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Poor bioavailability of oxytetracycline in sharpsnout sea bream Diplodus puntazzo
Aquaculture 235 (2004) 489 – 497 www.elsevier.com/locate/aqua-online
Poor bioavailability of oxytetracycline in sharpsnout sea bream Diplodus puntazzo G. Rigos a,b,*, Tyrpenou c, I. Nengas a, A.E.M. Alexis a, F. Athanassopoulou d, G.M. Troisi e a
Laboratory of Fish Nutrition and Pathology, National Centre for Marine Research, Aghios Kosmas 16604, Ellinikon, Attiki, Greece b School of Life Sciences, University of Kingston, Kingston upon Thames, KT1 2EE Surrey, London, UK c Department of Residue Research, HPLC Laboratory, Institute of Veterinary Research of Athens, National Agricultural Research Foundation, 25 Neapoleos Street, Agia Paraskevi 15310, Athens, Greece d Faculty of Veterinary Medicine, School of Health Sciences, University of Thessaly, Karditsa, 43100, Greece e Environmental Monitoring Unit, ETC Building, Brunel University, Uxbridge, UB8 3PH, Middlesex, UK Received 17 July 2003; received in revised form 15 October 2003; accepted 15 October 2003
Abstract The pharmacokinetics of oxytetracycline (OTC) were investigated following single intravascular injection (40 mg/kg) in sharpsnout sea bream (90 g) at 19 jC. The distribution half-life (t1/2a) and the elimination half-life (t1/2b) of OTC were calculated to be 1.4 and 35 h, respectively. The apparent volume of distribution of the drug at steady-state (Vd(ss)) was found to be 4 l/kg. The total clearance rate (CLT) of the drug was low (0.08 l/kg h). Repeated attempts to investigate the bioavailability of OTC following oral administration (75 mg/kg; forced-fed or via the feed), revealed undetectable levels of the drug in plasma and muscle samples. Poor tissue absorption is likely due to significant hepatic metabolism of OTC in sharpsnout sea bream, but this requires further investigation. Thus, oral administration of OTC in farmed sharpsnout sea bream should be discouraged in favour of other routes of administration or other antibiotic drugs. D 2003 Elsevier B.V. All rights reserved. Keywords: Oxytetracyline; Diplodus puntazzo; Pharmacokinetics; Absorption
1. Introduction During the last decade, there has been a need in the Mediterranean aquaculture industry to cultivate new fish species to overcome the problem arising from overproduction of the * Corresponding author. Tel.: +30-210-9829238; fax: +30-210-1-9811713. E-mail address: [email protected] (G. Rigos). 0044-8486/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2003.10.016
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two main farmed species, gilthead sea bream and European sea bass, by product diversification (FGM, 2000). Sharpsnout sea bream is a fast growing sparid and so far represents more than 60% of Greek mariculture production for all new farmed species (FGM, 2000). Up to now, as with other water warm fish species, oxytetracycline (OTC) therapeutic regimes for sharpsnout sea bream are extrapolated from pharmacokinetic information derived from salmonids. However, to optimise the treatment regimens for Mediterranean fish farming of sharpsnout sea bream, specific information on the pharmacokinetics of OTC in this species is needed. Therefore, the aim of this study was to investigate the kinetic profile of OTC in sharpsnout sea bream following intravascular and oral drug administration.
2. Materials and methods 2.1. Chemicals Oxytetracycline hydrochloride of high purity (>99%) and erythrosine were obtained from Sigma (USA). High-performance liquid chromatography (HPLC) grade solvents were obtained from Labscan (Ireland). The columns Isolute C18 used for solid phase extraction (SPE) were bought from International Technology (Sorben, UK). 2.2. Fish Two hundred healthy sharpsnout sea bream Diplodus puntazzo (90 F 20 g) were acclimated for 2 weeks in the facilities of the National Centre for Marine Research in Athens before starting the experiment. They were placed in cylindroconical fiberglass tanks (120 l) receiving water sea with salinity of 36xat a flow rate of 2 l/min. The temperature was maintained at 19 F 0.5 jC. Since it is a common practice in fish farms to starve fish to increase appetite and minimize stress prior to drug administration, fish were not fed for 2 days prior to commencing experiments and were kept at 19 F 0.5 jC. 2.3. Intravascular administration Prior to injection, fish were anaesthetized with quinaldine (2 ml/l salt water; 36x) and weighed. Intravenous injections of OTC (200 Al; 40 mg/kg fish) dissolved in sterile water were made into the caudal vein. To ensure the correct position of the needle (Microlance 23G 11/4 0.6 30, Becton Dickinson, Spain), blood was aspirated into the syringe prior to and following injection. Fish which were bled heavily were excluded and replaced (30%). Approximately 1 ml of blood was drawn from the caudal vein (away from the injection site) of several fish (six to eight individuals), at each of the following time points after dosing: 1, 2, 4, 8, 16, 24, 48, 72 and 150 h. After collecting blood samples, fish were killed by a blow to the head and 1 g of muscle devoid of skin or blood was taken from the dorsal area just above the lateral line. Plasma was prepared from blood samples by centrifugation
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at 3500 rpm for 10 min at 4 jC. Muscle and plasma samples were stored at 20 jC until analysis. 2.4. Oral administration Prior to oral administration of the substance, fish were anaesthetized with quinaldine as previously stated and weighed. Oxytetracycline (75 mg/kg fish) was administered via stomach tube (diameter 2 mm) initially dissolved in starch and sterile water (0.5 ml containing the red food stain erythrosine in order to detect possible regurgitation of the drug). Since significant regurgitation was evident (90%), the drug was re-administered in sterile aqueous suspensions (0.5 ml). Notwithstanding, OTC acceptance was improved with the different solution, regurgitation was still considerable (70%), thus the results were rejected (the analysis of samples from treated fish showing no regurgitation of OTC revealed undetectable levels). Then, it was decided to avoid force-feeding and administer a single dosage of drug mixed in feed pellets. One hundred fish of the same size were given control feed for a week at daily rate of 1% biomass. They were maintained at cylindroconical fiberglass tanks (880 l) under the aforementioned conditions. Following a 1-week acclimatization period, medicated pellets (75 mg/kg fish) were carefully delivered to minimise variation and readily consumed by the fish. Ten feed samples (100 Ag) were subjected to HPLC analysis for concentration confirmation. Approximately 1 ml of blood was drawn from the caudal vein of several fish (six to eight individuals), at each of the following time points after dosing: 4, 8, 16, 24, 48, 72 and 150 h. Plasma preparation and sample storage conditions were identical to the protocol followed for the intravascular administration. HPLC analysis of the plasma and muscle samples after administration of OTC-medicated pellets again revealed undetectable levels of the drug for all time points. To further investigate this discrepancy, the procedure was repeated by delivering medicated pellets to sharpsnout sea bream with the application of extent monitoring in glass aquaria (220 l) to permit regular observation of fish feeding behaviour and identify any possible digestive disorders that could have caused rejection of the medicated feed. Oxytetracyline-medicated feed was observed to be readily consumed by the 10 studied fish. Within 1 h following administration, no vomiting or other signs of digestive disorders were observed. Faecal release was normal and commenced approximately 25 min after the administration of the medicated pellets. Five of the fish were collected after 12 h and the remaining fish sampled after 24 h from the time of oral dosing. Plasma and muscle samples were taken from these fish and stored as stated previously. HPLC analysis of the plasma and muscle samples again revealed undetectable levels of the drug at both time points. To understand further the mechanisms of low OTC absorption in sharpsnout sea bream, a follow-up experiment was undertaken in 100 fish (200 g) kept in small fish cages (2 2 m) at 17 jC. Basically, the single oral OTC dosing (75 mg/kg) through pellets was repeated, but drug concentrations in liver samples not previously collected, were also analysed. This was to investigate any hepatic accumulation, which would indicate that most of the drug is metabolised prior to entering the general circulation. Muscle and liver samples from 20 fish were analysed. No plasma
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samples were available. Tissue samples from 20 fish were stored and treated as stated previously. 2.5. OTC analysis The analysis of OTC was performed according to the procedure of Tyrpenou (1995) with some modifications. Briefly, tissue samples (1 g) were homogenized with 10 ml McIlvaine buffer (citric acid 0.06, K2HPO42H2O 0.02 M) (68:32 v/v) and centrifuged for 10 min at 12,000 rpm. The supernatant was retained and the procedure repeated. To the combined supernatants, 2 ml of hexane were added and the solution centrifuged at 4000 rpm for 5 min. The organic layer containing lipid was discarded. The samples were cleaned up by a SPE procedure using Isolute C18 disposable columns (500 mg of 6 ml capacity). Columns were pre-conditioned with 5 ml methanol and 5 ml McIlvaine buffer. Samples were eluted with 10 ml methanol/ acetonitrile (50:50 v/v). After this, columns were cleaned using 10 ml HPLC-grade water and dried under vacuum for 15 min. The eluate was evaporated to dryness under nitrogen at 50 jC, resuspended in 1 ml of HPLC mobile phase solvent (oxalic acid 0.01 M: acetonitrile) (85:15 v/v), vortexed and filtered (0.45 Am). Exactly 100 Al of sample were injected into a Waters Alliance 2690MX HPLC system fitted with a Water 510 pump and a Waters 484 UV detector. Analytes were separated using an H&P Deactivated Hypersil ODS C18 (100 4 mm i.d.; pore size 5 Am). The detection wavelength was 360 nm and the flow rate 0.5 ml/min. Under these conditions, the retention time of OTC was 6 min and the detection limit of OTC was z 15 ng/ml (linear range to 1000 ppb in drug-free plasma ( P < 0.002) and tissue samples ( P < 0.005)). OTC concentrations were extrapolated from calibration curves of different concentrations of authentic high-purity OTC standard. For plasma samples (200 Al), the method was repeated by reducing all volumes by a factor of five. In the SPE procedure, smaller Isolute C18 disposable columns were used (100 mg of 1 ml capacity). The percentage of recoveries of OTC from all samples was calculated by comparing OTC concentrations in spiked drug-free plasma and muscle samples to a standard solution. OTC recovery through the method was found to be 69 F 4% for tissue samples and 62 F 5% for plasma samples. 2.6. Pharmacokinetic analysis The data were analysed for the best fit to a two- or three-compartment open pharmacokinetic model using non-linear regression analysis programs (NLREG 2001, P.H. Sherrod), following a semi-logarithmic plot of the data (curve EXPERT 1997, D. Hyams) and least square fitting (Ritschel, 1986). Diffusion processes were assumed to follow first order kinetics. Calculations of the apparent volume of distribution at steady state (Vdss) and total body clearance (CLT) were performed in a model independent way (Ritschel, 1986) to achieve objective comparison and to avoid overestimation by using the model slope b. The area under the concentration– time curve (AUC) was calculated using the trapezoidal rule and was extrapolated to infinity (Ritschel, 1986). Least square regression analysis was applied to calculate the elimina-
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Table 1 Plasma and muscle concentration of OTC in sharpsnout sea bream (90 g) at 19 jC following single intravascular injection (40 mg/kg) at each individual time point (mean F S.D.; n = 7) Time from dosing (h)
1 2 4 8 16 24 48 72 150
OTC concentration Plasma (Ag/ml)
Muscle (Ag/g)
18.6 F 6.8 14.1 F 4.3 12.4 F 2.8 9.9 F 2.6 7.7 F 1.2 5.8 F 1.4 4.0 F 0.5 1.6 F 0.2 0.9 F 0.2
4.5 F 1.4 5.5 F 2.2 4.6 F 0.7 10.1 F 2.1 6.6 F 1.4 6.1 F 1.6 11.1 F 3.2 10.2 F 2.6 3.0 F 1.1
tion slope b. The elimination half-lives were calculated from the equation t1/2 = ln2/b (Baggot, 1977).
3. Results Data analysis of the intravascular injection conformed better to a two-compartment model than a three-compartment model. Thus, the plasma concentration versus time
Table 2 Calculated pharmacokinetic parameters (compartmental; mean F S.D.) of intravascularly injected OTC (40 mg/ kg) in sharpsnout sea bream (90 g) at 19 jC Parameter
Values
Compartmental A (Ag/ml) Aˆ (Ag/ml) a (h 1) b (h 1) t1/2a (h) t1/2b (h) Vc (l/kg)
12.9 F 4.9 11.3 F 2.0 0.6 F 0.3 0.02 F 0.008 1.4 F 0.5 34.5 F 3.0 1.7 F 0.03
Non-compartmental Vd(ss) (l/kg) 4.0 AUC0 ! l (Ag/h ml) 549 0.08 CLT (l/kg h) MRT (h) 49 Abbreviations: Aa, Aˆ: zero-time plasma drug concentration intercepts of biphasic disposition curve; a, b: distribution and elimination rate constants; t1/2a: distribution half-life of the drug; t1/2b: elimination half-life of the drug; Vd(ss): apparent volume of distribution of the drug at steady-state; Vc: extensive apparent volume of the central compartment; AUC0 ! l: area under the drug concentration curve extrapolated to infinity; CLT: total body clearance of the drug; MRT: mean residence time.
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curve was calculated using the bi-exponential equation: C = Ae at + Be bt where C is the plasma concentration, t is the time, a and b are the slopes of mono-exponential declining curves, and A and B are the zero time plasma concentrations (Ritschel, 1986). Mean OTC concentrations following intravascular dosing decreased steadily in plasma from 18.6 to 0.9 Ag/ml over 150 h post-dosing. In contrast, muscle OTC concentrations were variable ranging from 3.0 to 11.1 Ag/g over the same time period (Table 1). The calculated pharmacokinetic parameters are presented in Table 2. The distribution half-life (t1/2a) was calculated to be fast but the elimination half-life (t1/2b) of OTC from plasma was found to be long. The apparent volume of distribution of the drug at steady-state (Vd(ss)) and the extensive apparent volume of the central compartment (Vc) were both large. The total clearance of the drug (CLT) was found to be low and the mean residence time (MRT) was long. In the follow-up experiment, analysis of muscle samples revealed extremely low or undetectable OTC concentrations. In contrast, OTC levels up to 8.4 Ag/g were observed in liver samples (Table 3) indicative of hepatic accumulation and metabolism of the drug. The concentration of OTC in the medicated diet was 110% (110 F 5; mean F S.E.) of the original dose.
Table 3 Mean muscle and liver concentrations (Ag/g) of OTC in sharpsnout sea bream (200 g) kept in situ (n = 20) at 17 jC, 24 h post-oral dosing (75 mg/kg) Fish sample
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Mean F S.D. n/d—not detectable.
OTC concentration (Ag/g) Muscle
Liver
n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d 0.039 0.040 0.030 0.028 0.035 n/d n/d n/d n/d n/d 0.008
4.5 3.7 5.1 7.4 2.8 4.8 5.8 5.8 4.5 5.1 8.2 8.4 8.1 7.1 6.9 5.9 6.1 6.2 9.0 9.1 6.2 F 1.8
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4. Discussion Following the injection, the distribution half-life (t1/2a) of OTC (1.4 h) indicated that the drug was distributed relatively fast to the tissues from the blood compartment. This value is shorter than that found in gilthead sea bream (2 h) maintained at the same water temperature (Rigos et al., 2003) but longer than that reported in European sea bass at 22 or 13 jC (0.2 –1 h) (Rigos et al., 2002a) and ayu kept at 18 jC (1 h) (Uno, 1996). Since experimental protocols were almost identical in the studies of the euryhaline fish, differences on OTC distribution may be due to inter-specific differences in their physiology. Repeated attempts to investigate the bioavailability of OTC in this species were unsuccessful since oral administration of OTC-medicated solution via a stomach tube or by-hand delivery of OTC-treated pellets revealed undetectable concentrations in plasma and muscle of sharpsnout sea bream. There was no evidence of any digestive disorders (vomiting or abnormal faecal release) that could have caused rejection of the drug. This is possibly the result of major hepatic metabolism of OTC in this species supported by OTC hepatic accumulation (follow-up experiment), but this assumption requires further investigation. The pertinent scientific literature report that first pass effect on OTC is considerable, also evidenced by the fact that considerable amounts of the drug are detected in the liver and bile soon after oral administration (Plakas et al., 1988; Bjorklund and Bylund, 1990). In addition, further OTC absorption can be further reduced due to the considerable potential of this drug to complex with cations (e.g. Mg2 + and Ca2 +) in the feed (diet used contained 1.2% Ca2 + with no addition of Mg2 +) and in the intestinal environment of the fish, possibly reducing membrane permeability (Clive, 1968). Published bioavailabilities ( F%) of OTC in other species of farmed fish, also indicate significantly reduced absorption of the drug after oral administration (0.6 – 9%) (Grondel et al., 1987; Bjorklund and Bylund, 1991; Elema et al., 1996; Uno, 1996; Haug and Hals, 2000; Rigos et al., 2003). It another study, it was found that 60% of OTC mixed in to the diet, passes unabsorbed in the faeces of sharpsnout sea bream (Rigos et al., 2002b). The amount of unabsorbed OTC is even higher (73%) in the faeces of other species, such as gilthead sea bream (Rigos et al., 2002b) and rainbow trout (90%) (Cravedi et al., 1987). As in mammals, the piscine hepatic microsomal cytochrome P450-dependent mixedfunction oxidase system (P450 monooxygenase) is known to be the primary mechanism for the oxidative metabolism of a variety of xenobiotics, including drugs (Stegeman, 1989). Unfortunately, to date there have been no studies of hepatic P450 monooxygenase enzymes in sharpsnout sea bream. Further investigation of CYP450 isozyme induction profiles and catalytic activity following OTC exposure is clearly warranted in this species. Furthermore, the determination of OTC metabolites in bile samples would also help to improve our understanding of OTC metabolism in sharpsnout sea bream. The elimination half-life (t1/2b) of OTC from sharpsnout sea bream plasma (35 h) is shorter than that found in gilthead sea bream (53 h) held at the same temperature (Rigos et al., 2003) and ayu held at 18 jC (52 h) (Uno, 1996). A faster elimination has also been observed in European sea bass kept at 22 jC (10 h) (Rigos et al., 2002a). Although
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temperature is reported to be a major factor affecting the rate of OTC elimination (Rigos et al., 2002a), the temperature variation between these studies is small, so differences in t1/2b of OTC between these species are likely the result of physiological differences between species and/or small differences in experimental design. The apparent volume of distribution of OTC at steady-state (Vd(ss)) in sharpsnout sea bream (4.0 l/kg) observed here is larger than that reported for gilthead sea bream (2.9 l/kg; Rigos et al., 2003) and European sea bass (2.6 l/kg; Rigos et al., 2002a). The experimental procedures were similar between this and the other reported studies; thus, these differences can again be attributed to species-specific differences. The observed large volume of distribution in sharpsnout sea bream indicates that the drug is adequately distributed throughout the body from the blood which is a favourable property, in terms of treating bacterial pathogens localised in poorly vascularised areas, such as the skin or muscle (Bjorklund and Bylund, 1991). However, intravascular administration is not the most practical preferred mode of dosing in fish farming. Poor OTC tissue absorption following oral administration shown in this study, suggest that possibly considerable OTC pollution could occur in the vicinity of aquatic farms using this drug, with potentially devastating consequences. These mainly include the development of antibiotic resistance in local bacterial populations (Bjorklund et al., 1991; Coyne et al., 1994; Herwig and Gray, 1997; Herwig et al., 1997) and the accumulation of drug residues in aquatic animals, including species used for human consumption, such as mussels and crabs (Capone et al., 1996; Coyne et al., 1997).
5. Conclusion In conclusion, the pharmacokinetic profile of OTC following intravascular injection in sharpsnout sea bream shows a fast distribution to tissues, a slow elimination from plasma and a large penetration throughout the body compartment. Oral administration was unsuccessful however, reflected by undetectable OTC levels in plasma and muscle samples. Thus, oral administration of OTC in sharpsnout sea bream should be discouraged in favour of other routes of administration, such as bathing fish in the drug-treated water, and where this is not practical, other antibiotics should be considered. Furthermore, this study has highlighted the paucity in our understanding on the mechanisms involved in OTC metabolism in this species and in sparids generally.
References Baggot, J.D., 1977. The Principles of Drug Disposition in Domestic Animals: The Basis of Veterinary Clinical Pharmacology. Saunders, Philadelphia. 238 pp. Bjorklund, H., Bylund, G., 1990. Temperature-related absorption and excretion of oxytetracycline in rainbow trout (Salmo gairdneri R.). Aquaculture 84, 363 – 372. Bjorklund, H.V., Bylund, G., 1991. Comparative pharmacokinetics and bioavailability of oxolinic acid and oxytetracycline in rainbow trout (Oncorhynchus mykiss). Xenobiotica 21, 1511 – 1520. Bjorklund, H.V., Rabergh, C.M.I., Bylund, G., 1991. Residues of oxolinic acid and oxytetracycline in fish and sediments from fish farms. Aquaculture 97, 85 – 96.
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Capone, D.G., Weston, D.P., Miller, V., Shoemaker, C., 1996. Anti-bacterial residues in marine sediments and invertebrates following chemotherapy in aquaculture. Aquaculture 145, 55 – 75. Clive, D.L.G., 1968. Chemistry of tetracyclines. Quart. Rev. Chem. Soc. 22, 435 – 456. Coyne, R., Hiney, M., O’Connor, B., Kerry, J., Cazabon, D., Smith, P., 1994. Concentration and persistence of oxytetracycline in sediments under a marine salmon farm. Aquaculture 123, 31 – 42. Coyne, R., Hiney, M., Smith, P., 1997. Transient presence of oxytetracycline inmussels (Mytilus edulis) following its therapeutic use at a marine Atlantic salmon farm. Aquaculture 149, 175 – 181. Cravedi, J.P., Choubert, G., 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. Elema, M.O., Hoff, K.A., Kristensen, H.G., 1996. Bioavailability of oxytetracycline from medicated feed administered to Atlantic salmon (Salmo salar) in sea water. Aquaculture 143, 7 – 14. FGM, 2000. Federation of Greek Mariculture. Annual Report. In Greek. Grondel, J.L., Nouws, J.F.M., De Jong, M., Schutte, A.R., Driessens, F., 1987. Pharmacokinetics and tissue distribution of oxytetracycline in carp, Cyprinus carpio L. following different routes of administartion. J. Fish Dis. 10, 153 – 163. Haug, T., Hals, P.A., 2000. Pharmacokinetics of oxytetracycline in Arctic charr (Salvelinus alpinus L.) in freshwater at low temperature. Aquaculture 186, 175 – 191. Herwig, R.P., Gray, J.P., 1997. Microbial response to antibacterial treatment in marine microcosms. Aquaculture 152, 139 – 154. Herwig, R.P., Gray, J.P., Weston, D.P., 1997. Antibacterial resistant bacteria in surficial sediments near salmon net-cage farms in Puget Sound Washington. Aquaculture 149, 263 – 283. Plakas, S.M., McPearson, R.M., Guarino, A.M., 1988. Disposition and bioavailability of 3H-tetracycline in the channel catfish (Ictalurus punctatus). Xenobiotica 18, 83 – 93. Rigos, G., Alexis, M., Andriopoulou, A., Nengas, I., 2002a. Pharmacokinetics and tissue distribution of oxytetracycline in sea bass (Dicentrarchus labrax) at two water temperatures. Aquaculture 210, 59 – 67. Rigos, G., Nengas, I., Alexis, M., Tyrpenou, A.E., Arbanitis, P., Troisi, G.M., 2002b. Digestibility of oxytetracycline and oxolinic acid in Sparidae farmed fish. 10th International Symposium on Nutrition and Feeding in Fish, Feeding for Quality, 2 – 7 June, Rhodes, Greece, p. 113. Rigos, G., Nengas, I., Tyrpenou, A.E., Alexis, M., Troisi, G.M., 2003. Pharmacokinetics and bioavailability of oxytetracycline in gilthead sea bream (Sparus aurata) after a single dose. Aquaculture 221, 75 – 83. Ritschel, W.A., 1986. Handbook of Basic Pharmacokinetic, Including Clinical Applications, 3rd ed. Drug Intelligence Publications, Hamilton. 544 pp. Stegeman, J.J., 1989. Cytochrome P-450 forms in fish: catalytic, immmunological and sequence similarities. Xenobiotica 19, 1093 – 1110. Tyrpenou, A.E., 1995. Tetracycline (OTC, TC, CTC) determination by HPLC-PDA. Quality assurance and validation procedures used at the EU-NRL of Athens-Greece. EU Workshop on Quality Assurance in HPLC-validation of HPLC Methods for Antimicrobial Residues in Meat. CNEVA-Laboratoire des Medicamentes Veterinaire. CRL-Community Reference Laboratory, 13 – 16 June, Fougeres-France. Uno, K., 1996. Pharmacokinetic study of oxytetracycline in healthy and vibriosis-infected ayu (Plecoglossus altivelis). Aquaculture 143, 33 – 42.
Poor bioavailability of oxytetracycline in sharpsnout sea bream Diplodus puntazzo G. Rigos a,b,*, Tyrpenou c, I. Nengas a, A.E.M. Alexis a, F. Athanassopoulou d, G.M. Troisi e a
Laboratory of Fish Nutrition and Pathology, National Centre for Marine Research, Aghios Kosmas 16604, Ellinikon, Attiki, Greece b School of Life Sciences, University of Kingston, Kingston upon Thames, KT1 2EE Surrey, London, UK c Department of Residue Research, HPLC Laboratory, Institute of Veterinary Research of Athens, National Agricultural Research Foundation, 25 Neapoleos Street, Agia Paraskevi 15310, Athens, Greece d Faculty of Veterinary Medicine, School of Health Sciences, University of Thessaly, Karditsa, 43100, Greece e Environmental Monitoring Unit, ETC Building, Brunel University, Uxbridge, UB8 3PH, Middlesex, UK Received 17 July 2003; received in revised form 15 October 2003; accepted 15 October 2003
Abstract The pharmacokinetics of oxytetracycline (OTC) were investigated following single intravascular injection (40 mg/kg) in sharpsnout sea bream (90 g) at 19 jC. The distribution half-life (t1/2a) and the elimination half-life (t1/2b) of OTC were calculated to be 1.4 and 35 h, respectively. The apparent volume of distribution of the drug at steady-state (Vd(ss)) was found to be 4 l/kg. The total clearance rate (CLT) of the drug was low (0.08 l/kg h). Repeated attempts to investigate the bioavailability of OTC following oral administration (75 mg/kg; forced-fed or via the feed), revealed undetectable levels of the drug in plasma and muscle samples. Poor tissue absorption is likely due to significant hepatic metabolism of OTC in sharpsnout sea bream, but this requires further investigation. Thus, oral administration of OTC in farmed sharpsnout sea bream should be discouraged in favour of other routes of administration or other antibiotic drugs. D 2003 Elsevier B.V. All rights reserved. Keywords: Oxytetracyline; Diplodus puntazzo; Pharmacokinetics; Absorption
1. Introduction During the last decade, there has been a need in the Mediterranean aquaculture industry to cultivate new fish species to overcome the problem arising from overproduction of the * Corresponding author. Tel.: +30-210-9829238; fax: +30-210-1-9811713. E-mail address: [email protected] (G. Rigos). 0044-8486/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2003.10.016
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two main farmed species, gilthead sea bream and European sea bass, by product diversification (FGM, 2000). Sharpsnout sea bream is a fast growing sparid and so far represents more than 60% of Greek mariculture production for all new farmed species (FGM, 2000). Up to now, as with other water warm fish species, oxytetracycline (OTC) therapeutic regimes for sharpsnout sea bream are extrapolated from pharmacokinetic information derived from salmonids. However, to optimise the treatment regimens for Mediterranean fish farming of sharpsnout sea bream, specific information on the pharmacokinetics of OTC in this species is needed. Therefore, the aim of this study was to investigate the kinetic profile of OTC in sharpsnout sea bream following intravascular and oral drug administration.
2. Materials and methods 2.1. Chemicals Oxytetracycline hydrochloride of high purity (>99%) and erythrosine were obtained from Sigma (USA). High-performance liquid chromatography (HPLC) grade solvents were obtained from Labscan (Ireland). The columns Isolute C18 used for solid phase extraction (SPE) were bought from International Technology (Sorben, UK). 2.2. Fish Two hundred healthy sharpsnout sea bream Diplodus puntazzo (90 F 20 g) were acclimated for 2 weeks in the facilities of the National Centre for Marine Research in Athens before starting the experiment. They were placed in cylindroconical fiberglass tanks (120 l) receiving water sea with salinity of 36xat a flow rate of 2 l/min. The temperature was maintained at 19 F 0.5 jC. Since it is a common practice in fish farms to starve fish to increase appetite and minimize stress prior to drug administration, fish were not fed for 2 days prior to commencing experiments and were kept at 19 F 0.5 jC. 2.3. Intravascular administration Prior to injection, fish were anaesthetized with quinaldine (2 ml/l salt water; 36x) and weighed. Intravenous injections of OTC (200 Al; 40 mg/kg fish) dissolved in sterile water were made into the caudal vein. To ensure the correct position of the needle (Microlance 23G 11/4 0.6 30, Becton Dickinson, Spain), blood was aspirated into the syringe prior to and following injection. Fish which were bled heavily were excluded and replaced (30%). Approximately 1 ml of blood was drawn from the caudal vein (away from the injection site) of several fish (six to eight individuals), at each of the following time points after dosing: 1, 2, 4, 8, 16, 24, 48, 72 and 150 h. After collecting blood samples, fish were killed by a blow to the head and 1 g of muscle devoid of skin or blood was taken from the dorsal area just above the lateral line. Plasma was prepared from blood samples by centrifugation
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at 3500 rpm for 10 min at 4 jC. Muscle and plasma samples were stored at 20 jC until analysis. 2.4. Oral administration Prior to oral administration of the substance, fish were anaesthetized with quinaldine as previously stated and weighed. Oxytetracycline (75 mg/kg fish) was administered via stomach tube (diameter 2 mm) initially dissolved in starch and sterile water (0.5 ml containing the red food stain erythrosine in order to detect possible regurgitation of the drug). Since significant regurgitation was evident (90%), the drug was re-administered in sterile aqueous suspensions (0.5 ml). Notwithstanding, OTC acceptance was improved with the different solution, regurgitation was still considerable (70%), thus the results were rejected (the analysis of samples from treated fish showing no regurgitation of OTC revealed undetectable levels). Then, it was decided to avoid force-feeding and administer a single dosage of drug mixed in feed pellets. One hundred fish of the same size were given control feed for a week at daily rate of 1% biomass. They were maintained at cylindroconical fiberglass tanks (880 l) under the aforementioned conditions. Following a 1-week acclimatization period, medicated pellets (75 mg/kg fish) were carefully delivered to minimise variation and readily consumed by the fish. Ten feed samples (100 Ag) were subjected to HPLC analysis for concentration confirmation. Approximately 1 ml of blood was drawn from the caudal vein of several fish (six to eight individuals), at each of the following time points after dosing: 4, 8, 16, 24, 48, 72 and 150 h. Plasma preparation and sample storage conditions were identical to the protocol followed for the intravascular administration. HPLC analysis of the plasma and muscle samples after administration of OTC-medicated pellets again revealed undetectable levels of the drug for all time points. To further investigate this discrepancy, the procedure was repeated by delivering medicated pellets to sharpsnout sea bream with the application of extent monitoring in glass aquaria (220 l) to permit regular observation of fish feeding behaviour and identify any possible digestive disorders that could have caused rejection of the medicated feed. Oxytetracyline-medicated feed was observed to be readily consumed by the 10 studied fish. Within 1 h following administration, no vomiting or other signs of digestive disorders were observed. Faecal release was normal and commenced approximately 25 min after the administration of the medicated pellets. Five of the fish were collected after 12 h and the remaining fish sampled after 24 h from the time of oral dosing. Plasma and muscle samples were taken from these fish and stored as stated previously. HPLC analysis of the plasma and muscle samples again revealed undetectable levels of the drug at both time points. To understand further the mechanisms of low OTC absorption in sharpsnout sea bream, a follow-up experiment was undertaken in 100 fish (200 g) kept in small fish cages (2 2 m) at 17 jC. Basically, the single oral OTC dosing (75 mg/kg) through pellets was repeated, but drug concentrations in liver samples not previously collected, were also analysed. This was to investigate any hepatic accumulation, which would indicate that most of the drug is metabolised prior to entering the general circulation. Muscle and liver samples from 20 fish were analysed. No plasma
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samples were available. Tissue samples from 20 fish were stored and treated as stated previously. 2.5. OTC analysis The analysis of OTC was performed according to the procedure of Tyrpenou (1995) with some modifications. Briefly, tissue samples (1 g) were homogenized with 10 ml McIlvaine buffer (citric acid 0.06, K2HPO42H2O 0.02 M) (68:32 v/v) and centrifuged for 10 min at 12,000 rpm. The supernatant was retained and the procedure repeated. To the combined supernatants, 2 ml of hexane were added and the solution centrifuged at 4000 rpm for 5 min. The organic layer containing lipid was discarded. The samples were cleaned up by a SPE procedure using Isolute C18 disposable columns (500 mg of 6 ml capacity). Columns were pre-conditioned with 5 ml methanol and 5 ml McIlvaine buffer. Samples were eluted with 10 ml methanol/ acetonitrile (50:50 v/v). After this, columns were cleaned using 10 ml HPLC-grade water and dried under vacuum for 15 min. The eluate was evaporated to dryness under nitrogen at 50 jC, resuspended in 1 ml of HPLC mobile phase solvent (oxalic acid 0.01 M: acetonitrile) (85:15 v/v), vortexed and filtered (0.45 Am). Exactly 100 Al of sample were injected into a Waters Alliance 2690MX HPLC system fitted with a Water 510 pump and a Waters 484 UV detector. Analytes were separated using an H&P Deactivated Hypersil ODS C18 (100 4 mm i.d.; pore size 5 Am). The detection wavelength was 360 nm and the flow rate 0.5 ml/min. Under these conditions, the retention time of OTC was 6 min and the detection limit of OTC was z 15 ng/ml (linear range to 1000 ppb in drug-free plasma ( P < 0.002) and tissue samples ( P < 0.005)). OTC concentrations were extrapolated from calibration curves of different concentrations of authentic high-purity OTC standard. For plasma samples (200 Al), the method was repeated by reducing all volumes by a factor of five. In the SPE procedure, smaller Isolute C18 disposable columns were used (100 mg of 1 ml capacity). The percentage of recoveries of OTC from all samples was calculated by comparing OTC concentrations in spiked drug-free plasma and muscle samples to a standard solution. OTC recovery through the method was found to be 69 F 4% for tissue samples and 62 F 5% for plasma samples. 2.6. Pharmacokinetic analysis The data were analysed for the best fit to a two- or three-compartment open pharmacokinetic model using non-linear regression analysis programs (NLREG 2001, P.H. Sherrod), following a semi-logarithmic plot of the data (curve EXPERT 1997, D. Hyams) and least square fitting (Ritschel, 1986). Diffusion processes were assumed to follow first order kinetics. Calculations of the apparent volume of distribution at steady state (Vdss) and total body clearance (CLT) were performed in a model independent way (Ritschel, 1986) to achieve objective comparison and to avoid overestimation by using the model slope b. The area under the concentration– time curve (AUC) was calculated using the trapezoidal rule and was extrapolated to infinity (Ritschel, 1986). Least square regression analysis was applied to calculate the elimina-
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Table 1 Plasma and muscle concentration of OTC in sharpsnout sea bream (90 g) at 19 jC following single intravascular injection (40 mg/kg) at each individual time point (mean F S.D.; n = 7) Time from dosing (h)
1 2 4 8 16 24 48 72 150
OTC concentration Plasma (Ag/ml)
Muscle (Ag/g)
18.6 F 6.8 14.1 F 4.3 12.4 F 2.8 9.9 F 2.6 7.7 F 1.2 5.8 F 1.4 4.0 F 0.5 1.6 F 0.2 0.9 F 0.2
4.5 F 1.4 5.5 F 2.2 4.6 F 0.7 10.1 F 2.1 6.6 F 1.4 6.1 F 1.6 11.1 F 3.2 10.2 F 2.6 3.0 F 1.1
tion slope b. The elimination half-lives were calculated from the equation t1/2 = ln2/b (Baggot, 1977).
3. Results Data analysis of the intravascular injection conformed better to a two-compartment model than a three-compartment model. Thus, the plasma concentration versus time
Table 2 Calculated pharmacokinetic parameters (compartmental; mean F S.D.) of intravascularly injected OTC (40 mg/ kg) in sharpsnout sea bream (90 g) at 19 jC Parameter
Values
Compartmental A (Ag/ml) Aˆ (Ag/ml) a (h 1) b (h 1) t1/2a (h) t1/2b (h) Vc (l/kg)
12.9 F 4.9 11.3 F 2.0 0.6 F 0.3 0.02 F 0.008 1.4 F 0.5 34.5 F 3.0 1.7 F 0.03
Non-compartmental Vd(ss) (l/kg) 4.0 AUC0 ! l (Ag/h ml) 549 0.08 CLT (l/kg h) MRT (h) 49 Abbreviations: Aa, Aˆ: zero-time plasma drug concentration intercepts of biphasic disposition curve; a, b: distribution and elimination rate constants; t1/2a: distribution half-life of the drug; t1/2b: elimination half-life of the drug; Vd(ss): apparent volume of distribution of the drug at steady-state; Vc: extensive apparent volume of the central compartment; AUC0 ! l: area under the drug concentration curve extrapolated to infinity; CLT: total body clearance of the drug; MRT: mean residence time.
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curve was calculated using the bi-exponential equation: C = Ae at + Be bt where C is the plasma concentration, t is the time, a and b are the slopes of mono-exponential declining curves, and A and B are the zero time plasma concentrations (Ritschel, 1986). Mean OTC concentrations following intravascular dosing decreased steadily in plasma from 18.6 to 0.9 Ag/ml over 150 h post-dosing. In contrast, muscle OTC concentrations were variable ranging from 3.0 to 11.1 Ag/g over the same time period (Table 1). The calculated pharmacokinetic parameters are presented in Table 2. The distribution half-life (t1/2a) was calculated to be fast but the elimination half-life (t1/2b) of OTC from plasma was found to be long. The apparent volume of distribution of the drug at steady-state (Vd(ss)) and the extensive apparent volume of the central compartment (Vc) were both large. The total clearance of the drug (CLT) was found to be low and the mean residence time (MRT) was long. In the follow-up experiment, analysis of muscle samples revealed extremely low or undetectable OTC concentrations. In contrast, OTC levels up to 8.4 Ag/g were observed in liver samples (Table 3) indicative of hepatic accumulation and metabolism of the drug. The concentration of OTC in the medicated diet was 110% (110 F 5; mean F S.E.) of the original dose.
Table 3 Mean muscle and liver concentrations (Ag/g) of OTC in sharpsnout sea bream (200 g) kept in situ (n = 20) at 17 jC, 24 h post-oral dosing (75 mg/kg) Fish sample
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Mean F S.D. n/d—not detectable.
OTC concentration (Ag/g) Muscle
Liver
n/d n/d n/d n/d n/d n/d n/d n/d n/d n/d 0.039 0.040 0.030 0.028 0.035 n/d n/d n/d n/d n/d 0.008
4.5 3.7 5.1 7.4 2.8 4.8 5.8 5.8 4.5 5.1 8.2 8.4 8.1 7.1 6.9 5.9 6.1 6.2 9.0 9.1 6.2 F 1.8
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4. Discussion Following the injection, the distribution half-life (t1/2a) of OTC (1.4 h) indicated that the drug was distributed relatively fast to the tissues from the blood compartment. This value is shorter than that found in gilthead sea bream (2 h) maintained at the same water temperature (Rigos et al., 2003) but longer than that reported in European sea bass at 22 or 13 jC (0.2 –1 h) (Rigos et al., 2002a) and ayu kept at 18 jC (1 h) (Uno, 1996). Since experimental protocols were almost identical in the studies of the euryhaline fish, differences on OTC distribution may be due to inter-specific differences in their physiology. Repeated attempts to investigate the bioavailability of OTC in this species were unsuccessful since oral administration of OTC-medicated solution via a stomach tube or by-hand delivery of OTC-treated pellets revealed undetectable concentrations in plasma and muscle of sharpsnout sea bream. There was no evidence of any digestive disorders (vomiting or abnormal faecal release) that could have caused rejection of the drug. This is possibly the result of major hepatic metabolism of OTC in this species supported by OTC hepatic accumulation (follow-up experiment), but this assumption requires further investigation. The pertinent scientific literature report that first pass effect on OTC is considerable, also evidenced by the fact that considerable amounts of the drug are detected in the liver and bile soon after oral administration (Plakas et al., 1988; Bjorklund and Bylund, 1990). In addition, further OTC absorption can be further reduced due to the considerable potential of this drug to complex with cations (e.g. Mg2 + and Ca2 +) in the feed (diet used contained 1.2% Ca2 + with no addition of Mg2 +) and in the intestinal environment of the fish, possibly reducing membrane permeability (Clive, 1968). Published bioavailabilities ( F%) of OTC in other species of farmed fish, also indicate significantly reduced absorption of the drug after oral administration (0.6 – 9%) (Grondel et al., 1987; Bjorklund and Bylund, 1991; Elema et al., 1996; Uno, 1996; Haug and Hals, 2000; Rigos et al., 2003). It another study, it was found that 60% of OTC mixed in to the diet, passes unabsorbed in the faeces of sharpsnout sea bream (Rigos et al., 2002b). The amount of unabsorbed OTC is even higher (73%) in the faeces of other species, such as gilthead sea bream (Rigos et al., 2002b) and rainbow trout (90%) (Cravedi et al., 1987). As in mammals, the piscine hepatic microsomal cytochrome P450-dependent mixedfunction oxidase system (P450 monooxygenase) is known to be the primary mechanism for the oxidative metabolism of a variety of xenobiotics, including drugs (Stegeman, 1989). Unfortunately, to date there have been no studies of hepatic P450 monooxygenase enzymes in sharpsnout sea bream. Further investigation of CYP450 isozyme induction profiles and catalytic activity following OTC exposure is clearly warranted in this species. Furthermore, the determination of OTC metabolites in bile samples would also help to improve our understanding of OTC metabolism in sharpsnout sea bream. The elimination half-life (t1/2b) of OTC from sharpsnout sea bream plasma (35 h) is shorter than that found in gilthead sea bream (53 h) held at the same temperature (Rigos et al., 2003) and ayu held at 18 jC (52 h) (Uno, 1996). A faster elimination has also been observed in European sea bass kept at 22 jC (10 h) (Rigos et al., 2002a). Although
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temperature is reported to be a major factor affecting the rate of OTC elimination (Rigos et al., 2002a), the temperature variation between these studies is small, so differences in t1/2b of OTC between these species are likely the result of physiological differences between species and/or small differences in experimental design. The apparent volume of distribution of OTC at steady-state (Vd(ss)) in sharpsnout sea bream (4.0 l/kg) observed here is larger than that reported for gilthead sea bream (2.9 l/kg; Rigos et al., 2003) and European sea bass (2.6 l/kg; Rigos et al., 2002a). The experimental procedures were similar between this and the other reported studies; thus, these differences can again be attributed to species-specific differences. The observed large volume of distribution in sharpsnout sea bream indicates that the drug is adequately distributed throughout the body from the blood which is a favourable property, in terms of treating bacterial pathogens localised in poorly vascularised areas, such as the skin or muscle (Bjorklund and Bylund, 1991). However, intravascular administration is not the most practical preferred mode of dosing in fish farming. Poor OTC tissue absorption following oral administration shown in this study, suggest that possibly considerable OTC pollution could occur in the vicinity of aquatic farms using this drug, with potentially devastating consequences. These mainly include the development of antibiotic resistance in local bacterial populations (Bjorklund et al., 1991; Coyne et al., 1994; Herwig and Gray, 1997; Herwig et al., 1997) and the accumulation of drug residues in aquatic animals, including species used for human consumption, such as mussels and crabs (Capone et al., 1996; Coyne et al., 1997).
5. Conclusion In conclusion, the pharmacokinetic profile of OTC following intravascular injection in sharpsnout sea bream shows a fast distribution to tissues, a slow elimination from plasma and a large penetration throughout the body compartment. Oral administration was unsuccessful however, reflected by undetectable OTC levels in plasma and muscle samples. Thus, oral administration of OTC in sharpsnout sea bream should be discouraged in favour of other routes of administration, such as bathing fish in the drug-treated water, and where this is not practical, other antibiotics should be considered. Furthermore, this study has highlighted the paucity in our understanding on the mechanisms involved in OTC metabolism in this species and in sparids generally.
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