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Pharmacokinetics and bioavailability of flumequine and oxolinic acid after various routes of administration to Atlantic salmon in seawater
207
Aquaculture, 110 (1993) 207-220 Elsevier Science Publishers B.V., Amsterdam AQUA 10004
Pharmacokinetics and bioavailability of flumequine and oxolinic acid after various routes of administration to Atlantic salmon in seawater Astri Rogstad, Odd F. Ellingsen and Christian Syvertsen Apothekernes Laboratorium AS, Skeyen, Oslo, Norway (Accepted
13 October 1992)
ABSTRACT Rogstad, A., Ellingsen, O.F. and Syvertsen, C., 1993. Pharmacokinetics and bioavailability of flumequine and oxolinic acid after various routes of administration to Atlantic salmon in seawater. Aquaculture, 110: 207-220. The present preclinical study was performed to investigate the pharmacokinetics of flumequine in Atlantic salmon (Safmo salar L. ) in seawater after administration of different doses and dosage formulations. Flumequine was administered intravenously (dose 4.9 mg/kg fish) and orally from the drug delivery system Aqualets as Apoquin 5 g/kg (dose 25 mg/kg) and 10 g/kg (dose 50 mg/kg), respectively. Experiments were carried out with oxolinic acid administered in the same way for the purpose of comparing the two compounds. The seawater temperature was 5 ? 0.2”C in all experiments. The pharmacokinetic calculations showed that the distribution half-life for flumequine was lia = 1.3 h and for oxolinic acid tla= 0.7 h. The drugs were absorbed rapidly, and flumequine reached a plasma concentration of C,, = 2.26 pgg/ml after a single oral dose of 25 mg/kg, whereas oxolinic acid reached C,.,=O.99 pg/ml. The apparent bioavailability of flumequine was found to be 40-45%, whereas the apparent bioavailability of oxolinic acid varied from 25% at a dose of 50 mg to 40°Yaat a dose of 25 mg/kg body weight of fish. The distribution profile of flumequine in the various compartments of fish appeared to be different from that of oxolinic acid. After a single oral dose (25 mg/kg) the areas under the concentration-time curves showed that flumequine was 2.3 times more concentrated in plasma and 2.6 times more concentrated in liver compared to oxolinic acid. in muscle the difference was less pronounced, flumequine being 1.4times more concentrated than oxolinic acid.
INTRODUCTION
Quinolones represent the latest generation of antibacterial agents. The compounds have broad spectrum activity particularly against Gram-negative bacteria, as well as fungi, protozoans and helminths. Oxolinic acid was introduced to Norwegian veterinary medicine in 1987. The drug has been applied C’orrespondenee to: A. Rogstad, Norway.
00448486/93/$06.00
Nycomed
Imaging A.S, P.O. Box 4220, Torshov. N-040 I Oslo,
0 1993 Elsevier Science Publishers
B.V. All rights reserved.
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A. ROGSTAD ET AL.
particularly in aquaculture in Norway and has appeared to be efficient against a number of Gram-negative bacteria that cause diseases such as furunculosis, yersiniosis, vibriosis and cold-water vibriosis. However, this antibiotic was reported to have been tested on fish (Endo et al., 1973a,b) as early as 1973, and over the last decade has been administered routinely to cultured fish in many countries both as a prophylactic and as a chemotherapeutic agent (Austin et al., 1983; Rodgers and Austin, 1983; Austin and Austin, 1987). The fluoroquinolones are second generation 4-quinolones. Flumequine is a representative of the fluoroquinolone derivatives, and its activity has been studied both in vitro and in vivo. In vivo studies have been carried out in both mammals (Dorrestein et al., 1983; Ziv et al., 1986; Mevius et al., 1989) and fish (Michel et al., 1980; Chevalier et al,. 198 1; O’Grady et al., 1988 ) during the last decade, and some recent in vitro studies on its activity against fishpathogenic Aeromonas sulmonicida were performed by Barnes et al. ( 199 1a,b ) . The latter studies showed that flumequine is preferable to oxolinic acid with regard to its microbiological activity. Studies on its pharmacokinetic properties in domestic animals showed that flumequine was absorbed well after oral administration. O’Grady et al. ( 1988) reported that flumequine was rapidly distributed to all tissues in high concentrations when administered to farmed fish. The aim of this study was to investigate the absorption, distribution, elimination and bioavailability of flumequine (FQ) in Atlantic salmon after oral administration of Apoquin Aqualets vet. 5 g/kg and 10 g/kg, respectively. The results have been compared with results from similar studies on oxolinic acid administered as Apoxolon Aqualets vet. 5 g/kg and as medicated feed 5 g/kg and 10 g/kg. Intravenous injections of flumequine and oxolinic acid to fish were also performed. Pharmacokinetic parameters and apparent bioavailability have been calculated. Our results were compared with pharmacokinetic values obtained for oxolinic acid by Hustvedt ( 199 1) and Bjiirklund (1991). MATERIALS
AND METHODS
Chemicals and medicatedfish pellets Flumequine and oxolinic acid were purchased from Welding Pharma A.G. (Hamburg, Germany). Tricaine methanesulphonate (MS-222) was obtained from Sandoz (Basel, Switzerland). Alcian Blue GS was supplied from Sigma Chemicals (St. Louis, MO, USA). The following five types of medicated products for fish were tested: - Apoquin Aqualets vet., medicinal pellets with flumequine 10 g/kg, batch no. 8905 1, d = 6 mm, Apothekemes Laboratorium (Oslo, Norway) - Apoquin Aqualets vet., medicinal pellets with flumequine 5 g/kg, batch no. 89057, d= 6 mm, Apothekemes Laboratorium (Oslo, Norway)
FLUMEQUINE AND OXOLINIC ACID ADMINISTERED TO ATLANTIC SALMON
209
-
Apoxolon Aqualets vet., medicinal pellets with oxolinic acid 5 g/kg, batch no. 89030, d = 6 mm, Apothekernes Laboratorium (Oslo, Norway) - Medicated feed with oxolinic acid 5 g/kg, Ewos Aqua (Lorenskog, Norway) - Medicated feed with oxolinic acid 10 g/kg, Ewos Aqua (Lorenskog, Norway). AqualetsTM is the trade name of a drug delivery system for fish developed by Apothekernes Laboratorium A.S (Ellingsen et al., 1989). The product consists of co-extruded pellets with a two-layer structure. The outer layer is made of nutritional components and binding agents. The drug filling in the inner core consists of a suspension of the antibiotic and pharmaceutical excipients in an appropriate solvent. Solutions for intravenous injection were prepared by dissolving flumequine and oxolinic acid, respectively, in 0.1 M NaOH to a concentration of 4.9 mg/ml and adjusting the pH of the solutions to 9 in the case of flumequine and to 10 in the case of oxolinic acid. Experimental conditions Atlantic salmon (Salmo salar L. ) (av. weight 425 g) were obtained from a local fish farm and held in six cylindrical libre glass tanks of ca. 1.8 m3 with a continuous flow of seawater (temp. 5 + 0.2”C, salinity 30%0, flow rate 10 l/min). Each tank contained 1O-20 fish. Fish were anaesthetized with tricaine methanesulphonate, weighed and individually marked with Alcian Blue on their ventral sides. The fish were then starved for 3 days, and anaesthetized before a dose of flumequine and oxolinic acid (Aqualets or medicated feed), respectively, corresponding to 0.5% of body weight of the fish was force fed into the stomach using a syringe. The doses were 25 and 50 mg antibiotic per kg fish. Intravenous injections of flumequine and oxolinic acid, respectively, were made into the caudal vein. The dose was 4.9 mg/ kg fish. The fish were starved for 3 days, anaesthetized and weighed. The drug was administered by aspirating blood into the syringe prior to the injection to ensure that the syringe with the drug solution was located in the caudal vein. While injecting, the syringe was held steady and the position of the needle was tested occasionally by aspirating blood into the syringe. Blood was sampled from the caudal vein using Venoject blood sampling equipment (0.9 x 4.0 mm needles) with heparin. In the case of injection, the first sampling was performed after 30 min and then at periodic intervals for 96 h, whereas in the case of oral administration, the first sample was taken after 4 h and then at periodic intervals for 96 h. Blood was collected from five fish at each sampling time. Plasma was isolated by centrifugation of the blood at 4000 rpm for 10 min. The samples were immediately frozen and stored at - 20” C until the analyses were performed.
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Fish were not fed ordinary (unmedicated) feed during the sampling period, to avoid interference between the drug and feed. Analyses ofjlumequine
and oxolinic acid
Plasma concentrations of flumequine and oxolinic acid were determined using high performance liquid chromatography following clean-up either on a solid phase extraction column or by column switching (Rasmussen et al., 1989). Tissue samples were pre-treated by liquid-liquid extraction and then analyzed by high performance liquid chromatography (Rogstad et al., 1989). Calculations
The pharmacokinetic calculations for intravascular injection were based on an open two-compartment model (Ritschel, 1986). The plasma concentration versus time curves were analyzed using non-linear regression analysis (Number Cruncher Statistical System, version 5.0, 1987, J.L. Hintze, Utah, USA) of the two-exponential equation: c, =A*e-“f+R.e-fi’, where c, is the plasma concentration, t is the time, cyand/.? are slopes of monoexponential declining curves, and A and B are the zero-time plasma concentrations. The diffusion processes were all assumed to follow first order kinetics. The areas under the concentration-time curves (AUC) were calculated by using the trapezoidal method. The absolute bioavailability (%) was estimated according to the formula: F= lOO.AUC,,, .dosei.V./AUCi.,. ~dose,,~, and determined after a single oral dose and a single intravascular (iv. ) of oxolinic acid and flumequine, respectively.
injection
RESULTS AND DISCUSSION
Intravascular injection
The mean plasma drug concentration values obtained from a single i.v. injection of flumequine and oxolinic acid, respectively, are listed in Table 1, and the calculated pharmacokinetic parameters are shown in Table 2. The plasma concentration values presented as a semi-logarithmic plot in Fig. 1 show that after a single intravenous dose of flumequine and oxolinic acid, respectively, there was a rapid decline of the drug concentration in the distribution phase. The calculated distribution half-lives ( tta) of flumequine and oxolinic acid were 1.3 and 0.7 h, respectively. More than 98% of the administered dose of flumequine was distributed over a period of 10 h. A very
211
FLUMEQUINE AND OXOLINIC ACID ADMINISTERED TO ATLANTIC SALMON
TABLE I Concentration of flumequine and oxolinic acid in salmon plasma after a single intravascular dose and after administration of a single oral dose. The average concentration value and concentration range at each sampling time are quoted. Seawater temperature 5 f 0.2%; fish weight 425 g Hours after administration
0.5 1 2 4 6 8 12 24 48 12 96
*Rg. ‘Av. Rg. Av. Rg. Av. Rg. Av. Rg. Av. Rg. Av. Rg. Av. Rg. Av. Rg. Av. Rg. Av. Rg. Av.
Flumequine, i.v. injection (Pgg/ml)
Oxolinic acid, i.v. injection @g/ml)
2.78-8.69 4.86 2.18-5.29 3.76 2.16-3.35 2.75 0.90-2.2 1 1.62 1.02-1.49 1.14 0.70-1.04 0.89 0.68-1.00 0.77 0.27-0.79 0.49 0.22-0.34 0.27 0.10-0.25 0.16 0.10-0.30 0.17
2.52-5.78 3.58 1.83-2.88 2.34 1.23-1.93 1.61 0.65-l .08 0.87 0.95-0.38 0.68 0.48-0.60 0.52 0.34-0.7 1 0.47 0.12-0.27 0.20 0.061-0.13 0.086 0.024-0.040 0.032 0.003-O. 13 0.067
Flumequine’, oral admin. 25 mg/kg @g/ml)
Oxolinic acid’, oral admin. 25 mg/kg @g/ml 1
0 0 -
0.000-0.016 0.005 0.1 l-0.39 0.20 0.37-0.92 0.68 0.92-l .02 0.99 0.45-0.75 0.56 0.15-0.59 0.28 0.1 l-O.29 0.20
0.18-0.60 0.37 1.08-3.45 2.26 1.32-2.09 1.81 0.85-2.10 1.21 0.55-0.96 0.70 0.74-1.32 0.96
‘Av. =mean plasma concentration, calculated from five fish. Rg. = range of plasma concentration values at each sampling time. ‘Aqualets 5 g antibiotic/kg.
thorough study of the pharmacokinetics of oxolinic acid in Atlantic salmon and rainbow trout in sea water has been carried out by Hustvedt and Salte ( 199 1). They administered a single intravascular dose which was twice that administered in the present study in the case of rainbow trout in freshwater and seawater and four times in the case of Atlantic salmon in seawater. Those studies also indicated a rapid distribution of oxolinic acid. Hustvedt and Salte ( 199 1) and Hustvedt et al. ( 199 la) estimated ttcr= 0.2 h for rainbow trout in seawater, 0.5 h for rainbow trout in freshwater and 0.3 h for Atlantic salmon in seawater. In the recent study by BjSrklund ( 199 1) it was concluded that the distribution half-life of oxolinic acid was 0.5 h for rainbow trout in brackish water. On the whole these distribution half-lives for oxolinic acid were shorter than that found for flumequine. The elimination rate of both flumequine and oxolinic acid after i.v. admin-
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ROGSAD
ET AL.
Pharmacokinetic parameters for flumequine and oxolinic acid following a single intravenous tion to Atlantic salmon. Seawater temperature 5 !z 0.2”C. fish weight 425 g
injec-
A.
TABLE 2
Parameter
Flumequine
Oxohnic acid
Dose (mg/kg b.w.)
4.9 0.52?0.04 0.030 * 0.006 1.340.2 2325 46.5 52.2 5.9 2.3 3.1 0.8
4.9 1.0&0.2 0.07 * 0.02 0.7kO.2 IOk4 23.0 24.0 5.2 4.9 2.9 0.9
; tr/za (h) ‘r/za (h) AU&, (pg*h/ml) AU&, (pg*h/ml) C(0) @g/ml) Clr (l/kg per 24 h) k’.,s (f/kg) P, (l/kg)
LY,8: Slopes of mono-exponential declining curves. t,,,: Half-lives of a drug in the different phases. AUC: Area under the drug-concentration curve. C(0): Drug concentration at time 0. Cl,: Total systemic clearance; Cl,=dose,,,,/AUC,_,. V,+ Apparent volume of distribution; V,,=CIr//I. FE:Volume of central plasma compartment; v== dosei_,/C (0 )
0
i 20
40
60
80
100
Hours Fig. 1. Semilogarithmic plot of concentration of flumequine and oxolinic acid in plasma of Atlantic salmon after intravascular administration of a single dose of 4.9 mg/kg body weight. Fish weight 425 g; seawater temperature 5°C. 0-O Fhrmequine in plasma; A-A oxolinic acid in plasma.
&ration was estimated by non-linear regression analysis. The deviation from linearity at the last sampling time (96 h) shown in Fig. 1 indicated that a more complex model had to be used to describe the terminal phase of elimination of both flumequine and oxolinic acid. A similar deviation from linearity was observed in pharmacokinetic studies of dosage regimes with flume-
FLUMEQUINE AND OXOLINIC ACID ADMINISTERED TO ATLANTIC SALMON
213
quine and oxolinic acid (Rogstad and Syvertsen, unpublished). The drugs were eliminated from plasma, muscle and liver at rates corresponding to the values of tt8 quoted in Table 2, whereas at low concentrations ( 1O-50 ng/ml) the drugs persisted for a longer period of time (up to 40 days). Hustvedt et al. ( 199 1a) used a three-compartment model to fit the data obtained after i.v. injection of oxolinic acid in Atlantic salmon and calculated the final half-life for elimination to be 60 h at 9°C. The extensive apparent volume of the central compartment indicated that flumequine ( V, = 0.8 l/kg) and oxolinic acid ( V, = 0.9 l/kg) were rapidly distributed to tissues outside the blood. Bjorklund ( 199 1) determined V,= 0.399 l/kg for oxolinic acid and V, = 0.198 l/kg for oxytetracycline in rainbow trout in brackish water. V, was estimated to be 0.19 l/kg for oxytetracycline in carp (Grondel et al., 1987) and the apparent volume of the central compartment was 1.04 l/kg for 14C-ormetoprim in rainbow trout in freshwater (Droy et al., 1990). The apparent volume of distribution for flumequine and oxolinic acid was almost identical and calculated as 3.0 l/kg and 2.9 l/kg, respectively. Hustvedt and Salte ( 199 1) and Hustvedt et al. ( 199 la) found an apparent volume of distribution at steady state for oxolinic acid in Atlantic salmon equal to 1.8 l/kg and in rainbow trout in seawater and freshwater to 2.6 and 2.9 l/kg, respectively. Clearance was calculated as 2.3 l/kg per 24 h for flumequine in Atlantic salmon and 4.9 l/kg per 24 h for oxolinic acid in the present study. Clearance was estimated to be 0.7 l/kg per 24 h for oxolinic acid in Atlantic salmon and 2.0 l/kg per 24 h in rainbow trout in seawater by Hustvedt and Salte ( 199 1) and Hustvedt et al. ( 199la). In comparison, Droy et al. ( 1990) found CIT= 5.47 l/kg per 24 h for “C-ormetoprim in rainbow trout, while clearance for oxytetracycline was 0.24 l/kg per 24 h in carp (Grondel et al., 1987) and 0.48 l/kg per 24 h in rainbow trout in brackish water (Bjiirklund, 1991). It was expected that distribution volume and clearance should be dependent on type of drug. Our results and those referred to show that the pharmacokinetic parameters for fish depend on water temperature and salinity, fish species, as well as health status of the individual fish (Bruno, 1989; Hustvedt, 199 1). Oral administration Fig. 2 shows the curves of plasma concentration versus time for Atlantic salmon given oral doses of 25 and 50 mg/kg of flumequine (Apoquin) and oxolinic acid (medicated feed), respectively. The pharmacokinetic parameters following oral administration are listed in Table 3. Plasma concentration values after administration of Aqualets are listed in Table 1, and the corresponding muscle and liver concentrations are listed in Table 4. A peak plasma concentration (C,,,) of flumequine of 2.26 &ml was obtained after oral administration of 25 mg/kg body weight. This was about twice that of oxolinic acid, C,,,= 0.99 pg/ml. The plasma concentration-
214
A. ROGSTAD ET AL.
Hours Fig. 2. Concentration of flumequine and oxolinic acid in plasma of Atlantic salmon following oral administration of Aqualets; Apoquin 5 g/kg, Apoquin 10 g/kg and medicated feed with oxolinic acid 5 g/kg and 10 g/kg. Fish weight 425 g; seawater temperature 5°C. A-A Medicated feed with 5 g oxolinic acid/kg. Dose: 25 mg oxolinic acid per kg fish. A-A Medicated feed with 10 g oxolinic acid/kg. Dose: 50 mg oxolinic acid per kg fish. O-O Apoquin Aqualets 5 g/kg. Dose: 25 mg flumequine per kg fish. 0-O Apoquin Aqualets 10 g/kg. Dose: 50 mg flumequine per kg fish.
time curves indicated a T,,,,, of 12 h following administration of flumequine and of 24 h for oxolinic acid. Hustvedt et al. ( 199 lb) observed in a similar study a r,,,,, of 24.3 h when administering a dose of 26 mg oxolinic acid per kg fish. For flumequine the r,,,, appeared to increase with dose in a similar way to that observed for oxolinic acid by Hustvedt et al. ( 199 1b). In liver, a T,,, of 12 h was observed for both flumequine and oxolinic acid, whereas the r,,,,, was apparently delayed by approximately 12 h in muscle. The muscle concentration of flumequine at peak maximum was about 2.5 times higher than the corresponding plasma concentration (C,,,), and the liver concentration at peak maximum was about 8 times the plasma concentration. Furthermore, the results show that the muscle concentration of oxolinic acid was about the same as that of liver at peak maximum and that AUCmuscrewas about the same as AUCii,er. Our dosage regime study (Syvertsen and Rogstad, 1988 ) confirmed that the observed differences between flumequine and oxolinic acid were less pronounced with regard to the AUC values for muscle than those for plasma and liver. The distribution curves of flumequine and oxolinic acid in muscle and liver are shown in Figs. 3 and 4, respectively, and demonstrate the difference in distribution pattern of oxolinic acid and flumequine in Atlantic salmon.
FLUMEQUINE AND OXOLINIC ACID ADMINISTERED TO ATLANTIC SALMON
215
TABLE 3 Pharmacokinetic parameters for flumequine and oxolinic acid in Atlantic salmon following a single oral dose of Apoquin and Apoxolon Aqualets and medicated feed with oxolinic acid. Seawater temperature 5? 0.2”C, fish weight 425 g
Plasma
Muscle
Parameter
Flumequine (Apoquin)
Oxolinic acid (Apoxolon)
Oxolinic acid (medicated feed)
Dose (mg/kg b.w.)
25
50
25
25
50
AU&, @g-h/ml) AUC+,/dose (h/l) C,, @g/ml) T,B, (h) F (%)
110
184 3.7 3.83 24 39
47.1 1.9 0.87 24 40
58.9 1.2 1.17 24 25
AU&, (/%-h/g)
402 16.1 6.58 24
286 11.4 5.72 24
729 29.2 19.5 12
280 11.2 6.83 12
AU&_,/dose Clnax @g/g) T,, (h) Liver
(h/l)
AU&-, (pg*h/g) AUC,,_,/dose (h/l) Cln., @g/g) T-(h)
AU&, (muscle) /AU&, (plasma) AU&_, (liver ) /AU&, (plasma )
4.4 2.26 12 46
3.1 6.6
46.6 1.9 0.99 24 40
6.1 6.0
AUC: Area under the concentration-time curve. C Inax. . Concentration at the maximum of the absorption curve. T_: Time for concentration maximum of the absorption curve. F: Apparent bioavailability.
Apparent bioavailability In the present study the area under the concentration-time curve (AUC) for flumequine was about twice that of oxolinic acid at an oral dose of 25 mg/ kg body weight. Increase of the dose to 50 mg/kg enhanced the AUC value by 67% for flumequine, but by only 25% for oxolinic acid. The apparent bioavailability (F) after an oral dose of 25 mg/kg was 46% for flumequine and 25% for oxolinic acid, The difference in bioavailability between the two drugs is more pronounced at the high dose of 50 mg/kg, being F= 39% for flumequine and 25O/6for oxolinic acid. The intravascular dose of the drugs was 1/5th and 1/ 10th of that of the oral doses, respectively, due to their low solubilities in aqueous solvents (pH < 10). An increase of the pH of the intravascular solution to 1 l- 12 will dissolve more drug. However, it was considered unfavourable to the fish to have a drug solution of pH 12 injected intravenously.
216
A. ROGSTAD ET AL.
TABLE 4 Concentration of flumequine and oxolinic acid in muscle and liver of Atlantic salmon after implantation of a single dose of 25 m&kg of flumequine and oxolinic acid in Aqualets dosage form. Sea water temperature 5 + 0.1 a C; fish weight 425 g Hours after administration
4
*Rg. *Av. Rg. Av. Rg. Av. Rg. Av. Rg. Av. Rg. Av. Rg. Av.
8 12 24 48 72 96
Flumequine in muscle (P&?/P)
Oxolinic acid in muscle (M/g)
Flumequine in liver kg/Id
Oxolinic acid in liver (M/P)
0.00 0.18-1.47 0.57 1.73-10.87 5.26 4.74-8.03 6.58 3.36-6.74 4.94 1.46-4.41 3.25 2.01-6.20 3.55
0.00 0.083-0.76 0.33 1.06-2.40 1.88 4.32-6.61 5.72 3.09-5.60 3.98 1.93-3.01 2.33 0.76-2.74 1.55
0.00-0.3 1 0.13 2.24-14.6 5.35 4.89-40.2 19.5 7.14-17.4 9.97 3.25-9.00 6.37 3.30-8.23 6.25 3.59-l 1.2 7.17
0.00 1.39-5.2 1 2.40 2.76-8.50 6.83 4.41-5.11 4.74 I .93-3.23 2.64 1.71-2.48 2.14 0.78-2.42 1.31
‘Av. =mean tissue concentration, calculated from five fish. Rg. = range of tissue concentration values.
y ;.
a-.-
20
40
60
80
100
Fig. 3. Concentration of flumequine and oxolinic acid in muscle of Atlantic salmon following oral administration of Aqualets. Fish weight 425 g; seawater temperature 5°C. 0-O Apoquin Aqualets 5 g/kg; A-A Apoxolon Aqualets 5 g/kg.
FLUMEQUINE AND OXOLINIC ACID ADMINISTERED TO ATLANTIC SALMON
217
20r
01 0
20
40
60
80
I 100
Hours Fig. 4. Concentration of flumequine and oxolinic acid in liver of Atlantic salmon following oral administration of Aqualets. Fish weight 425 g; seawater temperature 5°C. 0-O Apoquin Aqualets 5 g/kg; A- A Apoxolon Aqualets 5 g/kg.
The apparent bioavailability of drugs in Atlantic salmon is generally low. Hustvedt et al. ( 199 1b ) found F = 2 1% after a single oral dose of oxolinic acid and huge inter-individual variations; F ranged from 12 to 44%. Cravedi et al. ( 1987) calculated F to be 40% in rainbow trout after an oral dose of 20 mg oxolinic acid per kg body weight and F= 12% when administered at a dose of 100 mg. Bjiirklund and Bylund ( 199 1) estimated the apparent oral bioavailability of oxolinic acid to be 13.6% after a dose of 75 mg/kg to rainbow trout in freshwater. Microbiologicalactivity Flumequine has been found to be preferable to oxolinic acid in terms of both MIC and bactericidal activity. Investigations of the activity of flumequine and oxolinic acid against fish pathogenic bacteria such as Vibrio spp. and Aeromonas spp. showed low and almost identical MIC values. Barnes et al. ( 199 1a,b ) found that flumequine was only marginally more active against Aeromonas salmonicida than oxolinic acid in terms of MIC. However, flumequine was bactericidal at concentrations slightly above the MIC after 6 h exposure whereas oxolinic acid was merely bacteriostatic. The bactericidal activity increased with the duration of exposure at concentrations much lower than those measured in plasma and tissue. Furthermore, they showed that the
218
A. ROGSTAD ET AL.
mutation frequency of A. sulmonicidu strains that developed in the presence of flumequine was 1/ 10th of that observed with oxolinic acid. CONCLUSION
The present results on pharmacokinetics of flumequine following administration of a single intravascular dose of 4.9 mg/kg and single oral doses of 25 mg/kg or 50 mg/kg, respectively, were compared with those of oxolinic acid. They show that flumequine was favourable to oxolinic acid for use as a therapeutic drug in fish. Flumequine was absorbed more rapidly and to a higher concentration than oxolinic acid, as demonstrated by both plasma and tissue concentration values following oral administration. The apparent bioavailability was more favourable for flumequine than for oxolinic acid at high dose (50 mg/kg). The distribution profile in the various compartments was different for the two compounds. ACKNOWLEDGEMENTS
We are very grateful to L. Bjelland and A. Aanesrud for technical assistance, and to A.L. Christiansen and V. Holm for inspiring discussions on pharmacokinetics of ,drugs in fish.
REFERENCES Austin, B. and Austin, D.A. (Editors), 1987. Control of bacterial fish diseases. In: Bacterial Fish Pathogens: Disease in Farmed and Wild Fish. Ellis Horwood, Chichester, pp. 33 l-353. Austin, B., Rayment, J. and Alderman, D.J., 1983. Control of furunculosis by oxolinic acid. Aquaculture, 31: 101-108. Barnes, A.C., Lewin, C.S., Hastings, T.S. and Amyes, S.G.B., 1991a. Bactericidal activity of quinolones, including flumequine, against Aeromonas salmonicida. Proceedings O.I.E. Meeting Problems of Chemotherapy in Aquaculture: from Theory to Reality. Paris, pp. 287293. Barnes, A.C., Lewin, C.S., Hastings, T.S. and Amyes, S.G.B., 199 lb. In vitro susceptibility of the fish pathogen Aeromonas salmonicida to flumequine. Antimicrob. Agents Chemother.. 35: 2634-2635. Bjorklund, H.V., 1991. Oxytetracycline and oxolinic acid as antibacterials in aquaculture analysis, pharmacokinetics and environmental impacts. Thesis for academic dissertation, Abe Akademi University. BjorkIund, H.V. and Bylund, G., 199 1. Comparative pharmacokinetics and bioavailability of oxolinic acid and oxytetracycline in rainbow trout (Oncorhynchus mykiss). Xenobiotica, 2 1: 1511-1520. Bruno, D.W., 1989. An investigation into oxytetracycline residues in Atlantic salmon, Salmo salar L. J. Fish Dis., 12: 77-86. Chevalier, R., Gerard, J.P. and Michel, C., 198 1. Distribution et cinetique tissulaire de la flumequine chez la truite arc-en-ciel (Salmo gairdneri, Richardson). Recherche de residus. Rev. Med. Vet.. 132: 831-837.
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Cravedi, J-P., Choubert, G. and Delous, G., 1987. Digestibility of chloramphenicol, oxolinic acid and oxytetracycline in rainbow trout and influence of these antibiotics on lipid digestibility. Aquaculture, 60: 133- 141. Dorrestein, G.M., Van Gogh, H., Buitelaar, M.N. and Nouws, J.F.M., 1983. Clinical pharmacology and pharmacokinetics of flumequine after intravenous, intramuscular and oral administration in pigeons (Columba fivea). J. Vet. Pharmacol. Therap., 6: 281-292. Droy, B.F., Goodrich, MS., Lech, J.J. and Kleinow, K.M., 1990. Bioavailability, disposition and pharmacokinetics of i4C-ormetoprim in rainbow trout (Salmo gairdneri). Xenobiotica, 20: 147-157. Ellingsen, O.F., Nordby, K-E. and Rasmussen, K.E., 1989. Pharmaceutical dosage form for the medication of fish. Intemat. Patent Appl. no. PCT/N089/00059. Endo, T., Ogishima, K., Hayasaki, S., Kaneko, S. and Ohshima, S., 1973a. Application of oxolinic acid as a chemotherapeutic agent against infectious diseases in fish. I. Antibacterial activity, chemotherapeutic effect and pharmacokinetic effect of oxolinic acid in fish. Bull. Jpn. Sot. Sci. Fish., 39: 165-l 7 1. Endo, T., Sakuma, M., Tanaka, H., Ogishima, K. and Hara, T., 1973b. Application of oxolinic acid as chemotherapeutic agent against infectious diseases in fish. 11. Study of chemotherapeutic effect by whole body autobacteriography. Bull. Jpn. Sot. Sci. Fish., 39: 173-l 77. Grondel, J.L., Nouws, J.F.M., De Jong, M., Schutte, R. and Driessens, F., 1987. Pharmacokinetics and tissue distribution of oxytetracycline in carp (Cyprinus carpio L). following different routes of administration. J. Fish Dis., 10: 153- 163. Hustvedt, SO., 199 1. Pharmacokinetics of oxolinic acid in Atlantic salmon (Salmo salar L. ) and rainbow trout (Uncorhynchus mykiss Walbaum). Thesis, dr. scient, University of Oslo. Hustvedt, S.O. and Salte, R., 1991. Distribution and elimination of oxolinic acid in rainbow trout (Onchorhynchus mykiss Walbaum) after a single rapid intravascular injection. Aquaculture, 92: 297-303. Hustvedt, S.O., Salte, R. and Vassvik, V., 1991a. Absorption, distribution and elimination of oxolinic acid in Atlantic salmon (Salmo salar L. ) after various routes of administration. Aquaculture, 95: 193-l 99. Hustvedt, S.O., Salte, R., Kvendset, 0. and Vassvik, V., 199 1b. Bioavailability of oxolinic acid in Atlantic salmon (Salmo salar L. ) from medicated feed. Aquaculture, 97: 305-3 10. Mevius, D.J., Breukink, H.J., Jansen, T., Guelen, P.J.M. and De G&e, B., 1989. Oral absorption and bioavailability of flumequine in veal calves. Vet. Q., 11: 232-241. Michel, C., Gerard, J-P., Fourbet, B., Collas, R. and Chevalier, R., 1980. Emploi de la flumequine contre la furonculose des salmonides: essais therapeutiques et perspectives pratiques. Bull. Fr. Piscic., 52: 154-162. O’Grady, P., Moloney, M. and Smith, P.R., 1988. Bath administration of the quinolone antibiotic ilumequine to brown trout Salmo trutta and Atlantic salmon S. salar. Dis. Aquat. Organ., 4: 27-33. Rasmussen, K.E., Tonnesen, F., Thanh, H.H., Rogstad, A. and Aanesrud, A., 1989. Solid-phase extraction and high-performance liquid chromatographic determination of flumequine and oxolinic acid in salmon plasma. J. Chromatogr., 496: 355-364. Ritschel, W.A., 1986. Handbook of Basic Pharmacokinetics, including Clinical Applications, 3rd edn. Drug Intelligence Publications Inc., Hamilton, USA, pp. 177-l 90. Rodgers, C.J. and Austin, B., 1983. Oxolinic acid for control of enteric red-mouth disease in rainbow trout. Vet. Rec., 112: 83. Rogstad, A. and Syvertsen, C., unpublished. Free dosage to salmon of AqualetsTM containing oxolinic acid. Internal Report, Apothekernes Laboratorium A.S. Rogstad, A., Hormazabal, V. and Yndestad, M., 1989. Simultaneous extraction and determination of oxolinic acid and flumequine in fish tissues by high performance liquid chromatography. J. Liquid Chromatogr., 12: 3073-3086.
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Syvertsen, C. and Rogstad, A., 1988. Dosage regimen for flumequine administered to salmon. Report included in the registration file for Apoquin Aqualets vet. 5 g/kg. Ziv, G., Soback, S., Bor, A. and Kurts, B., 1986. Clinical pharmacokinetics of flumequine in calves. J. Vet. Pharmacol. Therap., 9: 17 I - 182.
Aquaculture, 110 (1993) 207-220 Elsevier Science Publishers B.V., Amsterdam AQUA 10004
Pharmacokinetics and bioavailability of flumequine and oxolinic acid after various routes of administration to Atlantic salmon in seawater Astri Rogstad, Odd F. Ellingsen and Christian Syvertsen Apothekernes Laboratorium AS, Skeyen, Oslo, Norway (Accepted
13 October 1992)
ABSTRACT Rogstad, A., Ellingsen, O.F. and Syvertsen, C., 1993. Pharmacokinetics and bioavailability of flumequine and oxolinic acid after various routes of administration to Atlantic salmon in seawater. Aquaculture, 110: 207-220. The present preclinical study was performed to investigate the pharmacokinetics of flumequine in Atlantic salmon (Safmo salar L. ) in seawater after administration of different doses and dosage formulations. Flumequine was administered intravenously (dose 4.9 mg/kg fish) and orally from the drug delivery system Aqualets as Apoquin 5 g/kg (dose 25 mg/kg) and 10 g/kg (dose 50 mg/kg), respectively. Experiments were carried out with oxolinic acid administered in the same way for the purpose of comparing the two compounds. The seawater temperature was 5 ? 0.2”C in all experiments. The pharmacokinetic calculations showed that the distribution half-life for flumequine was lia = 1.3 h and for oxolinic acid tla= 0.7 h. The drugs were absorbed rapidly, and flumequine reached a plasma concentration of C,, = 2.26 pgg/ml after a single oral dose of 25 mg/kg, whereas oxolinic acid reached C,.,=O.99 pg/ml. The apparent bioavailability of flumequine was found to be 40-45%, whereas the apparent bioavailability of oxolinic acid varied from 25% at a dose of 50 mg to 40°Yaat a dose of 25 mg/kg body weight of fish. The distribution profile of flumequine in the various compartments of fish appeared to be different from that of oxolinic acid. After a single oral dose (25 mg/kg) the areas under the concentration-time curves showed that flumequine was 2.3 times more concentrated in plasma and 2.6 times more concentrated in liver compared to oxolinic acid. in muscle the difference was less pronounced, flumequine being 1.4times more concentrated than oxolinic acid.
INTRODUCTION
Quinolones represent the latest generation of antibacterial agents. The compounds have broad spectrum activity particularly against Gram-negative bacteria, as well as fungi, protozoans and helminths. Oxolinic acid was introduced to Norwegian veterinary medicine in 1987. The drug has been applied C’orrespondenee to: A. Rogstad, Norway.
00448486/93/$06.00
Nycomed
Imaging A.S, P.O. Box 4220, Torshov. N-040 I Oslo,
0 1993 Elsevier Science Publishers
B.V. All rights reserved.
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A. ROGSTAD ET AL.
particularly in aquaculture in Norway and has appeared to be efficient against a number of Gram-negative bacteria that cause diseases such as furunculosis, yersiniosis, vibriosis and cold-water vibriosis. However, this antibiotic was reported to have been tested on fish (Endo et al., 1973a,b) as early as 1973, and over the last decade has been administered routinely to cultured fish in many countries both as a prophylactic and as a chemotherapeutic agent (Austin et al., 1983; Rodgers and Austin, 1983; Austin and Austin, 1987). The fluoroquinolones are second generation 4-quinolones. Flumequine is a representative of the fluoroquinolone derivatives, and its activity has been studied both in vitro and in vivo. In vivo studies have been carried out in both mammals (Dorrestein et al., 1983; Ziv et al., 1986; Mevius et al., 1989) and fish (Michel et al., 1980; Chevalier et al,. 198 1; O’Grady et al., 1988 ) during the last decade, and some recent in vitro studies on its activity against fishpathogenic Aeromonas sulmonicida were performed by Barnes et al. ( 199 1a,b ) . The latter studies showed that flumequine is preferable to oxolinic acid with regard to its microbiological activity. Studies on its pharmacokinetic properties in domestic animals showed that flumequine was absorbed well after oral administration. O’Grady et al. ( 1988) reported that flumequine was rapidly distributed to all tissues in high concentrations when administered to farmed fish. The aim of this study was to investigate the absorption, distribution, elimination and bioavailability of flumequine (FQ) in Atlantic salmon after oral administration of Apoquin Aqualets vet. 5 g/kg and 10 g/kg, respectively. The results have been compared with results from similar studies on oxolinic acid administered as Apoxolon Aqualets vet. 5 g/kg and as medicated feed 5 g/kg and 10 g/kg. Intravenous injections of flumequine and oxolinic acid to fish were also performed. Pharmacokinetic parameters and apparent bioavailability have been calculated. Our results were compared with pharmacokinetic values obtained for oxolinic acid by Hustvedt ( 199 1) and Bjiirklund (1991). MATERIALS
AND METHODS
Chemicals and medicatedfish pellets Flumequine and oxolinic acid were purchased from Welding Pharma A.G. (Hamburg, Germany). Tricaine methanesulphonate (MS-222) was obtained from Sandoz (Basel, Switzerland). Alcian Blue GS was supplied from Sigma Chemicals (St. Louis, MO, USA). The following five types of medicated products for fish were tested: - Apoquin Aqualets vet., medicinal pellets with flumequine 10 g/kg, batch no. 8905 1, d = 6 mm, Apothekemes Laboratorium (Oslo, Norway) - Apoquin Aqualets vet., medicinal pellets with flumequine 5 g/kg, batch no. 89057, d= 6 mm, Apothekemes Laboratorium (Oslo, Norway)
FLUMEQUINE AND OXOLINIC ACID ADMINISTERED TO ATLANTIC SALMON
209
-
Apoxolon Aqualets vet., medicinal pellets with oxolinic acid 5 g/kg, batch no. 89030, d = 6 mm, Apothekernes Laboratorium (Oslo, Norway) - Medicated feed with oxolinic acid 5 g/kg, Ewos Aqua (Lorenskog, Norway) - Medicated feed with oxolinic acid 10 g/kg, Ewos Aqua (Lorenskog, Norway). AqualetsTM is the trade name of a drug delivery system for fish developed by Apothekernes Laboratorium A.S (Ellingsen et al., 1989). The product consists of co-extruded pellets with a two-layer structure. The outer layer is made of nutritional components and binding agents. The drug filling in the inner core consists of a suspension of the antibiotic and pharmaceutical excipients in an appropriate solvent. Solutions for intravenous injection were prepared by dissolving flumequine and oxolinic acid, respectively, in 0.1 M NaOH to a concentration of 4.9 mg/ml and adjusting the pH of the solutions to 9 in the case of flumequine and to 10 in the case of oxolinic acid. Experimental conditions Atlantic salmon (Salmo salar L. ) (av. weight 425 g) were obtained from a local fish farm and held in six cylindrical libre glass tanks of ca. 1.8 m3 with a continuous flow of seawater (temp. 5 + 0.2”C, salinity 30%0, flow rate 10 l/min). Each tank contained 1O-20 fish. Fish were anaesthetized with tricaine methanesulphonate, weighed and individually marked with Alcian Blue on their ventral sides. The fish were then starved for 3 days, and anaesthetized before a dose of flumequine and oxolinic acid (Aqualets or medicated feed), respectively, corresponding to 0.5% of body weight of the fish was force fed into the stomach using a syringe. The doses were 25 and 50 mg antibiotic per kg fish. Intravenous injections of flumequine and oxolinic acid, respectively, were made into the caudal vein. The dose was 4.9 mg/ kg fish. The fish were starved for 3 days, anaesthetized and weighed. The drug was administered by aspirating blood into the syringe prior to the injection to ensure that the syringe with the drug solution was located in the caudal vein. While injecting, the syringe was held steady and the position of the needle was tested occasionally by aspirating blood into the syringe. Blood was sampled from the caudal vein using Venoject blood sampling equipment (0.9 x 4.0 mm needles) with heparin. In the case of injection, the first sampling was performed after 30 min and then at periodic intervals for 96 h, whereas in the case of oral administration, the first sample was taken after 4 h and then at periodic intervals for 96 h. Blood was collected from five fish at each sampling time. Plasma was isolated by centrifugation of the blood at 4000 rpm for 10 min. The samples were immediately frozen and stored at - 20” C until the analyses were performed.
210
A. ROGSTAD ET AL.
Fish were not fed ordinary (unmedicated) feed during the sampling period, to avoid interference between the drug and feed. Analyses ofjlumequine
and oxolinic acid
Plasma concentrations of flumequine and oxolinic acid were determined using high performance liquid chromatography following clean-up either on a solid phase extraction column or by column switching (Rasmussen et al., 1989). Tissue samples were pre-treated by liquid-liquid extraction and then analyzed by high performance liquid chromatography (Rogstad et al., 1989). Calculations
The pharmacokinetic calculations for intravascular injection were based on an open two-compartment model (Ritschel, 1986). The plasma concentration versus time curves were analyzed using non-linear regression analysis (Number Cruncher Statistical System, version 5.0, 1987, J.L. Hintze, Utah, USA) of the two-exponential equation: c, =A*e-“f+R.e-fi’, where c, is the plasma concentration, t is the time, cyand/.? are slopes of monoexponential declining curves, and A and B are the zero-time plasma concentrations. The diffusion processes were all assumed to follow first order kinetics. The areas under the concentration-time curves (AUC) were calculated by using the trapezoidal method. The absolute bioavailability (%) was estimated according to the formula: F= lOO.AUC,,, .dosei.V./AUCi.,. ~dose,,~, and determined after a single oral dose and a single intravascular (iv. ) of oxolinic acid and flumequine, respectively.
injection
RESULTS AND DISCUSSION
Intravascular injection
The mean plasma drug concentration values obtained from a single i.v. injection of flumequine and oxolinic acid, respectively, are listed in Table 1, and the calculated pharmacokinetic parameters are shown in Table 2. The plasma concentration values presented as a semi-logarithmic plot in Fig. 1 show that after a single intravenous dose of flumequine and oxolinic acid, respectively, there was a rapid decline of the drug concentration in the distribution phase. The calculated distribution half-lives ( tta) of flumequine and oxolinic acid were 1.3 and 0.7 h, respectively. More than 98% of the administered dose of flumequine was distributed over a period of 10 h. A very
211
FLUMEQUINE AND OXOLINIC ACID ADMINISTERED TO ATLANTIC SALMON
TABLE I Concentration of flumequine and oxolinic acid in salmon plasma after a single intravascular dose and after administration of a single oral dose. The average concentration value and concentration range at each sampling time are quoted. Seawater temperature 5 f 0.2%; fish weight 425 g Hours after administration
0.5 1 2 4 6 8 12 24 48 12 96
*Rg. ‘Av. Rg. Av. Rg. Av. Rg. Av. Rg. Av. Rg. Av. Rg. Av. Rg. Av. Rg. Av. Rg. Av. Rg. Av.
Flumequine, i.v. injection (Pgg/ml)
Oxolinic acid, i.v. injection @g/ml)
2.78-8.69 4.86 2.18-5.29 3.76 2.16-3.35 2.75 0.90-2.2 1 1.62 1.02-1.49 1.14 0.70-1.04 0.89 0.68-1.00 0.77 0.27-0.79 0.49 0.22-0.34 0.27 0.10-0.25 0.16 0.10-0.30 0.17
2.52-5.78 3.58 1.83-2.88 2.34 1.23-1.93 1.61 0.65-l .08 0.87 0.95-0.38 0.68 0.48-0.60 0.52 0.34-0.7 1 0.47 0.12-0.27 0.20 0.061-0.13 0.086 0.024-0.040 0.032 0.003-O. 13 0.067
Flumequine’, oral admin. 25 mg/kg @g/ml)
Oxolinic acid’, oral admin. 25 mg/kg @g/ml 1
0 0 -
0.000-0.016 0.005 0.1 l-0.39 0.20 0.37-0.92 0.68 0.92-l .02 0.99 0.45-0.75 0.56 0.15-0.59 0.28 0.1 l-O.29 0.20
0.18-0.60 0.37 1.08-3.45 2.26 1.32-2.09 1.81 0.85-2.10 1.21 0.55-0.96 0.70 0.74-1.32 0.96
‘Av. =mean plasma concentration, calculated from five fish. Rg. = range of plasma concentration values at each sampling time. ‘Aqualets 5 g antibiotic/kg.
thorough study of the pharmacokinetics of oxolinic acid in Atlantic salmon and rainbow trout in sea water has been carried out by Hustvedt and Salte ( 199 1). They administered a single intravascular dose which was twice that administered in the present study in the case of rainbow trout in freshwater and seawater and four times in the case of Atlantic salmon in seawater. Those studies also indicated a rapid distribution of oxolinic acid. Hustvedt and Salte ( 199 1) and Hustvedt et al. ( 199 la) estimated ttcr= 0.2 h for rainbow trout in seawater, 0.5 h for rainbow trout in freshwater and 0.3 h for Atlantic salmon in seawater. In the recent study by BjSrklund ( 199 1) it was concluded that the distribution half-life of oxolinic acid was 0.5 h for rainbow trout in brackish water. On the whole these distribution half-lives for oxolinic acid were shorter than that found for flumequine. The elimination rate of both flumequine and oxolinic acid after i.v. admin-
212
ROGSAD
ET AL.
Pharmacokinetic parameters for flumequine and oxolinic acid following a single intravenous tion to Atlantic salmon. Seawater temperature 5 !z 0.2”C. fish weight 425 g
injec-
A.
TABLE 2
Parameter
Flumequine
Oxohnic acid
Dose (mg/kg b.w.)
4.9 0.52?0.04 0.030 * 0.006 1.340.2 2325 46.5 52.2 5.9 2.3 3.1 0.8
4.9 1.0&0.2 0.07 * 0.02 0.7kO.2 IOk4 23.0 24.0 5.2 4.9 2.9 0.9
; tr/za (h) ‘r/za (h) AU&, (pg*h/ml) AU&, (pg*h/ml) C(0) @g/ml) Clr (l/kg per 24 h) k’.,s (f/kg) P, (l/kg)
LY,8: Slopes of mono-exponential declining curves. t,,,: Half-lives of a drug in the different phases. AUC: Area under the drug-concentration curve. C(0): Drug concentration at time 0. Cl,: Total systemic clearance; Cl,=dose,,,,/AUC,_,. V,+ Apparent volume of distribution; V,,=CIr//I. FE:Volume of central plasma compartment; v== dosei_,/C (0 )
0
i 20
40
60
80
100
Hours Fig. 1. Semilogarithmic plot of concentration of flumequine and oxolinic acid in plasma of Atlantic salmon after intravascular administration of a single dose of 4.9 mg/kg body weight. Fish weight 425 g; seawater temperature 5°C. 0-O Fhrmequine in plasma; A-A oxolinic acid in plasma.
&ration was estimated by non-linear regression analysis. The deviation from linearity at the last sampling time (96 h) shown in Fig. 1 indicated that a more complex model had to be used to describe the terminal phase of elimination of both flumequine and oxolinic acid. A similar deviation from linearity was observed in pharmacokinetic studies of dosage regimes with flume-
FLUMEQUINE AND OXOLINIC ACID ADMINISTERED TO ATLANTIC SALMON
213
quine and oxolinic acid (Rogstad and Syvertsen, unpublished). The drugs were eliminated from plasma, muscle and liver at rates corresponding to the values of tt8 quoted in Table 2, whereas at low concentrations ( 1O-50 ng/ml) the drugs persisted for a longer period of time (up to 40 days). Hustvedt et al. ( 199 1a) used a three-compartment model to fit the data obtained after i.v. injection of oxolinic acid in Atlantic salmon and calculated the final half-life for elimination to be 60 h at 9°C. The extensive apparent volume of the central compartment indicated that flumequine ( V, = 0.8 l/kg) and oxolinic acid ( V, = 0.9 l/kg) were rapidly distributed to tissues outside the blood. Bjorklund ( 199 1) determined V,= 0.399 l/kg for oxolinic acid and V, = 0.198 l/kg for oxytetracycline in rainbow trout in brackish water. V, was estimated to be 0.19 l/kg for oxytetracycline in carp (Grondel et al., 1987) and the apparent volume of the central compartment was 1.04 l/kg for 14C-ormetoprim in rainbow trout in freshwater (Droy et al., 1990). The apparent volume of distribution for flumequine and oxolinic acid was almost identical and calculated as 3.0 l/kg and 2.9 l/kg, respectively. Hustvedt and Salte ( 199 1) and Hustvedt et al. ( 199 la) found an apparent volume of distribution at steady state for oxolinic acid in Atlantic salmon equal to 1.8 l/kg and in rainbow trout in seawater and freshwater to 2.6 and 2.9 l/kg, respectively. Clearance was calculated as 2.3 l/kg per 24 h for flumequine in Atlantic salmon and 4.9 l/kg per 24 h for oxolinic acid in the present study. Clearance was estimated to be 0.7 l/kg per 24 h for oxolinic acid in Atlantic salmon and 2.0 l/kg per 24 h in rainbow trout in seawater by Hustvedt and Salte ( 199 1) and Hustvedt et al. ( 199la). In comparison, Droy et al. ( 1990) found CIT= 5.47 l/kg per 24 h for “C-ormetoprim in rainbow trout, while clearance for oxytetracycline was 0.24 l/kg per 24 h in carp (Grondel et al., 1987) and 0.48 l/kg per 24 h in rainbow trout in brackish water (Bjiirklund, 1991). It was expected that distribution volume and clearance should be dependent on type of drug. Our results and those referred to show that the pharmacokinetic parameters for fish depend on water temperature and salinity, fish species, as well as health status of the individual fish (Bruno, 1989; Hustvedt, 199 1). Oral administration Fig. 2 shows the curves of plasma concentration versus time for Atlantic salmon given oral doses of 25 and 50 mg/kg of flumequine (Apoquin) and oxolinic acid (medicated feed), respectively. The pharmacokinetic parameters following oral administration are listed in Table 3. Plasma concentration values after administration of Aqualets are listed in Table 1, and the corresponding muscle and liver concentrations are listed in Table 4. A peak plasma concentration (C,,,) of flumequine of 2.26 &ml was obtained after oral administration of 25 mg/kg body weight. This was about twice that of oxolinic acid, C,,,= 0.99 pg/ml. The plasma concentration-
214
A. ROGSTAD ET AL.
Hours Fig. 2. Concentration of flumequine and oxolinic acid in plasma of Atlantic salmon following oral administration of Aqualets; Apoquin 5 g/kg, Apoquin 10 g/kg and medicated feed with oxolinic acid 5 g/kg and 10 g/kg. Fish weight 425 g; seawater temperature 5°C. A-A Medicated feed with 5 g oxolinic acid/kg. Dose: 25 mg oxolinic acid per kg fish. A-A Medicated feed with 10 g oxolinic acid/kg. Dose: 50 mg oxolinic acid per kg fish. O-O Apoquin Aqualets 5 g/kg. Dose: 25 mg flumequine per kg fish. 0-O Apoquin Aqualets 10 g/kg. Dose: 50 mg flumequine per kg fish.
time curves indicated a T,,,,, of 12 h following administration of flumequine and of 24 h for oxolinic acid. Hustvedt et al. ( 199 lb) observed in a similar study a r,,,,, of 24.3 h when administering a dose of 26 mg oxolinic acid per kg fish. For flumequine the r,,,, appeared to increase with dose in a similar way to that observed for oxolinic acid by Hustvedt et al. ( 199 1b). In liver, a T,,, of 12 h was observed for both flumequine and oxolinic acid, whereas the r,,,,, was apparently delayed by approximately 12 h in muscle. The muscle concentration of flumequine at peak maximum was about 2.5 times higher than the corresponding plasma concentration (C,,,), and the liver concentration at peak maximum was about 8 times the plasma concentration. Furthermore, the results show that the muscle concentration of oxolinic acid was about the same as that of liver at peak maximum and that AUCmuscrewas about the same as AUCii,er. Our dosage regime study (Syvertsen and Rogstad, 1988 ) confirmed that the observed differences between flumequine and oxolinic acid were less pronounced with regard to the AUC values for muscle than those for plasma and liver. The distribution curves of flumequine and oxolinic acid in muscle and liver are shown in Figs. 3 and 4, respectively, and demonstrate the difference in distribution pattern of oxolinic acid and flumequine in Atlantic salmon.
FLUMEQUINE AND OXOLINIC ACID ADMINISTERED TO ATLANTIC SALMON
215
TABLE 3 Pharmacokinetic parameters for flumequine and oxolinic acid in Atlantic salmon following a single oral dose of Apoquin and Apoxolon Aqualets and medicated feed with oxolinic acid. Seawater temperature 5? 0.2”C, fish weight 425 g
Plasma
Muscle
Parameter
Flumequine (Apoquin)
Oxolinic acid (Apoxolon)
Oxolinic acid (medicated feed)
Dose (mg/kg b.w.)
25
50
25
25
50
AU&, @g-h/ml) AUC+,/dose (h/l) C,, @g/ml) T,B, (h) F (%)
110
184 3.7 3.83 24 39
47.1 1.9 0.87 24 40
58.9 1.2 1.17 24 25
AU&, (/%-h/g)
402 16.1 6.58 24
286 11.4 5.72 24
729 29.2 19.5 12
280 11.2 6.83 12
AU&_,/dose Clnax @g/g) T,, (h) Liver
(h/l)
AU&-, (pg*h/g) AUC,,_,/dose (h/l) Cln., @g/g) T-(h)
AU&, (muscle) /AU&, (plasma) AU&_, (liver ) /AU&, (plasma )
4.4 2.26 12 46
3.1 6.6
46.6 1.9 0.99 24 40
6.1 6.0
AUC: Area under the concentration-time curve. C Inax. . Concentration at the maximum of the absorption curve. T_: Time for concentration maximum of the absorption curve. F: Apparent bioavailability.
Apparent bioavailability In the present study the area under the concentration-time curve (AUC) for flumequine was about twice that of oxolinic acid at an oral dose of 25 mg/ kg body weight. Increase of the dose to 50 mg/kg enhanced the AUC value by 67% for flumequine, but by only 25% for oxolinic acid. The apparent bioavailability (F) after an oral dose of 25 mg/kg was 46% for flumequine and 25% for oxolinic acid, The difference in bioavailability between the two drugs is more pronounced at the high dose of 50 mg/kg, being F= 39% for flumequine and 25O/6for oxolinic acid. The intravascular dose of the drugs was 1/5th and 1/ 10th of that of the oral doses, respectively, due to their low solubilities in aqueous solvents (pH < 10). An increase of the pH of the intravascular solution to 1 l- 12 will dissolve more drug. However, it was considered unfavourable to the fish to have a drug solution of pH 12 injected intravenously.
216
A. ROGSTAD ET AL.
TABLE 4 Concentration of flumequine and oxolinic acid in muscle and liver of Atlantic salmon after implantation of a single dose of 25 m&kg of flumequine and oxolinic acid in Aqualets dosage form. Sea water temperature 5 + 0.1 a C; fish weight 425 g Hours after administration
4
*Rg. *Av. Rg. Av. Rg. Av. Rg. Av. Rg. Av. Rg. Av. Rg. Av.
8 12 24 48 72 96
Flumequine in muscle (P&?/P)
Oxolinic acid in muscle (M/g)
Flumequine in liver kg/Id
Oxolinic acid in liver (M/P)
0.00 0.18-1.47 0.57 1.73-10.87 5.26 4.74-8.03 6.58 3.36-6.74 4.94 1.46-4.41 3.25 2.01-6.20 3.55
0.00 0.083-0.76 0.33 1.06-2.40 1.88 4.32-6.61 5.72 3.09-5.60 3.98 1.93-3.01 2.33 0.76-2.74 1.55
0.00-0.3 1 0.13 2.24-14.6 5.35 4.89-40.2 19.5 7.14-17.4 9.97 3.25-9.00 6.37 3.30-8.23 6.25 3.59-l 1.2 7.17
0.00 1.39-5.2 1 2.40 2.76-8.50 6.83 4.41-5.11 4.74 I .93-3.23 2.64 1.71-2.48 2.14 0.78-2.42 1.31
‘Av. =mean tissue concentration, calculated from five fish. Rg. = range of tissue concentration values.
y ;.
a-.-
20
40
60
80
100
Fig. 3. Concentration of flumequine and oxolinic acid in muscle of Atlantic salmon following oral administration of Aqualets. Fish weight 425 g; seawater temperature 5°C. 0-O Apoquin Aqualets 5 g/kg; A-A Apoxolon Aqualets 5 g/kg.
FLUMEQUINE AND OXOLINIC ACID ADMINISTERED TO ATLANTIC SALMON
217
20r
01 0
20
40
60
80
I 100
Hours Fig. 4. Concentration of flumequine and oxolinic acid in liver of Atlantic salmon following oral administration of Aqualets. Fish weight 425 g; seawater temperature 5°C. 0-O Apoquin Aqualets 5 g/kg; A- A Apoxolon Aqualets 5 g/kg.
The apparent bioavailability of drugs in Atlantic salmon is generally low. Hustvedt et al. ( 199 1b ) found F = 2 1% after a single oral dose of oxolinic acid and huge inter-individual variations; F ranged from 12 to 44%. Cravedi et al. ( 1987) calculated F to be 40% in rainbow trout after an oral dose of 20 mg oxolinic acid per kg body weight and F= 12% when administered at a dose of 100 mg. Bjiirklund and Bylund ( 199 1) estimated the apparent oral bioavailability of oxolinic acid to be 13.6% after a dose of 75 mg/kg to rainbow trout in freshwater. Microbiologicalactivity Flumequine has been found to be preferable to oxolinic acid in terms of both MIC and bactericidal activity. Investigations of the activity of flumequine and oxolinic acid against fish pathogenic bacteria such as Vibrio spp. and Aeromonas spp. showed low and almost identical MIC values. Barnes et al. ( 199 1a,b ) found that flumequine was only marginally more active against Aeromonas salmonicida than oxolinic acid in terms of MIC. However, flumequine was bactericidal at concentrations slightly above the MIC after 6 h exposure whereas oxolinic acid was merely bacteriostatic. The bactericidal activity increased with the duration of exposure at concentrations much lower than those measured in plasma and tissue. Furthermore, they showed that the
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mutation frequency of A. sulmonicidu strains that developed in the presence of flumequine was 1/ 10th of that observed with oxolinic acid. CONCLUSION
The present results on pharmacokinetics of flumequine following administration of a single intravascular dose of 4.9 mg/kg and single oral doses of 25 mg/kg or 50 mg/kg, respectively, were compared with those of oxolinic acid. They show that flumequine was favourable to oxolinic acid for use as a therapeutic drug in fish. Flumequine was absorbed more rapidly and to a higher concentration than oxolinic acid, as demonstrated by both plasma and tissue concentration values following oral administration. The apparent bioavailability was more favourable for flumequine than for oxolinic acid at high dose (50 mg/kg). The distribution profile in the various compartments was different for the two compounds. ACKNOWLEDGEMENTS
We are very grateful to L. Bjelland and A. Aanesrud for technical assistance, and to A.L. Christiansen and V. Holm for inspiring discussions on pharmacokinetics of ,drugs in fish.
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