a Department of Pharmacy Practice and b Department of Pharmaceutical and Administrative Sciences, Creighton University School of Pharmacy & Allied Health Professions, 2500 California Plaza, Omaha, NE 68178, USA
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Abstract |
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Introduction |
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The human pharmacokinetics of levofloxacin indicate wide distribution after iv administration. The volume of distribution (Vd) averages 1.3 L/kg. Peak serum concentrations average 6.2 mg/L after a 500 mg iv dose, 7.1 mg/L after 750 mg orally and 8.9 mg/L after a 1 g oral dose.4 The elimination half-life ranges from 6 to 8 h after administration, allowing for once-daily dosage.4 Penetration across the bloodbrain barrier for quinolones in general averages 3550% depending on the specific compound.57 Friedland et al.5 used an iv bolus followed by a 10 mg/kg continuous infusion over 7 h to calculate levofloxacin cerebrospinal fluid (CSF) penetration.
Given the MIC of levofloxacin for the pneumococcus, we postulated that levofloxacin could be an alternative agent in the treatment of bacterial meningitis, specifically pneumococcal meningitis. Therefore, we sought to determine the penetration of levofloxacin in an experimental rabbit meningitis model, using microdialysis as the CSF sampling tool. Using this method, we determined the area under the CSF concentrationtime curve (AUCCSF) and compared it with the area under the plasma concentrationtime curve (AUCplasma).
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Materials and methods |
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The MBC was defined as the lowest concentration showing 99.9% bactericidal effect on the organisms after streaking 10 µL from each clear tube, on to the surface of blood agar plates and incubating overnight at 35°C.
The experimental meningitis animal model of Dacey & Sande9 was used in this study. New Zealand white rabbits weighing 2.03.0 kg each were allowed to adapt to the animal centre for 4872 h before surgery. On the day of surgery, the animal was weighed and a penicillin G dose equivalent to 300 000 units was injected im. Ten minutes later, the animal was anaesthetized with ketamine (40 mg/kg) and xylazine (4 mg/kg) im in the contralateral thigh. Animals had the surgical area shaved and were then placed into a stereotaxic frame equipped with a rabbit adapter (Kopf Instruments, Tujunga, CA, USA) and securely positioned. The head of the animal was immobilized and the skull denuded. Betadine solution was applied to the area. The animal was draped with sterile towels. A midline incision made parallel to the sagittal suture exposed the cranium. Sterile gauze was applied to control bleeding. Tissue covering the skull was averted. Using a dental drill, two holes were drilled, in the frontal and parietal bones. Screws (1 x 8 mm) were placed in the skull to anchor the dental acrylic helmet. Then, a hole (2 mm diameter) was drilled for the microdialysis guide cannula. The microdialysis guide cannula was implanted into the subarachnoid space using the following stereotaxic coordinates: anterior 1.2 mm, lateral 2.4 mm and ventral 5.7 mm.10 A dental acrylic helmet was attached to the skull. Care of the animal during the surgical procedure and recovery period was in accordance with the Animal Care Policies of Creighton University Health Sciences (Approval ARC# 97-0242). After implantation of the guide cannula, the tissue outside the dental helmet was sutured together leaving only the dental helmet exposed. The animal was allowed to recover for a minimum of 72 h after implantation.
Experimental procedure
Calibration of microdialysis probes was performed in vivo by the use of retrodialysis.11 A known amount of tosufloxacin (2.0 mg/L; Abbott Laboratories, Chicago, IL, USA) was placed in the perfusing syringe along with the artificial CSF. The microdialysis probe dwelled in the CSF of the animals and the effluent was collected and analysed for levofloxacin, tosufloxacin and the internal standard (phenacetin). The amount of tosufloxacin obtained from the effluent determined the in vivo recovery of levofloxacin by the probe.
Rabbits were inoculated with 104 cfu in 0.3 mL saline injected intracisternally and returned to their cages for 1618 h. The animals were anaesthetized with multiple doses of ketamine (40 mg/kg) and xylazine (4 mg/kg) given subcutaneously. A competent veterinary technician monitored anaesthesia throughout the experiment.
Groups of animals (n = 5) received levofloxacin at doses of 7, 10.5 and 14 mg/kg iv (equivalent to adult doses of 500, 750 and 1000 mg) over 10 min.
At the start of the experiment, the microdialysis guide cannula was replaced with a CMA-11 microdialysis probe (CMA, Boston, MA, USA) connected to the microinfusion pump by PE10 tubing. Simulated CSF (1.1 mM Mg, 1.35 mM Ca, 3.0 mM K, 144.48 mM Na, 20 mM HCO3, 0.242 mM HPO4 and 131.9 mM Cl, pH 7.6) was used for these experiments. Artificial CSF was delivered to the CSF at a flow rate of 1.5 µL/min for at least 15 min to correct for volume in the tubing before collecting any samples containing levofloxacin. The effluent from the dialysis probe for levofloxacin concentrations were collected in amber glass vials at half-hour intervals from the beginning of the experiment to 8 h. Blood was obtained from a cannulated ear vein at baseline, 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 6 and 8 h after the levofloxacin administration. CSF effluent was analysed for levofloxacin and tosufloxacin while plasma was analysed for levofloxacin using a high performance liquid chromatographic (HPLC) method. Phenacetin was the internal standard for both procedures.
Since the microdialysis membrane only allows free drug to pass through the membrane, the rabbit plasma specimens were analysed for the free fraction of the drug. Briefly, uninfected, untreated rabbit plasma was spiked with 2.5 and 5.0 mg/L levofloxacin at room temperature and centrifuged through a 5000 MWCO centrifuge concentrator membrane (Life Science Products, Denver, CO, USA) at 5000 rpm for 25 min. The protein-free portion of the samples was assayed for levofloxacin according to the HPLC procedures that were already developed.
The HPLC method used an isocratic system with UV detection at 280 nm operated at ambient temperature and the mobile phase flow rate was 0.2 mL/min. The mobile phase consisted of methanol:acetonitrile:phosphate buffer (20:15:65, by vol). Chromatographic separation was achieved on a BDS C18 microbore column (150 x 2.5 mm, Hypersil, 5µ, pH stable; Keystone Scientific, Bellefonte, PA, USA). Levofloxacin within-day and day-to-day relative standard deviation (R.S.D.) for the assay precision was 0.64.8% and 1.36.5%, respectively. The R.S.D. for the accuracy measurements ranged from 3.1 to 5.7%. The lower limit of detection for levofloxacin was 0.157 ± 0.031 mg/L.
Pharmacokinetic parameters [elimination half-life and area under the concentrationtime curve (AUC08)] in plasma and CSF were calculated using non-linear regression where appropriate. The pharmacokinetic model was selected using Akaike Information Criteria.12 Maximum levofloxacin concentrations (Cmax) in plasma and CSF were determined by inspection of the concentrationtime curves. The volume of distribution was calculated using a standard pharmacokinetic equation.13 CSF penetration was determined by effluent concentrations expressed as a percentage {area under the CSF concentration time curve [AUCCSF(08)] to the AUCserum(08) x 100 after probe calibration}. All values were expressed as mean ± S.D.
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Results |
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The protein binding of levofloxacin in the rabbit plasma averaged 25%. Plasma concentration results were fitted to a two-compartment iv administration model. The protein-free plasma concentrationtime curves for the three different dosages used in these experiments are depicted in Figure 1. The free peak plasma concentrations of levofloxacin were 3.9, 6.4 and 10.3 mg/L, respectively. All three concentrations occurred at 0.25 h after the end of the infusion. There was no difference in the plasma elimination half-lives or the volume of distribution for the three doses studied. The plasma elimination half-lives were then calculated and found to be 7.6 ± 3.5, 7 ± 1.6 and 9.5 ± 3.5 h. There was a significant increase in the plasma AUC08 (29.7 ± 6.3, 49.1 ± 19.1 and 67.6 ± 8.9 mgh/L) for the three different doses investigated. Based on the data, the plasma AUC08 appears to increase proportionately slightly more than dose.
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Discussion |
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These results are similar to other reports that have used levofloxacin and other quinolones. Pefloxacin CNS penetration was found to be 51% in infected rabbits.14 In patients treated with iv amoxycillin and ofloxacin 200 mg orally twice daily, the CSF concentration was reported to reach levels between 50 and 60% of concurrent serum levels.15,16 Finally, diagnostic taps in 17 patients revealed 4787% penetration of ofloxacin into the CSF.17
The pharmacodynamics of levofloxacin have been investigated in humans. For infections other than meningitis, the antibacterial effect of levofloxacin has been reported to be dependent on plasma concentrations being above the MIC of the organism.18 The bactericidal properties of levofloxacin in the CSF have been characterized in a similar rabbit model by Nau et al.19 In this study, levofloxacin showed a positive correlation between bactericidal rates in the CSF and the CSF concentration, relative to the MBC. Studies of ciprofloxacin and gatifloxacin have also demonstrated that bactericidal effect is proportional to the dose of the drug administered.6,20
Studies of newer quinolones have included those on gatifloxacin in which the percentage penetration of three doses ranged from 56% for 7.5 mg/kg to 46% for 30 mg/kg in a rabbit meningitis model.20 The percentage penetration of the iv form of trovafloxacin averaged 25% in healthy volunteers in the absence of meningeal inflammation.21 Higher concentrations would be expected in the presence of meningeal inflammation, due to the antibiotic lipophilicity and molecular size.22 Finally, three dosages (2.5, 10 and 20 mg/kg/h) of moxifloxacin given as a continuous infusion produced a CSF concentration to serum ratio that ranged from 36% to 47% (16) in rabbits.23
It appears from these data that the penetration of levofloxacin into the CSF averages 66% for the doses and dosing intervals studied; however, it is not FDA approved for thrice-daily dosing. The empirical treatment of pneumococcal meningitis in adults and children is still a ß-lactam agent with vancomycin. Further work may be necessary to determine whether dexamethasone influences levofloxacin CSF penetration.
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Acknowledgments |
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Notes |
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References |
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Schmidt, H., Dalhoff, A., Stuertz, K., Trostdorf, F., Chien, V., Schneider, O. et al. (1998). Moxifloxacin in the therapy of experimental pneumococcal meningitis. Antimicrobial Agents and Chemotherapy 42, 1397401.
Received 6 September 2000; returned 6 November 2000; revised 7 December 2000; accepted 29 January 2001