Fluoroquinolone-susceptible reference strain E. coli ATCC 25922 was also tested.

2.11. Statistical analyses

UBTs were transformed into ordinal data using a scale of dilution steps of 0 for a UBT of 0, and from 1 for a UBT of 1 up to 11 for UBTs of 1:≥1024. The AUBT was calculated as the sum of the products of the UBT steps and the respective time intervals for each test organism and for each drug. Laboratory, pharmacokinetic, UBT and AUBT data for the two drugs were compared for each individual by the paired t-test. Application of the paired t-test appears to be adequate according to our previous analysis of the respective residuals [6]. An α value equal to 0.05 was chosen to determine statistical significance. Owing to the high number of tests performed, the results are of descriptive nature only. Statistical calculations were performed using the Documenta Geigy program [13].

3. Results

3.1. Volunteers

The volunteers included seven men and seven women. The mean age was 23 years (median 21 years; range 18–40 years), the mean body weight was 64.7 kg (median 65.0 kg; range 54.0–85.0 kg), the mean height was 172 cm (median 171 cm; range 160–184 cm) and the mean body mass index was 21.9 kg/m² (median 22.1 kg/m²; range 18.5–25.9 kg/m²).

3.2. Safety and laboratory test results

Levofloxacin and ciprofloxacin were well tolerated by all volunteers. No serious adverse events occurred during the study. Neither drug caused clinically significant changes in the results of routine tests of blood and serum.

3.3. Concentrations in plasma and pharmacokinetic parameters

The concentrations in plasma and the pharmacokinetic indices for levofloxacin and ciprofloxacin are given in Table 1 and Table 2, respectively.

3.4. Urinary pHs, volumes and drug concentrations in urine

The medians and ranges of urinary pH and volume, drug concentration in urine and cumulative renal excretion obtained in the two study phases are given in Table 3. The data for the corresponding collection periods of the respective study phases showed minor but significant differences in urinary pH values for the collection periods 12–24 h, 36–48 h and 72–96 h. However, in both study phases the median pH for the collection period 6–12 h (afternoon) was higher than those for the other collection periods.

Urinary concentrations of levofloxacin were significantly higher than those of ciprofloxacin throughout the study period (Fig. 1). The median proportion of cumulative renal excretion of the administered dose of the parent drug up to 120 h was 81.2% for levofloxacin (range 69.6–86.7%; mean ± standard deviation (S.D.), 81.2 ± 5.2%) and 36.2% for ciprofloxacin (range 25.9–50.3%; mean ± S.D., 37.2 ± 8.1%). The cumulative renal excretion in all collection periods showed statistically significant differences between the two drugs. Within the first 24 h, >90% of the total excretion of both drugs was completed. None of the urine samples collected prior to drug administration had detectable drug concentrations.

3.5. MICs and MBCs

The MICs of levofloxacin and ciprofloxacin, respectively, were as follows: for E. coli ATCC 25922, 0.03 mg/L and 0.008 mg/L; for nalidixic acid-resistant E. coli clinical strain 523, 0.25 mg/L and 0.125 mg/L; for K. pneumoniae clinical strain 595, 0.03 mg/L and 0.008 mg/L; for P. mirabilis clinical strain 414, 0.06 mg/L and 0.03 mg/L; for P. aeruginosa clinical strain 568, 2 mg/L and 0.5 mg/L; for S. aureus clinical strain 161, 2 mg/L and 4 mg/L; for S. aureus clinical strain 636, 8 mg/L and 32 mg/L; for S. saprophyticus clinical strain Ho94, 0.25 mg/L and 0.25 mg/L; for E. faecalis clinical strain 60, 1 mg/L and 1 mg/L; and for E. faecalis clinical strain 55, 16 mg/L and 32 mg/L. The MBCs of levofloxacin and ciprofloxacin were equal to the corresponding MICs for all strains tested.

3.6. UBTs and AUBTs

The UBTs (Fig. 2) and AUBTs of both study drugs for the test organisms are given in Table 4. The UBTs measured within the first 12 h for both quinolones were found to be between 0 (no bactericidal activity of undiluted urine) and 1:≥1024, correlating with the MICs for the strains. For Gram-negative strains, the UBTs within the first 12 h were comparable for both quinolones, despite the lower MICs of ciprofloxacin. Thereafter, however, the UBTs of levofloxacin were significantly higher than those of ciprofloxacin. For Gram-positive strains, for which the MICs of levofloxacin were equal to or lower than those of ciprofloxacin, the UBTs of levofloxacin were already significantly higher from the beginning. The AUBTs of levofloxacin were significantly higher than those of ciprofloxacin, including for P. aeruginosa.