3. Results
3.1. Method of analysis By reducing the washing volume and the elution volume in the SPE method, the
recovery for OTC and TC from plasma was increased from 45.3 12.4 and 40.1 Ž
. 12.0 to 83.0 8.9 and 78.5 8.7 n s 10 , respectively. The retention time for
OTC and TC in the HPLC system was 3.07 and 3.59 min, respectively. The LOD was Ž
. calculated to be 0.05 mg OTCrml. The response of the UV detector peak height
Ž .
correlated well with OTC concentrations r s 0.9985 and was found to be linear over the range from 0.1 to 5.0 mg OTCrml; thus, 0.1 mg OTCrml was used as LOQ. Three
samples from fish given 50 mgrkg of OTC in agar and one sample from fish given 100 mgrkg of OTC in liposomeralginate contained concentrations above LOD but below
LOQ, and here the concentrations were quantitated by extrapolation of the standard curve. Plasma samples with concentrations above 5.0 mg OTCrml were diluted in water
and reanalysed.
Table 1 Ž
. Pharmacokinetic parameters mean valueS.D. for OTC after i.v. administration to Arctic charr
Abbrevations: A, B, C: zero-time plasma drug concentration intercepts of triphasic disposition curve; C :
zero-time plasma concentration of OTC; a , b : distribution rate constants; g : elimination rate constant; t
, t : distribution half-lives and elimination half-life; AUC: area under the concentration–time curve;
1r 2 a , b 1r2g
AUMC: area under the curve of a plot of the product of time and plasma drug concentration–time; Cl : total
T
body clearance; V : apparent volume of distribution; V
: apparent volume of distribution at steady state;
dŽa rea. dŽss.
V : apparent volume of the central compartment; MRT: mean residence time.
c
Parameter Value
Ž . Fish weight g
454149 37621
Number of fish 6
6 Ž
. Dose mgrkg
10 20
Ž .
A mgrml 65.6923.75
160.3022.26 Ž
. B mgrml
9.283.48 31.3523.12
Ž .
C mgrml 3.571.55
5.341.32 Ž
. C
mgrml 78.5320.66
196.9826.84
y1
Ž .
a h 0.4680.083
0.4080.126
y1
Ž .
b h 0.0640.035
0.0820.052
y1
Ž .
g h 0.0030.001
0.0020.000 Ž .
t h
1.50.3 1.80.5
1r 2 a
Ž . t
h 16.514.5
12.28.3
1r 2 b
Ž . t
h 266.361.7
326.960.4
1r 2g
Ž .
AUC mg hrml
1591.4369.5 3321.0708.3
0 – ` 2
Ž .
AUMC mg h rml
478,667147,033 1,237,080574,494
0 – `
AUCrDose 159.137.0
166.135.4
y1 y1
Ž .
Cl ml kg
h 6.541.33
6.271.39
T
Ž .
V lrkg
2.571.03 2.900.61
dŽa rea.
Ž .
V lrkg
1.980.60 2.140.32
dŽs s.
Ž .
V lrkg
0.130.03 0.100.02
c
Ž . MRT h
301.249.2 357.197.6
3.2. The i.Õ. administration The plasma concentration–time curves for both doses were best described with the
tri-exponential three-compartment open model: C s Ae
ya t
q Be
yb t
q Ce
yg t
where C
p p
is the concentration of OTC in plasma; A, B and C are zero-time plasma drug concentration intercepts of the triphasic concentration–time curve; a and b are
distribution rate constants; g is the elimination rate constant; t is time; and e represents the base of the natural logarithm. The observed individual plasma drug concentrations of
OTC after i.v. administration are depicted in Figs. 1 and 2. The pharmacokinetic parameters for OTC after i.v. administration are listed in Table 1.
Ž .
The volume of distribution V was 2.57 1.03 and 2.90 0.61 lrkg at doses
dŽa rea.
of 10 and 20 mgrkg, respectively. The volume of distribution of the central compart- Ž
. Ž
. ment V
ranged from 0.10 to 0.13 lrkg. Total body clearance Cl was 6.54 1.33
c T
ml kg
y1
h
y1
at the lower dose and 6.27 1.39 ml kg
y1
h
y1
at the higher dose. The Ž
. elimination half-life t
of the terminal part of the elimination phase was estimated
1r2g
to be 266.3 61.7 and 326.9 60.4 h at 10 and 20 mgrkg, respectively. The MRT was calculated to be 301.2 49.2 and 357.1 97.6 h in the low and high doses, respec-
tively.
Ž .
Fig. 3. Semilogarithmic plot of plasma OTC concentration vs. time in Arctic charr ns 7
after p.o. Ž
. administration of a single dose of 50 mgrkg body weight agar formulation . Each curve represents data from
one fish.
3.3. The p.o. administration The observed individual plasma drug concentrations of OTC after p.o. administration
of agar formulations and the liposomeralginate formulation are depicted in Figs. 3–5. Ž
. The maximum plasma concentration of OTC C
, 1.51 0.86 mgrml, was achieved
max
30.3 33.3 h after p.o. administration of 50 mgrkg in agar. At 100 mgrkg, the C
ma x
Ž .
3.93 0.99 mgrml
was achieved
17.8 17.4 h
after administration.
The Ž
. Ž
. liposomeralginate formulation 100 mgrkg delayed the C
0.97 0.33 mgrml to
max
136.0 60.4 h after administration. A large individual variation in the C and in the
ma x
Ž .
time to reach C T
was observed. With the agar formulations, the T ranged
max max
max
from 4 to 96 h, while with the liposomeralginate formulation, the T ranged from 72
max
to 192 h. Ž
. The mean bioavailability of OTC was estimated to be 4.2 1.3 50 mgrkg
and Ž
. 7.3 4.0 100 mgrkg
in the agar formulations, respectively. The mean bioavailabil- Ž
. ity of the liposomeralginate formulation 100 mgrkg was estimated to be 3.2 0.9.
Ž .
The elimination half-life t after p.o. administration was apparently longer than the
1r2
t after i.v. administration, with values ranging from 367.0 to 468.5 h. The pharma-
1r2
cokinetic parameters for OTC after p.o. administration are given in Table 2.
Ž .
Fig. 4. Semilogarithmic plot of plasma OTC concentration vs. time in Arctic charr ns9
after p.o. Ž
. administration of a single dose of 100 mgrkg body weight agar formulation . Each curve represents data from
one fish.
Ž .
Fig. 5. Semilogarithmic plot of plasma OTC concentration vs. time in Arctic charr ns 3
after p.o. Ž
. administration of a single dose of 100 mgrkg body weight liposomeralginate formulation . Each curve
represents data from one fish.
It was observed that feed intake and swimming activity were reduced during the experimental period in all test groups.
Table 2 Ž
. Pharmacokinetic parameters mean valueS.D. for OTC after p.o. administration to Arctic charr
Abbrevations: C : maximum concentration; T
: time to reach maximum concentration; k : elimination
ma x max
e
rate constant; t : elimination half-life; AUC: area under the concentration–time curve; F: bioavalability.
1r 2
Parameter Value
Ž . Fish weight g
541107 562179
707107 Number of fish
7 9
3 Formulation
agar agar
liposomeralginate Ž
. Dose mgrkg
50 100
100 Ž
. C
mgrml 1.510.86
3.930.99 0.970.33
ma x
Range 0.82–3.03
2.66–5.36 0.61–1.27
Ž . T
h 30.333.3
17.817.4 136.060.4
ma x
Range 8–96
4–72 72–192
y1
Ž .
k h
0.0020.001 0.0020.001
0.0020.000
e
Ž . t
h 367.0175.8
444.2133.3 468.585.1
1r 2
Ž .
AUC mg hrml
341.9105.2 1188.1643.4
510.6160.5
0 – `
AUCrDose 6.82.1
11.96.4 5.21.5
Ž . F
4.21.3 7.34.0
3.20.9
4. Discussion
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