safe withdrawal periods, and to minimize the environmental effects of the drugs used in aquaculture. The pharmacokinetics of drugs may be affected by parameters such as fish
species, age, water temperature and salinity, route of administration and other experi- mental conditions. Therefore, the extrapolation of pharmacokinetic data obtained in one
species to another species, living under different conditions, should be done with caution.
The aim of the present study was twofold; firstly, to describe the pharmacokinetics of Ž
. Ž
. OTC in Arctic charr, living at low temperatures, after intravascular i.v. and oral p.o.
administration, and secondly, to determine the relative bioavailability of OTC after p.o. administration of a liposomeralginate formulation compared to the drug dissolved in
water with agar as a viscosity-increasing agent.
2. Materials and methods
2.1. Chemicals Ž
. Acetonitrile Rathburn Chemicals, Walkerburn, Scotland and N, N-dimethylform-
Ž .
amide Fluka, Buchs, Switzerland were both of high-performance liquid chromatogra- Ž
. phy HPLC -grade. All other chemicals were of analytical grade. The water used was
Ž .
distilled and purified with a Milli-Q reagent grade water system Millipore, MA, USA . OTC hydrochloride and tetracycline hydrochloride were purchased from the Norwe-
Ž .
gian Medicinal Depot Oslo, Norway . The formulation of OTC for i.v. administration Ž
. was made by solving OTC–HCl in 0.9 saline
10 and 20 mgrml . The oral Ž
formulations were made either by suspending the drug in 0.5 Bacto-Agar Difco .
Laboratories, Detroit, MI, USA or by encapsulating it into liposomeralginate particles Ž
. see below . Dosing solutions of OTC in agar were prepared at 50 and 100 mgrml.
Methanolic stock solutions of the two tetracyclines for analytical purposes were pre- pared at a concentration of 1 mgrml and stored in the dark at y208C. Working
standards were prepared daily by dilution from the stock solutions.
2.2. Preparation of OTC liposome r alginate particles Liposomeralginate particles containing OTC were prepared by mixing 6 g OTC with
Ž Ž .
. 6 g soya bean phospholipids Pro-Lipo S ; Lucas Meyer, Hamburg, Germany and
adding 20 ml of water dropwise. After 15 min of stirring, 80 ml of water was added, and the solution was set for sedimentation for 16 h at q48C. The excess of OTC was
removed from the preparation by centrifugation at 27,000 = g for 1 h and the super- natant was discarded. A volume of 140 ml of water was added to the liposome pellet,
the solution was centrifuged at 27,000 = g for 1.5 h, and the supernatant was discarded.
Ž .
Ž The pellet 9.2 g was mixed with 9.2 ml 2 Na-alginate Protanal LF 10r40 RB;
. Protan Biopolymer, Drammen, Norway . The solution was stirred and allowed to stand
for 10 min in order to remove air bubbles from the preparation. The suspension was loaded into a 10-ml syringe with a 27-gauge needle attached and sprayed down into
a solution of 0.5 M CaCl , which induced gelation of the alginate. In this manner,
2
0.5–2.0 mm diameter liposomeralginate particles were formed. After 2 h, the particles were washed in water in order to remove salt and free OTC, and stored at q48C until
administration. The concentration of OTC in the particles was determined by suspending crushed particles in water and analyzing the material by SPE and HPLC. The concentra-
Ž .
tion of OTC was found to be 79.2 7.9 mg OTCrg particles n s 4 . The particles were suspended in water to a concentration of 100 mg OTCrml before administration.
2.3. Experimental fish Ž
. Ž
. Healthy Arctic charr S. alpinus L. weighing 514 154 g mean S.D. , obtained
Ž .
from the Aquaculture Research Station Tromsø, Norway , were used. The fish were held in fibre-glass circulation tanks, supplied with aerated freshwater at a temperature
ranging from 4.28C to 8.68C, with a mean value of 6.38C. The fish were fed a commercial pelleted fish diet.
2.4. Experimental procedure Dorsal aorta cannulation was performed by a modification of the procedures of
Ž .
Ž .
Houston 1971 and Soivio et al. 1972,1975 . The fish were anaesthetized in well-aerated Ž
. water containing 100 mgrl tricaine methane sulphonate MS-222, Sigma and 200 mgrl
Ž .
sodium hydrogen carbonate NaHCO , Sigma . When the fish showed no respiratory
3
activity, they were taken out of the water, weighed, and placed on an operating table. Ž
The cannula consisted of a 35-cm polyethylene tubing PE 50, intramedic non-radio- .
Ž .
opaque, non-toxic; Clay Adams, NJ, USA . The top ca. 0.5 mm of a 1-ml pipette tip was pulled outside the tubing, ca. 5 cm from the end, which was to be inserted into the
aorta. Ž
. A guitar string d ; 0.5 mm was placed inside the cannula, and the cannula was
inserted through the palate between the first and second gill arches and pushed ca. 2 cm into the dorsal aorta. Upon successful entry into the aorta, blood filled the cannula as the
guitar string was removed. A tube clip was put on the cannula to prevent blood loss. A 17-gauge = 2 in. needle was inserted through the snout and the distal end of the cannula
was guided outside through the needle. The pipette tip prevented the cannula from being pulled out of the fish snout and the dorsal aorta. The needle was then removed and the
Ž .
cannula was flushed and filled with heparinized 200 IErml 0.9 saline, and melted at the end with a flame. The cannula was sutured to the roof of the mouth.
During the experiments, the fish were individually housed in flowthrough chambers Ž
. as described by Soivio et al. 1975 , which allowed the i.v. administration of OTC via
the aortic cannula without removing the fish from their free-swimming state. The chambers were made of brown polyethylene tubing with a diameter of 15 cm, a length
of 60 cm, and had a nylon net with a 15-mm mesh at each end. Fish for i.v. administration were allowed to recover from surgery and adapt to the chambers for 1
week before the drug was administered. Fish for p.o. administration were allowed to recover for 1 week in a 500-l tank before drug administration and placing in the
sampling chambers. Fish with a non-functional cannula were excluded from the study prior to drug administration.
2.5. Drug administration and sampling The fish were fasted for 2 days before administration of the drug. Dosing formula-
tions were administered at 1 mlrkg body weight. The i.v. administration was made Ž
. Ž
. directly into the dorsal aorta via the cannula at doses of 10 n s 6 or 20 n s 6 mg
OTCrkg body weight. The fish given OTC orally were lightly sedated with MS-222 before dosing. The drug formulation was given by gavage into the stomach, using a tube
Ž .
Ž which was connected to a disposable syringe. The doses were 50 n s 7 and 100 agar:
. n s 9, liposomeralginate: n s 3 mg OTCrkg body weight. The fish were placed in the
sampling chambers immediately after drug administration. Ž
. Blood ca. 600 ml was sampled via the aortic cannula at 1, 4, 8, 12, 24 and 48 h, and
at regular time intervals between 3 and 20 days after i.v. administration. In the fish given OTC orally, blood samples were taken at 4, 8, 12, 24, 48, 72 and 96 h, and at regular
intervals between 6 and 24 days after administration. In the group given 50 mgrkg OTC, p.o., additional samples were taken from four fishes after 33 days. The heparin–
saline solution was removed from the cannula before each sampling and discarded. The blood was centrifuged for 2 min at 10,000 = g and the obtained plasma was stored at
y208C until analysis.
2.6. Analytical procedures Ž
. The analysis of OTC in plasma was performed by the method of Bjorklund 1988 ,
¨
with minor modifications. Due to different analytical equipment, the injection volume and the mobile phase of the HPLC method had to be adjusted in order to obtain
Ž symmetric peaks and satisfactory resolution between OTC and tetracycline TC; used as
. Ž
. Ž
. internal standard . The solid phase extraction SPE method used by Bjorklund 1988
¨
gave a relatively low recovery of the tetracyclines on Bond Elut C18 columns. In order to increase the recovery, the washing volume was reduced from 30 to 5 ml, and the
elution volume was reduced from 10 to 1 ml. By reducing the elution volume, a time-consuming evaporation step was also eliminated. Thus, the analytical procedure
used in the present study was as follows. Plasma samples were spiked with 0.5 mg TC,
Ž .
and 10 ml of cold q48C extraction buffer, containing 0.1 M citric acid and 0.2 M Ž
. Ž .
disodium hydrogenphosphate 62:38 pH 4.0 , was added. The samples were shaken
and sonicated for 5 min and allowed to stand for 15 min at q48C. The plasma samples Ž
were then transferred to 1 ml Bond Elut C18 SPE columns Varian, Harbor City, CA, .
Ž .
USA , which had been activated by flushing with 5 ml methanol and 10 ml 5 wrv disodium EDTA. After the samples had run through the columns, they were washed
with 5 ml water. OTC and TC were eluted with 1 ml of 0.01 M methanolic oxalic acid, and 10 ml aliquots of the eluate were subjected to reverse-phase HPLC. The analysis of
each plasma sample was performed in duplicates.
Ž .
The HPLC system Waters Assosiates, Millipore, MA, USA consisted of a model 600E system controller, a model 717 autosampler, and a model 484 variable wavelength
UV detector set at 355 nm. The response was recorded using a model 745 Data Module Ž
. integrator. Separation was achieved on a Nova-Pak RP-C18 column 100 = 3.9 mm i.d.
Ž .
Ž .
using a Guard-Pak 4.0 mm i.d. pre-column . The mobile phase consisted of 14 vrv
Ž .
Ž .
Ž acetonitrile, 6 vrv N, N-dimethylformamide and 80 vrv 0.01 M oxalic acid pH
. 2.1 and had a flow rate of 1 mlrmin. The HPLC system was operated at 258C.
Quantification of OTC was accomplished using a standard curve constructed on the basis of extracted OTCrTC ratio. Peak height was used to compare OTC and TC levels
in the plasma samples. The extraction recoveries of OTC and TC were determined by comparing peak heights from the analysis of plasma spiked with 0.1–5.0 mgrml with
peak heights resulting from direct injection of methanolic standards. The detection limit Ž
. LOD was defined as the concentration of OTC in plasma which gave an HPLC–UV
signal three times greater than the signal-to-noise ratio. The lowest calibration standard, Ž
. i.e., 0.1 mg OTCrml, was used as the limit of quantitation LOQ .
2.7. Pharmacokinetic and statistical analysis The plasma OTC concentration vs. time data were analysed for each fish and
treatment, using non-linear least-squares regression. The pharmacokinetic computer Ž
program, SAAM II, version 1.1 SAAM Institute, University of Washington, Seattle, .
WA, USA was used for the calculations. The data obtained in the groups given 10 and 20 mgrkg, i.v., were best described by a three-compartment open model. The model
was selected on the basis of the residual sum of squares and the minimum Akaike’s Ž
. information criterion AIC . All diffusion processes were assumed to follow first-order
Ž .
Fig. 1. Semilogarithmic plot of plasma OTC concentration vs. time in Arctic charr ns6
after i.v. administration of a single dose of 10 mgrkg body weight. Each curve represents data from one fish.
Ž .
kinetics. The area under the concentration–time curve AUC from zero to infinity was calculated using the formula: AUC s Ara q Brb q Crg . The mean residence time
Ž .
MRT was determined as: MRT s AUMCrAUC, where AUMC is the AUC of a plot of the product of time and plasma drug concentration vs. time, from zero to infinity
Ž .
Ritschel, 1992 . The serum concentration–time curves after oral dosing could not be fitted by the
non-linear least squares method using compartment models with first-order absorption. Ž
. The elimination rate constant
k after p.o. administration was estimated from the
e
Ž .
terminal part of the elimination curve using AIC , including at least the six last data Ž
. points. The elimination half-life t
of OTC was calculated using: t s ln 2rk . The
1r2 1r2
e
AUC was determined by the trapezoidal rule, including the terminal portion. The percent Ž .
oral bioavailability F
was determined by comparing the areas under the plasma Ž
. Ž
concentration vs. time curves AUC
using the equation: F s 100 = AUC =
p.o.
. Ž .
Dose r AUC
= Dose
, where Dose rAUC
is the mean value from both the
i.v. i.v.
p.o. i.v.
i.v.
Ž .
Ž .
10 and 20 mgrkg, i.v., study. The peak OTC concentration C and peak time T
ma x max
for each fish were read directly from the concentration vs. time curve. All statistical analyses were performed with Mann–Whitney’s non-parametric test,
Ž using the computer program, Statviewe SE q , version 1.04 Abacus Concepts, Berke-
. ley, CA, USA . The level of significance was chosen to be p s 0.05. The pharmacoki-
netic data are presented as mean S.D.
Ž .
Fig. 2. Semilogarithmic plot of plasma OTC concentration vs. time in Arctic charr ns6
after i.v. administration of a single dose of 20 mgrkg body weight. Each curve represents data from one fish.
3. Results
