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Thiamphenicol disposition in pigs
Research in Veterinary Science 1999, 66, 219–222 Article No. rvsc.1998.0263, available online at http://idealibrary.com on
Thiamphenicol disposition in pigs G. CASTELLS*, C. PRATS, G. EL KORCHI, B. PÉREZ, M. ARBOIX, C. CRISTÒFOL. Department de Farmacologia i de Terapèutica. Facultat de Veterinària. Universitat Autònoma de Barcelona, 08193, Bellaterra, Spain, G. MARTÌ, Laboratori JAER S.A. Sant Vicenç dels Horts, Barcelona, Spain
SUMMARY Pharmacokinetic parameters of thiamphenicol (TAP) were determined after intravenous (i.v.) and intramuscular (i.m.) administration of 30 mg kg–1 of TAP in pigs. Plasma drug concentrations were determined by high performance liquid chromatography (HPLC) Intravenous TAP kinetics were fitted to a bi-exponential equation, with a first rapid disposition phase followed by a slower disposition phase. Elimination half-life was short, at 59·3 (29·4) minutes; volume of distribution at steady state was 0·62 (0·24) 1 kg–1; and plasma clearance was 13·4 (4·5) ml min–1 kg–1. After i.m. administration, the peak plasma concentration (Cmax = 4·1 µg ml–1) was reached in about 60 minutes; these concentrations are lower than those reported in other species. The TAP elimination half-life after i.m. administration, 250·2 (107·1) minutes was longer after than i.v. administration, probably due to the slow rate of absorption from the muscle. The mean bioavailability value for i.m. administration was 76 (12) per cent.
THIAMPHENICOL (TAP) is a bacteriostatic broad-spectrum antibiotic, an analogue of chloramphenicol with similar antibacterial activity (Neu and Fu 1980). The use of chloramphenicol has been strictly regulated because it is possibly responsible for severe adverse reactions in humans and animals (Yunis 1981, Paape et al 1990). The main adverse irreversible and non-dose-related effect related to chloramphenicol in humans is aplastic anaemia (Ferrari and Pajola 1981). The presence of the p-nitro group in the chloramphenicol molecule has been reported as the main factor responsible for this (Yunis 1988). That this structure is substituted by a sulpho-group TAP and floryfenicol molecules would explain the lack of such an adverse reaction after TAP and floryfenicol treatment (Powers et al 1988). Both these antibiotics, with similar spectra of activity, appear to be viable substitutes for chloramphenicol in veterinary medicine. The pharmacokinetic profile of TAP has been studied in cattle (Signorini et al 1986, Gámez et al 1992, Mestorino et al 1993) and sheep (Abdennebi et al 1994a), showing a good absorption and distribution when administered via the intramuscular (i.m.) route, with bioavailability values of 74 to 100 per cent. It also has a high distribution into the aqueous humour, central nervous system, lung tissue, kidney and mammary gland, among others (Cambieri et al 1970, Ferrari 1984, Mestorino et al 1993), and a low protein binding of about 10 per cent (Kawabe et al 1966). The elimination of the drug is rapid, showing an elimination half-life of about one to three hours in all studied species, and it is mainly excreted renally in its active form (McChesney et al 1960, Nakagawa et al 1975). There is little information about the pharmacokinetic profile of TAP in monogastric animals, although it may be of use in the treatment of infectious disease in pigs and chickens
*Corresponding author 0034-5288/99/030219 + 04 $12.00/0
among others. Recently, the use of TAP has been approved, with definitive MRL values of 50 ng g–1 for cattle and poultry. In anticipation of an MRL value being established for other food-producing animal species, the aim of this study was to analyse the pharmacokinetic behaviour of TAP in pigs after i.v. and i.m. administration of a single dose, to consider its use in the treating the most common bacterial infections.
MATERIALS AND METHODS Chemicals Thiamphenicol dimethylacetamide (30 per cent aqueous solution) provided by Lab. JAER S.A. (Barcelona, Spain), was used to treat the pigs. Florfenicol, provided by Lab. Zambon (Milan, Italy), was used as an internal standard. Animals Five healthy, male, castrated Large White pigs, weighting 26·6 (4·1) kg, were studied. The study was performed using a cross-over design. Three pigs were treated i.v. and two i.m. (in the gluteal region), with a dose of 30 mg kg–1TAP. Three weeks later, the same animals were given a second dose via the other route. Blood samples were collected at selected times following administration between three minutes and 24 hours, through a catheter placed in the jugular vein. Plasma, obtained by centrifugation of the blood samples (1932 g, 20 minutes), was kept at –20°C until analysis. Analytical procedures Thiamphenicol plasma concentration was determined by high performance liquid chromatography (HPLC) as described below. Plasma samples (1 ml) were extracted with ethylacetate (5 ml) in a 0·01 M sodium phosphate (pH 7·0) solution (1 ml). Samples were shaken in an horizontal mixer © 1999 W. B. Saunders Company Ltd
G. Castells, C. Prats, G. El Korchi, B. Pérez, M. Arboix, C. Cristòfol, G. Marti
for 20 minutes and centrifuged (1932 xg, 20 minutes). The aqueous phase was discarded and the organic phase was evaporated to dryness in a rotary evaporator. The extract was re-dissolved in the mobile phase, which was a mixture of a 0·05 M sodium acetate solution (pH 4·3) and acetonitrile (80:20 v/v). Chromatographic separation was performed with a reverse phase C18 column (200 × 4·6, 5 µm) at a flow rate of 1·2 ml min–1. Ultraviolet absorbance (λ = 223 nm) was used for drug detection. The retention times for TAP and florfenicol were 4·5 and 10·9 minutes, respectively. The quantification limit of the method was 20 ng ml–1. The recovery rates for this method were 80 to 97 per cent for the different TAP concentrations assayed. The precision of the method was expressed as the coefficient of variation (CV) of the results obtained from different analyses (CV < 8%). The accuracy of the method was defined as the difference between the actual and the theoretical values, expressed according to mean per cent error (ME < 15 per cent). The linearity of the method was assayed between 20 ng ml–1 and 15 µg ml–1.
100 Plasma concentration (µg ml–1)
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10
1
0.1
0
Pharmacokinetic and statistical analysis The plasma concentration-time curves were fitted and analysed by a non-linear least-squares program, PCNONLIN (Metzler and Weiner 1992), and the pharmacokinetic parameters were calculated from the model. For statistical analysis of goodness of fit of the kinetic model, we used Akaike’s criterion (Yamaoka et al 1978). Multiple dose simulation was by a non-compartmental data analysis computer program, PK Solutions (Farrier 1997). Following i.v. administration, all curves were described by a bi-exponential equation (Cp = B1e–αt + B2e–βt). The equation that best described the curves obtained after i.m administration was: Cp = B2e–βt – B3e–K01t. The area under the plasma concentration-time curve (AUC) was calculated using the trapezoidal rule. Bioavailability (F) after i.m. administration of TAP was determined by F = AUCIM (0–24 h) × 100/AUCIV (0–24 h). The analysis of the differences between i.v. and i.m. kinetic parameters was by a non-parametric Wilcoxon’s test and statistical significance was set at P < 0·05.
200
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Time (minutes) FIG 1: Mean (SD) plasma concentrations (semi-logarithmic plot) of TAP in five pigs after i.v. (● ● ) and i.m. (■) administration of a dose of 30 mg kg–1.
TABLE 1: Mean (SD) of the pharmacokinetic parameters for TAP after a single i.v. and i.m. dose of 30 mg kg–1 to pigs (n = 5) Parameter 1 2α 1 2β 1 2 K01
t (minutes) t (minutes) (minutes) t tmax (minutes) Cmax (µg ml–1) Vdss (I kg–1) CI (ml min–1 kg–1) AUC024 h (µg min ml–1) F (per cent)
i.v. 2·4 (1·9) 59·3 (29·4)* – – – 0·62 (0·24) 13·4 (4·5) 1958 (633)* –
i.m. – 250·2 (107·1)* 6·6 (8·3) 62·5 (39·9) 4·1 (2·1) – – 1454 (348)* 76 (12)
t 12 α = distribution phase half-life (harmonic mean); t 12 β = terminal phase half-life (harmonic mean); Vdss = volume of distribution at steady state; CI = total plasma clearance of the drug; AUC024 h = area under the plasma concentrationtime curve; t 12 K01 = initial phase half-life (harmonic mean); t 12 β = terminal phase half-life (harmonic mean); tmax = time of peak plasma concentration; Cmax = peak plasma concentration; F = bioavaliability. *P < 0·05.
RESULTS TAP plasma concentration curves versus time, after i.v. and i.m. administration of 30 mg kg–1 TAP, are shown in Fig. 1. After i.v. administration, the plasma concentration reached a value of 168·0 (92·0) µg ml–1. The thiamphenicol plasma disposition was best represented by a bi-compartmental model, and detectable levels were observed up to 14 hours post-administration. After i.m. administration, a biexponential equation was used to best describe the plasma disposition of TAP, with a maximum plasma concentration (Cmax) of 4·1 (2·1) µg ml–1, reached at about 60 minutes after drug administration. Plasma levels of TAP were detectable up to 24 hours after administration. The pharmacokinetic parameters after i.v. and i.m. administration, obtained from analysis of the plasma concentration-time curves, are summarized in Table 1. After i.v. administration, the elimination half-life (t 12β) was 59·3 (29·4)
minutes, and the volume of distribution (Vdss) and clearance (CI) values were 0·62 (0·24) 1 kg–1 and 13·4 (4·5) ml min–1 kg–1, respectively. After i.m. administration, measurable TAP levels were detected in the first collected plasma sample (five minutes after administration). The initial half-life of TAP (t 12K01) was 6·6 (8·2) minutes. The t 12β after i.m. administration (250.2 minutes) was about four times longer than that observed after i.v. administration (59·3 minutes). The AUC value after i.v. administration was statistically different (P < 0·05) from that found after i.m. administration, yielding a mean bioavailability value for i.m. administration of 76 (12) per cent. A simulation was performed to estimate the steady state parameters in a multiple dose of 30 mg kg–1 at 24 hour intervals. The predicted Css (max), Css (min) and Css (av) were 3·7, 0·3 and 1·4 µg ml–1, respectively.
Thiamphenicol disposition in pigs
DISCUSSION Understanding the kinetic pattern of TAP is of interest in establishing the dosage of this antibiotic in pigs. The TAP disposition study after a single i.v. dose of 30 mg kg–1 revealed that it was best described by a bi-exponential equation with an initial rapid disposition phase and a slower second disposition phase, clearly emphasised by the high ratio between constants (α/β < 9). A similar bi-compartmental profile has been found by Gámez et al (1992) in cattle, Abdennebi et al (1994a) in sheep, and Lavy et al (1991) in goats, thus suggesting that TAP is rapidly distributed from blood to tissues. This result is in concordance with the high liposolubility, low protein binding (8 to 10 per cent) and pKa value (7·2) of TAP, which furthermore suggest a good penetration and distribution of the drug in the body fluids, as described in some studies where TAP reached higher levels in the liver and kidney than in plasma (Zambon Group Report 1978). The data are also in agreement with those from humans (Cambieri et al 1970, Ferrari 1984), in which higher concentrations of TAP than plasma levels were found in the lungs and kidneys after an oral dose of 15 mg kg–1. Studies in lactating cows (Signorini et al 1986, Mestorino et al 1993) and goats (Lavy et al 1991) have shown that TAP reached the mammary gland, as therapeutic levels of antibiotic (≥ 5 µg ml–1) were found in milk after different i.m. doses (25, 20 and 50 mg kg–1) of the drug. Thiamphenicol may remain stored in extravascular tissue, which could explain the relatively high distribution values, 0·62 (0·24) 1 kg–1. Similar values of Vdss were observed in calves (0·68 1 kg–1), sheep (0·80 1 kg–1) and goats (0·70 1 kg–1), by Abdennebi et al (1994b) and Lavy et al (1991), respectively. Some studies using chloramphenicol in pigs (Rao and Clarenburg 1977) showed that tissular levels in kidney and bile were respectively four and two times higher than those observed in plasma at the same time point after administration. The Vdss value obtained in the present study suggests that TAP might easily reach well perfused tissues, possibly in levels high enough to reach the minimum inhibitory concentration (MIC) for some of the micro-organisms responsible for infectious disease. The short t 12β (59·3 minutes) and high clearance (13·4 ml min–1 kg–1) values agree with the rapid elimination of TAP from the body by renal excretion of the active form (50 to 80 per cent of administered dose) described by some authors in humans (McChesney et al 1960, Nakagawa et al 1975) and in rats (Gazzaniga et al 1973). This rapid elimination suggests that the withdrawal period may be short. However, it should be noted that the plasma half-life does not allow estimation of the tissue half-life as the drug could accumulate in tissue. In order to establish the withdrawal period, it is necessary to study the depletion rate of the drug in tissues destined for human consumption. After i.m. administration, the TAP levels reached a Cmax of 4·1 (2·1) µg ml–1 in about 60 minutes. These plasma concentration values were considerably lower than those reported in ruminants. After administration of an i.m. dose of 20 or 25 mg kg–1 (Signorini et al 1986, Mestorino et al 1993, Abdennebi et al 1994a,b) to calves and sheep, and an i.m. dose of 30 mg kg–1 of the same concentration as in the present study to calves (Gámez et al 1992), the values achieved
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Cmax were between four and eight times higher than those found here in pigs. This could be due to interspecific differences, but it could also be attributable to retention of the drug at the injection site and delayed absorption. The i.m. elimination half-life (250·2 minutes) was higher than that observed after i.v. administration (59·3 minutes). The absorption process probably continued for several hours, which would affect the drug elimination process (absorption rate-dependent elimination). Moreover, Davis et al (1972) observed similar behaviour with chloramphenicol disposition in pigs when the drug was administered by either the i.v. or the i.m. routes. The bioavailability value for the i.m. route (76 per cent) was rather lower than those previously reported for ruminants (Gámez et al 1992, Mestorino et al 1993) Abdennebi et al 1994a,b, and humans (Ferrari 1984), which reported F values of between 84 and 100 per cent. Studies carried out by Inamoto et al (1994a,b) and Gutiérrez et al (1993) have shown that the MIC value of TAP for some ethiological agents of infectious disease in swine, such as Actinobacillus pleuropneumoniae, Pasteurella multocida and Mycoplasma hyopneumoniae, were between 0·1 and 1.56 µg ml–1. Other studies have showed a ratio of tissue to serum concentration of ≥ 1 in tissues such as the kidney, lung and mammary gland (Cambieri et al 1970, Matsumoto et al 1975, Zambon Report 1978). The plasma levels and the distribution and elimination pattern obtained in the present study, as well as the simulated predicted concentrations in the steady state, suggest that a 30 per cent aqueous solution of thiamphenicol dimethylacetamide administered i.m. at 30 mg kg–1 could be sufficient to achieve the MIC of pathogenic bacteria involved in the commonest infections in pigs, and that the interval between each administered dose could be extended up to 24 hours.
ACKNOWLEDGEMENTS This study was supported by Lab. JAER S.A. and CIRIT-CIDEM. We thank Francisco Pérez for his technical assistance.
REFERENCES ABDENNEBI, E.H., KHALES, N., SAWCHUK, R.J. & STOWE, C.M. (1994a) Thiamphenicol pharmacokinetics in sheep. Journal of Veterinary Pharmacology and Therapeutics 17, 12–16 ABDENNEBI, E.H., SAWCHUK, R.J. & STOWE, C.M. (1994b) Thiamphenicol pharmacokinetics in beef and dairy cattle. Journal of Veterinary Pharmacology and Therapeutics 17, 365–368 CAMBIERI, F., GAMBINI, A. & LODOLA, E. (1970) Diffusion of thiamphenicol in the bronchial secretions and in the lung tissue. Chemotherapy 15, 336–341 DAVIS, L.E., NEFF, C.A., BAGGOT, J.E. & POWERS, T.E. (1972) Pharmacokinetics of chloramphenicol in domesticated animals. American Journal of Veterinary Research 33, 2259–2266 FARRIER, D.S. (1997) PK Solutions 2.0.2. Noncompartmental Pharmacokinetics Data Analysis. Ashland, Summit Research Services. FERRARI, V. (1984) Introductory address. Salient features of thiamphenicol: review of clinical pharmacokinetics and toxicity. Sexually Transmitted Disease 11, 336–339 FERRARI, V. & PAJOLA, E. (1981) Types of haemopoietic inhibition by chloramphenicol and thiamphenicol. In: Safety Problems Related to Chloramphenicol and Thiamphenicol Therapy. Eds. Y. Najean, G. Tognoni & A. A. Yunis. New York: Raven Press, pp 43–59 GÁMEZ, A., PÉREZ, Y., MARTÍ, G., CRISTÒFOL, C. & ARBOIX, M. (1992) Pharmacokinetics of thiamphenicol in veal calves. British Veterinary Journal 148, 535–539
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GAZZANIGA, A., PEZZOTTI, E. & COTTA RAMUSINO, A. (1973) A rapid gas chromatographic method for the determination of thiamphenicol in body fluids and tissues. Journal of Chromatography 81, 71–77 GUTIÉRREZ, C. B., PÍRIZ, S., VADILLO, S. & RODRÍGUEZ FERRI, E. F. (1993). In vitro susceptibility of Actinobacillus pleuropneumoniae strains to 42 antimicrobial agents. American Journal of Veterinary Research 54, 546–550 INAMOTO, T., KIKUCHI, K., IIJIMA, H., KAWASHIMA, Y., NAKAI, Y. & OGIMOTO, K. (1994) Antibacterial activity of tilmicosin against Pasteurella multocida and Actinobacillus pleuropneumoniae isolated from pneumonic lesions in swine. Journal of Veterinary Medical Science 56, 917–921 INAMOTO, T., TAKAHASHI, H., YAMAMOTO, K., NAKAI, Y. & OGIMOTO, K. (1994) Antibiotic susceptibility of Mycoplasma hyopneumoniae isolated from swine. Journal of Veterinary Medical Science 56, 393–394 KAWABE, K., YAMAMOTO, S., KANAZAWA, T., TAKIMOTO, G., MYAMOTO, A. & IKEDA, T. (1966) Basic studies on thiamphenicol, a new chemotherapeutic. Chemotherapy 14, 421–426 LAVY, E., ZIV, G., GLICKMAN, A. & BEN-ZVI, Z. (1991) Single-dose pharmacokinetics of thiamphenicol in lactating goats. Acta Veterinaria Scandinavica 87 (suppl.), 133–136 MATSUMOTO, K. & UZUKA, Y. (1975) Concentrations of antibiotics in bronchiolar secretions of the patients with chronic respiratory infections. In 9th International Congress of Chemotherapy. Eds. J. D. Williams and A.M. Geddes. London, 4, 73–79. MCCHESNEY, E.W., KOSS, R.F., SHEROSKY, J.M. & DEITZ W.H. (1960) Metabolism of dextrosulphenidol in several animal species. Journal of the American Pharmaceutical Association 49, 762–766 MESTORINO, N., LANDONI, M.F., ALT, M. & ERRECALDE, J.O. (1993) The pharmacokinetics of thiamphenicol in lactating cows. Veterinary Research Communications 17, 295–303 METZLER, C.M. & WEINER, D.L. (1992) PCNONLIN User’s Guide. Lexington, MA: Statistical Consultants Inc. NAKAGAWA, T., MASADA, M. & UNO, T. (1975) Gas chromatographic determination and gas chromatographic mass spectometric analysis of chloramphenicol, thiamphenicol and their metabolites. Journal of Chromatography 111, 335–340
NEU, H.C. & FU, K.P. (1980) In vitro activity of chloramphenicol and thiamphenicol analogs. Antibacterial Agents and Chemotherapy 18, 311–316 PAAPE, M.J., MILLER, R.H. & ZIV, G. (1990) Effects of florfenicol, chloramphenicol, and thiamphenicol on phagocytosis, chemoluminiscence, and morphology of bovine polymorphonuclear neutrophil leukocytes. Journal of Dairy Science 73, 1734–1744 POWERS, T.E., VARMA, K.J. & POWERS, J.D. (1988) Clinical pharmacology of a new antibiotic for veterinary medicine: Florfenicol. In Veterinary Pharmacology, Toxicology and Therapy in Food Producing Animals. Proceedings of the 4th Congress of European Association for Veterinary and Toxicology. Eds. Ferenc Simon, Peter Lees & Gábor Semjén. Budapest: University of Veterinary Science, pp 3–6 RAO V. & CLARENBURG, R. (1977) Pharmacokinetics of intravenously injected chloramphenicol in baby pigs. Drug Metabolism and Disposition 5, 253–258 SIGNORINI, G., FERRARI, A., BALLARINI, G., BONANOMI, L. & MONDELLINI, A. (1986) Livelli plasmatici ed escrezione mammaria del tiamfenicolo nel bovino. Documenti Veterinari 7, 61–63 WATSON, A.D.J. (1991) Chloramphenicol 2. Clinical pharmacology in dogs and cats. Australian Veterinary Journal 68, 2–5 YAMAOKA, K., NAKAGAWA, T. & UNO, T. (1978) Application of Akaike’s Information Criterion (AIC) in the evaluation of pharmacokinetic equations. Journal of Pharmacokinetics and Biopharmaceutics 6, 164–175 YUNIS, A.A. (1981) Chloramphenicol toxicity and the role of the p-NO2 in aplastic anemia. In Safety Problems Related to Chloramphenicol and Thiamphenicol Therapy. Eds. Y. Najean, G. Tognoni & A.A. Yunis. New York: Raven Press, pp 17–29 YUNIS, A.A. (1988) Chloramphenicol: relation of structure to activity and toxicity. Annual Review of Pharmacology and Toxicology 28, 83–100 ZAMBON GROUP REPORT (1978) Serum, Tissue and Body Fluid Levels of Thiamphenicol. Zambon Documentation Medical Centre, Milan, Italy.
Accepted December 2, 1998
Thiamphenicol disposition in pigs G. CASTELLS*, C. PRATS, G. EL KORCHI, B. PÉREZ, M. ARBOIX, C. CRISTÒFOL. Department de Farmacologia i de Terapèutica. Facultat de Veterinària. Universitat Autònoma de Barcelona, 08193, Bellaterra, Spain, G. MARTÌ, Laboratori JAER S.A. Sant Vicenç dels Horts, Barcelona, Spain
SUMMARY Pharmacokinetic parameters of thiamphenicol (TAP) were determined after intravenous (i.v.) and intramuscular (i.m.) administration of 30 mg kg–1 of TAP in pigs. Plasma drug concentrations were determined by high performance liquid chromatography (HPLC) Intravenous TAP kinetics were fitted to a bi-exponential equation, with a first rapid disposition phase followed by a slower disposition phase. Elimination half-life was short, at 59·3 (29·4) minutes; volume of distribution at steady state was 0·62 (0·24) 1 kg–1; and plasma clearance was 13·4 (4·5) ml min–1 kg–1. After i.m. administration, the peak plasma concentration (Cmax = 4·1 µg ml–1) was reached in about 60 minutes; these concentrations are lower than those reported in other species. The TAP elimination half-life after i.m. administration, 250·2 (107·1) minutes was longer after than i.v. administration, probably due to the slow rate of absorption from the muscle. The mean bioavailability value for i.m. administration was 76 (12) per cent.
THIAMPHENICOL (TAP) is a bacteriostatic broad-spectrum antibiotic, an analogue of chloramphenicol with similar antibacterial activity (Neu and Fu 1980). The use of chloramphenicol has been strictly regulated because it is possibly responsible for severe adverse reactions in humans and animals (Yunis 1981, Paape et al 1990). The main adverse irreversible and non-dose-related effect related to chloramphenicol in humans is aplastic anaemia (Ferrari and Pajola 1981). The presence of the p-nitro group in the chloramphenicol molecule has been reported as the main factor responsible for this (Yunis 1988). That this structure is substituted by a sulpho-group TAP and floryfenicol molecules would explain the lack of such an adverse reaction after TAP and floryfenicol treatment (Powers et al 1988). Both these antibiotics, with similar spectra of activity, appear to be viable substitutes for chloramphenicol in veterinary medicine. The pharmacokinetic profile of TAP has been studied in cattle (Signorini et al 1986, Gámez et al 1992, Mestorino et al 1993) and sheep (Abdennebi et al 1994a), showing a good absorption and distribution when administered via the intramuscular (i.m.) route, with bioavailability values of 74 to 100 per cent. It also has a high distribution into the aqueous humour, central nervous system, lung tissue, kidney and mammary gland, among others (Cambieri et al 1970, Ferrari 1984, Mestorino et al 1993), and a low protein binding of about 10 per cent (Kawabe et al 1966). The elimination of the drug is rapid, showing an elimination half-life of about one to three hours in all studied species, and it is mainly excreted renally in its active form (McChesney et al 1960, Nakagawa et al 1975). There is little information about the pharmacokinetic profile of TAP in monogastric animals, although it may be of use in the treatment of infectious disease in pigs and chickens
*Corresponding author 0034-5288/99/030219 + 04 $12.00/0
among others. Recently, the use of TAP has been approved, with definitive MRL values of 50 ng g–1 for cattle and poultry. In anticipation of an MRL value being established for other food-producing animal species, the aim of this study was to analyse the pharmacokinetic behaviour of TAP in pigs after i.v. and i.m. administration of a single dose, to consider its use in the treating the most common bacterial infections.
MATERIALS AND METHODS Chemicals Thiamphenicol dimethylacetamide (30 per cent aqueous solution) provided by Lab. JAER S.A. (Barcelona, Spain), was used to treat the pigs. Florfenicol, provided by Lab. Zambon (Milan, Italy), was used as an internal standard. Animals Five healthy, male, castrated Large White pigs, weighting 26·6 (4·1) kg, were studied. The study was performed using a cross-over design. Three pigs were treated i.v. and two i.m. (in the gluteal region), with a dose of 30 mg kg–1TAP. Three weeks later, the same animals were given a second dose via the other route. Blood samples were collected at selected times following administration between three minutes and 24 hours, through a catheter placed in the jugular vein. Plasma, obtained by centrifugation of the blood samples (1932 g, 20 minutes), was kept at –20°C until analysis. Analytical procedures Thiamphenicol plasma concentration was determined by high performance liquid chromatography (HPLC) as described below. Plasma samples (1 ml) were extracted with ethylacetate (5 ml) in a 0·01 M sodium phosphate (pH 7·0) solution (1 ml). Samples were shaken in an horizontal mixer © 1999 W. B. Saunders Company Ltd
G. Castells, C. Prats, G. El Korchi, B. Pérez, M. Arboix, C. Cristòfol, G. Marti
for 20 minutes and centrifuged (1932 xg, 20 minutes). The aqueous phase was discarded and the organic phase was evaporated to dryness in a rotary evaporator. The extract was re-dissolved in the mobile phase, which was a mixture of a 0·05 M sodium acetate solution (pH 4·3) and acetonitrile (80:20 v/v). Chromatographic separation was performed with a reverse phase C18 column (200 × 4·6, 5 µm) at a flow rate of 1·2 ml min–1. Ultraviolet absorbance (λ = 223 nm) was used for drug detection. The retention times for TAP and florfenicol were 4·5 and 10·9 minutes, respectively. The quantification limit of the method was 20 ng ml–1. The recovery rates for this method were 80 to 97 per cent for the different TAP concentrations assayed. The precision of the method was expressed as the coefficient of variation (CV) of the results obtained from different analyses (CV < 8%). The accuracy of the method was defined as the difference between the actual and the theoretical values, expressed according to mean per cent error (ME < 15 per cent). The linearity of the method was assayed between 20 ng ml–1 and 15 µg ml–1.
100 Plasma concentration (µg ml–1)
220
10
1
0.1
0
Pharmacokinetic and statistical analysis The plasma concentration-time curves were fitted and analysed by a non-linear least-squares program, PCNONLIN (Metzler and Weiner 1992), and the pharmacokinetic parameters were calculated from the model. For statistical analysis of goodness of fit of the kinetic model, we used Akaike’s criterion (Yamaoka et al 1978). Multiple dose simulation was by a non-compartmental data analysis computer program, PK Solutions (Farrier 1997). Following i.v. administration, all curves were described by a bi-exponential equation (Cp = B1e–αt + B2e–βt). The equation that best described the curves obtained after i.m administration was: Cp = B2e–βt – B3e–K01t. The area under the plasma concentration-time curve (AUC) was calculated using the trapezoidal rule. Bioavailability (F) after i.m. administration of TAP was determined by F = AUCIM (0–24 h) × 100/AUCIV (0–24 h). The analysis of the differences between i.v. and i.m. kinetic parameters was by a non-parametric Wilcoxon’s test and statistical significance was set at P < 0·05.
200
400
600
800
1000
Time (minutes) FIG 1: Mean (SD) plasma concentrations (semi-logarithmic plot) of TAP in five pigs after i.v. (● ● ) and i.m. (■) administration of a dose of 30 mg kg–1.
TABLE 1: Mean (SD) of the pharmacokinetic parameters for TAP after a single i.v. and i.m. dose of 30 mg kg–1 to pigs (n = 5) Parameter 1 2α 1 2β 1 2 K01
t (minutes) t (minutes) (minutes) t tmax (minutes) Cmax (µg ml–1) Vdss (I kg–1) CI (ml min–1 kg–1) AUC024 h (µg min ml–1) F (per cent)
i.v. 2·4 (1·9) 59·3 (29·4)* – – – 0·62 (0·24) 13·4 (4·5) 1958 (633)* –
i.m. – 250·2 (107·1)* 6·6 (8·3) 62·5 (39·9) 4·1 (2·1) – – 1454 (348)* 76 (12)
t 12 α = distribution phase half-life (harmonic mean); t 12 β = terminal phase half-life (harmonic mean); Vdss = volume of distribution at steady state; CI = total plasma clearance of the drug; AUC024 h = area under the plasma concentrationtime curve; t 12 K01 = initial phase half-life (harmonic mean); t 12 β = terminal phase half-life (harmonic mean); tmax = time of peak plasma concentration; Cmax = peak plasma concentration; F = bioavaliability. *P < 0·05.
RESULTS TAP plasma concentration curves versus time, after i.v. and i.m. administration of 30 mg kg–1 TAP, are shown in Fig. 1. After i.v. administration, the plasma concentration reached a value of 168·0 (92·0) µg ml–1. The thiamphenicol plasma disposition was best represented by a bi-compartmental model, and detectable levels were observed up to 14 hours post-administration. After i.m. administration, a biexponential equation was used to best describe the plasma disposition of TAP, with a maximum plasma concentration (Cmax) of 4·1 (2·1) µg ml–1, reached at about 60 minutes after drug administration. Plasma levels of TAP were detectable up to 24 hours after administration. The pharmacokinetic parameters after i.v. and i.m. administration, obtained from analysis of the plasma concentration-time curves, are summarized in Table 1. After i.v. administration, the elimination half-life (t 12β) was 59·3 (29·4)
minutes, and the volume of distribution (Vdss) and clearance (CI) values were 0·62 (0·24) 1 kg–1 and 13·4 (4·5) ml min–1 kg–1, respectively. After i.m. administration, measurable TAP levels were detected in the first collected plasma sample (five minutes after administration). The initial half-life of TAP (t 12K01) was 6·6 (8·2) minutes. The t 12β after i.m. administration (250.2 minutes) was about four times longer than that observed after i.v. administration (59·3 minutes). The AUC value after i.v. administration was statistically different (P < 0·05) from that found after i.m. administration, yielding a mean bioavailability value for i.m. administration of 76 (12) per cent. A simulation was performed to estimate the steady state parameters in a multiple dose of 30 mg kg–1 at 24 hour intervals. The predicted Css (max), Css (min) and Css (av) were 3·7, 0·3 and 1·4 µg ml–1, respectively.
Thiamphenicol disposition in pigs
DISCUSSION Understanding the kinetic pattern of TAP is of interest in establishing the dosage of this antibiotic in pigs. The TAP disposition study after a single i.v. dose of 30 mg kg–1 revealed that it was best described by a bi-exponential equation with an initial rapid disposition phase and a slower second disposition phase, clearly emphasised by the high ratio between constants (α/β < 9). A similar bi-compartmental profile has been found by Gámez et al (1992) in cattle, Abdennebi et al (1994a) in sheep, and Lavy et al (1991) in goats, thus suggesting that TAP is rapidly distributed from blood to tissues. This result is in concordance with the high liposolubility, low protein binding (8 to 10 per cent) and pKa value (7·2) of TAP, which furthermore suggest a good penetration and distribution of the drug in the body fluids, as described in some studies where TAP reached higher levels in the liver and kidney than in plasma (Zambon Group Report 1978). The data are also in agreement with those from humans (Cambieri et al 1970, Ferrari 1984), in which higher concentrations of TAP than plasma levels were found in the lungs and kidneys after an oral dose of 15 mg kg–1. Studies in lactating cows (Signorini et al 1986, Mestorino et al 1993) and goats (Lavy et al 1991) have shown that TAP reached the mammary gland, as therapeutic levels of antibiotic (≥ 5 µg ml–1) were found in milk after different i.m. doses (25, 20 and 50 mg kg–1) of the drug. Thiamphenicol may remain stored in extravascular tissue, which could explain the relatively high distribution values, 0·62 (0·24) 1 kg–1. Similar values of Vdss were observed in calves (0·68 1 kg–1), sheep (0·80 1 kg–1) and goats (0·70 1 kg–1), by Abdennebi et al (1994b) and Lavy et al (1991), respectively. Some studies using chloramphenicol in pigs (Rao and Clarenburg 1977) showed that tissular levels in kidney and bile were respectively four and two times higher than those observed in plasma at the same time point after administration. The Vdss value obtained in the present study suggests that TAP might easily reach well perfused tissues, possibly in levels high enough to reach the minimum inhibitory concentration (MIC) for some of the micro-organisms responsible for infectious disease. The short t 12β (59·3 minutes) and high clearance (13·4 ml min–1 kg–1) values agree with the rapid elimination of TAP from the body by renal excretion of the active form (50 to 80 per cent of administered dose) described by some authors in humans (McChesney et al 1960, Nakagawa et al 1975) and in rats (Gazzaniga et al 1973). This rapid elimination suggests that the withdrawal period may be short. However, it should be noted that the plasma half-life does not allow estimation of the tissue half-life as the drug could accumulate in tissue. In order to establish the withdrawal period, it is necessary to study the depletion rate of the drug in tissues destined for human consumption. After i.m. administration, the TAP levels reached a Cmax of 4·1 (2·1) µg ml–1 in about 60 minutes. These plasma concentration values were considerably lower than those reported in ruminants. After administration of an i.m. dose of 20 or 25 mg kg–1 (Signorini et al 1986, Mestorino et al 1993, Abdennebi et al 1994a,b) to calves and sheep, and an i.m. dose of 30 mg kg–1 of the same concentration as in the present study to calves (Gámez et al 1992), the values achieved
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Cmax were between four and eight times higher than those found here in pigs. This could be due to interspecific differences, but it could also be attributable to retention of the drug at the injection site and delayed absorption. The i.m. elimination half-life (250·2 minutes) was higher than that observed after i.v. administration (59·3 minutes). The absorption process probably continued for several hours, which would affect the drug elimination process (absorption rate-dependent elimination). Moreover, Davis et al (1972) observed similar behaviour with chloramphenicol disposition in pigs when the drug was administered by either the i.v. or the i.m. routes. The bioavailability value for the i.m. route (76 per cent) was rather lower than those previously reported for ruminants (Gámez et al 1992, Mestorino et al 1993) Abdennebi et al 1994a,b, and humans (Ferrari 1984), which reported F values of between 84 and 100 per cent. Studies carried out by Inamoto et al (1994a,b) and Gutiérrez et al (1993) have shown that the MIC value of TAP for some ethiological agents of infectious disease in swine, such as Actinobacillus pleuropneumoniae, Pasteurella multocida and Mycoplasma hyopneumoniae, were between 0·1 and 1.56 µg ml–1. Other studies have showed a ratio of tissue to serum concentration of ≥ 1 in tissues such as the kidney, lung and mammary gland (Cambieri et al 1970, Matsumoto et al 1975, Zambon Report 1978). The plasma levels and the distribution and elimination pattern obtained in the present study, as well as the simulated predicted concentrations in the steady state, suggest that a 30 per cent aqueous solution of thiamphenicol dimethylacetamide administered i.m. at 30 mg kg–1 could be sufficient to achieve the MIC of pathogenic bacteria involved in the commonest infections in pigs, and that the interval between each administered dose could be extended up to 24 hours.
ACKNOWLEDGEMENTS This study was supported by Lab. JAER S.A. and CIRIT-CIDEM. We thank Francisco Pérez for his technical assistance.
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Accepted December 2, 1998