However, this seems to be a considerably excessive dosing because of the high value of the Ž
. average steady-state serum concentration Css: 55.4 mgrml . q 2001 Elsevier Science B.V. All
rights reserved.
Keywords: Pharmacokinetics; Metabolism; Eel; Miloxacin; Bioavailability
1. Introduction
Ž w
x Miloxacin
5,8-dihydro-5-methoxy-8-oxo-2 H-1,3-dioxolo- 4,5-g quinoline-7-carbo- .
xylic acid which is closely related to oxolinic acid in structure, has exhibited a broad spectrum of antibacterial activity and is especially active against gram-negative bacteria.
Fig. 1 shows the chemical structure of miloxacin. The drug has been used for treatment of edwardsiellosis in cultured eel and is regulated in conformity with the Pharmaceutical
Law in Japan. However, there are few papers concerning miloxacin in cultured eel,
Ž .
although there have been some studies of miloxacin in yellowtail Ueno et al., 1985a,b . The present paper deals with the pharmacokinetics and metabolism of miloxacin after
administration in cultured eel. Miloxacin and its metabolite 5,8-dihydro-8-oxo-2 H-1,3- w
x Ž
. dioxolo- 4,5-g quinoline-7-carboxylic acid M-1 , which also has antibacterial activity
Ž .
Izawa et al., 1978 , were simultaneously determined with our newly developed high- Ž
. performance liquid chromatographic HPLC method.
2. Materials and methods
2.1. Fish Japanese eel Anguilla japonica were obtained from the Fisheries Research Institute
in Aichi Prefecture, Japan. The average body weight was 175 g. The fish were kept in tanks with running filtered water. The average water temperature was 278C.
2.2. Chemicals Ž
. Miloxacin and M-1 were obtained from Sumitomo Pharmaceutical, Osaka, Japan .
Ž .
b-Glucuronidase bovine liver, 78,000 Fishman unitsrg was from Tokyo Kasei Kogyo Ž
. Tokyo, Japan . Other chemicals were of analytical or HPLC grade.
Fig. 1. Chemical structure of miloxacin.
2.3. Drug administration Fish were anesthetized by placing them in ice-water for 5 min. For intravascular
Ž .
administration, miloxacin was dissolved in sterilized saline 100 mgrml and injected into the caudal vein at a dose of 30 mgrkg of body weight. The drug was mixed with a
fish diet and was orally given to the fish by a catheter at a dosage of 60 mgrkg body weight. The drug was given either in a single dose in 1 day or in six doses over a 6-day
period. Then, five fish were sampled at intervals ranging from 0.5 or 1 h to 20 days after the administration. The blood was sampled from the caudal vein with a syringe. The
serum was obtained by centrifugation of the blood after storage overnight in a refrigerator and kept frozen at y408C until analysis. Each sample was analyzed by
HPLC. The muscle, liver, kidney, and bile were also collected. Samples of each type were pooled and stored at y408C.
2.4. Assay procedure Ž
. Tissue samples 1 g muscle or 1 ml serum were homogenized for 2 min in 30 ml of
Ž .
0.1 M citrate buffer, pH 3.0: N, N-dimethylformamide 29:1
using a Physcotron Ž
. Nichi-On K.K., Tokyo, Japan . After centrifugation at 15,000 rpm for 20 min, the
supernatant was transferred into a 500 ml-separatory funnel, and 5.0 g of NaCl and 15 ml of ethyl acetate were added to the solution. The funnel was then gently shaken for 5
min. After standing for a few minutes, the organic layer was pooled. The ethyl acetate treatment was repeated two more times, and then the aqueous layer was adjusted to pH
11–12 by 6 N NaOH and re-extracted twice with 15 ml of ethyl acetate. The aqueous layer was discarded. The pooled ethyl acetate layer was evaporated to dryness. The
residue was dissolved in 1 ml of 1 Na CO and the solution was injected into the
2 3
HPLC. Ž
. Tissue samples 0.5 g liver or kidney, 0.3 ml bile was homogenized and extracted by
the same ethyl acetate treatment as described above. The aqueous layer was discarded. The pooled ethyl acetate layer was concentrated to ca. 30 ml in vacuo. Fifteen milliliters
of saturated NaCl was added to the concentrate. The sample was shaken vigorously for 5 min and centrifuged. The aqueous layer was discarded, and the resulting organic layer
was evaporated to dryness. The residue was dissolved in a mixture of 5 ml of 1 Na CO , 25 ml of 0.1 M citrate buffer, pH 3.0 and 2.0 g of NaCl. Fifteen milliliters of
2 3
n-hexane was added to the solution. The sample was shaken vigorously for 5 min and centrifuged at 3,000 rpm for 5 min. The organic layer was discarded. The resulting
Ž .
aqueous layer was poured into a Sep-Pak C cartridge Waters, Milford, MA, USA ,
18
which had previously been washed and wetted with 10 ml of methanol and 15 ml of water. The cartridge was washed with 10 ml of water, and then miloxacin was eluted
with 20 ml of methanol. The eluate was evaporated to dryness, and the residue was dissolved in 1 ml of 1 Na CO and the solution was injected into the HPLC.
2 3
The HPLC system consisted of a Gilson Model 802 pump and 311A UV detector Ž
. Ž
Gilson, France and a Chromatopac C-R3A integrator Shimadzu Seisakusho, Kyoto, .
Ž Japan . The analytical column was a YMC-Pack C
A-303 prepacked column 25
18
. cm = 4.6 mm I.D., Yamamura Chemical Lab., Kyoto, Japan . The mobile phase was
Ž .
0.1 trifluoroacetic acid: N, N-dimethylformamide:acetonitrile 72:1:27 . The flow rate was 1.0 mlrmin, and the UV detector was set at 254 nm. The injection volume was 20
ml. The column temperature was 308C. A standard solution containing of 100 mgrml miloxacin and 100 mgrml of M-1 was
prepared in 1 Na CO . The solution was diluted to the required concentration with 1
2 3
Na CO before use.
2 3
2.5. Pharmacokinetic analysis The most common method of pharmacokinetic evaluation is to assume that the drug
concentration-time data can be described by one of several compartment models and to fit the data to an equation consistent with the assumed model using a non-linear
least-squares regression. In our study, a pharmacokinetic analysis was applied assuming a one- or two-compartment model using the non-linear least-squares program MULTI
Ž
. Yamaoka et al., 1981 . Selection of models was judged by Akaike’s information
Ž .
criterion Yamaoka et al., 1978 . Ž
. Wagner and Nelson 1964 reported that the drug absorption rate could be calculated
from serum level vs. time data using the following equation when the behavior of the drug is expressed by a one-compartment model:
t
C q Ke Cd t
H
t
A
t
Fraction absorbed s s
`
A
`
Ke Cd t
H
where A is the cumulative amount of the drug absorbed up to time t, A is the amount
t `
of drug ultimately absorbed. C is the concentration at time t, and Ke is the first-order
t
Ž .
elimination rate constant the value for the drug following intravascular administration . This equation relates the cumulative amount of drug absorbed after a certain time to
the amount of drug ultimately absorbed, rather than to the dose administered. 2.6. ConsecutiÕe oral administration
Ž .
The serum level during multiple oral dosing of a constant dose Cn and average Ž
. steady-state serum concentration Css can be estimated according to the formula:
Ž .
Ž .
ynPKePt ynPKaPt
F P DoseP Ka 1 y e
1 y e
Ž .
Ž .
yKePt yKaPt
Cn s e
y e
Ž .
Ž .
yKePt yKaPt
Vd Ka y Ke 1 y e
1 y e
Ž .
F P Dose Css s
Vd P Ke P t where F is the bioavailability, Ka is the first-order absorption rate constant, Vd is the
apparent volume of distribution, t is the dosage interval, n is the dosage time, and Ke is Ž
the first-order elimination rate constant the value for the drug following oral administra- .
tion .
2.7. Statistical moment analysis Ž
. The area under the concentration-time curve AUC was calculated by using the
Ž .
trapezoid rule including the terminal portion. The mean residence time MRT of the drug was obtained by a non-compartment analysis based on the statistical moment
Ž .
theory Yamaoka and Tanigawara, 1983 . The bioavailability was calculated from the following equation:
AUC P dose
p .o . i . v .
F s 100
Ž .
AUC P dose
i . v . p .o .
where p.o. represents the oral administration, and i.v. represents the intravascular administration.
2.8. Presence of glucuronide conjugate Some of the residues that were previously prepared for miloxacin and M-1 extraction
were used for determination of glucuronide conjugates. These residues were from samples that were taken at the time of maximum serum concentration of miloxacin
Ž .
T and at 20 days post dosing. Each residue was dissolved in 100 ml of 0.1 M citrate
max
buffer, pH 3.0, and homogenized for 1 min. Five milligrams of b-glucuronidase and 1 ml of toluene were added to the homogenate as an antiseptic. The mixture was incubated
at 378C overnight. After incubation, the reaction mixture was concentrated to ca. 30 ml in vacuo. The released miloxacin and M-1 were re-extracted from the concentrate as
described in the text.
3. Results
