No effect of protein binding ratio of levofloxacin in hemodialysis patients
Materials
LVFX and pazufloxacin (PZFX) were provided by Daiichi Sankyo Co., Ltd. (Tokyo, Japan)
and Taisho-Toyama Pharmaceuticals Co., Ltd. (Tokyo, Japan), respectively. Oasis HLB
1-cm
3
(30-mg) extraction cartridges were obtained commercially from Waters Corporation (Milford,
MA). Human albumin and globulin were purchased from Wako Pure Chemical Industries
(Osaka, Japan). All other chemicals were of analytical grade and commercially available.
Subjects
Clinical laboratory data were collected from 13 patients, including 7 non-HD patients
and 6 HD patients, who were undergoing LVFX therapy for pneumonia, urinary tract infection,
osteomyelitis, and unidentified infections from June 2012 to December 2013. Non-HD
patients received LVFX 500 mg once daily orally, and HD patients received LVFX 500 mg
initially, followed by 250 mg after each HD session orally. All subjects were administered
LVFX therapy for 4–48 days. HD patients underwent thrice-weekly HD in the LVFX treatment
period. Written informed consent was obtained from all subjects before enrollment
in this study. This study and its protocol were approved by the Ethics Committee of
Kaetsu Hospital.
Blood sampling
To determine the serum concentration of LVFX and to obtain other laboratory data,
blood samples were obtained just before HD and on non-HD days in HD patients and just
before LVFX administration and 12 h after administration in non-HD patients. Blood
sampling times were different in order to obtain appropriate clinical blood samples
to evaluate infectious diseases. Blood samples were obtained 2–18 days after the administration
of LVFX was initiated. Both total and free fraction concentrations of LVFX were measured
in the 13 patients to determine the correlations between serum albumin, serum globulin,
serum concentration of LVFX, and protein binding ratio of LVFX. To clarify the relationship
between protein concentration and the protein binding ratio of LVFX in vitro and in
vivo, the effect of human albumin and globulin on the protein binding ratio of LVFX
in phosphate buffer (pH 7.4) was investigated.
Preparation of sample solutions
To determine the relationship between the serum concentration (1, 2, 4, 8, 10, and
20 mg/L as the general serum concentration and 50 and 100 mg/L as the high serum concentration)
and the protein binding ratio of LVFX in vitro, the effect of the protein binding
ratio was investigated using 0.025 M phosphate buffer solution (pH 7.5). To determine
the relationship between the protein concentration and the protein binding ratio of
LVFX in vitro, the effect of human albumin and globulin on the binding ratio was investigated
using 0.025 M phosphate buffer solution (pH 7.5). After the samples were incubated
for 30 min at 37 °C, the free fraction of the samples was obtained by ultrafiltration
(molecular weight cutoff, 10,000 Da).
Assay
Serum LVFX concentrations were determined by HPLC using PZFX as the internal standard
(IS). Briefly, sample processing was performed using a solid phase extraction cartridge.
The cartridges were conditioned with methanol and water. After the sample (100 ?L
serum sample?+?100 ?L IS?+?900 ?L 0.025 M phosphate buffer solution; pH 7.5) was loaded,
the cartridge was washed with 1 mL of 5 % methanol in water. The sample was eluted
with 1 mL of mobile phase, and 50 ?L was injected onto the HPLC column for analysis.
The HPLC system consisted of a reverse-phase column (Shim-pack, CLC-CN, Shimadzu Corp.,
Kyoto, Japan), and the fluorescence of the mobile phase was monitored (excitation
wavelength, 278 nm; emission wavelength, 445 nm). The mobile phase consisted of a
mixture (pH 3.0) of phosphate buffer, acetonitrile, and triethylamine (171:29:1 by
volume), and the flow rate was 1.0 mL/min. The retention times of the IS and LVFX
were 6.0 and 6.8 min, respectively. The lower limit of quantification of this system
was 0.05 ?g/mL in 0.1 mL of serum. Inter- and intra-day variations were 5.0 %. The
free fractions of the serum and spiked samples were obtained by ultrafiltration using
a disposable ultrafilter (Kurabo Industries, Ltd., Osaka, Japan). The protein binding
ratio of LVFX was calculated from the total and free concentration of LVFX.
Statistical analysis
Data are expressed as mean?±?standard deviation. Statistical analysis was performed
using the Student’s t test or Pearson’s correlation coefficient test, and significance was set at p??0.05. JMP 9 Software (SAS Institute Inc., Cary, NC) was used for all statistical
analyses.