Pharmacokinetics of Tetracycline in Chickens After Intravenous Administration

ENVIRONMENT AND HEALTH Pharmacokinetics of Tetracycline in Chickens After Intravenous Administration A. ANADON Departamento de Farmacologia y Toxicologia, Faculted de Veterinaria, Universidad de Leon, 24007 Leon, Spain M. R. MARTINEZ-LARRANAGA and M. J. DIAZ Instituto de Farmacologia y Toxicologia, Consejo Juperior de Investigaciones Cientificas, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid-9, Spain (Received for publication December 4, 1984)

1985 Poultry Science 64:2273-2279 INTRODUCTION

In recent years, it has become common practice to incorporate antibiotics in rations fed to poultry and other domestic animals, this incorporation has resulted in healthier and faster-growing animals. The tetracycline antibiotics have been used in poultry as prophylactic and therapeutic agents. Drug dosage regimens are derived from knowledge of the pharmacodynamics and pharmacokinetics of the drug. Rational use of an antimicrobial drug in the treatment of an infectious disease, caused by a bacterial agent, requires knowledge not only of the susceptibility of the pathogen to the drug but also of the concentration of the drug that can be achieved in the body fluids of the animal being treated. Information is available on the pharmacokinetic parameters of iv administered tetracyclines in cattle, swine, dogs, and horses (Schach Von Wittenau and Yeary, 1963; Pilloud, 1973; Ziv and Sulman, 1974; Baggot et al, 1977; Schifferli et al, 1982; Xia et al, 1983). However, limited information is available on the routes of excretion and on the pharmacokinetic parameters after iv administration of tetracyclines in avian species. Considerable research has been done on the administration of these antibiotics to poultry in the feed in order to evaluate serum levels

(Williams-Smith, 1954), dietary modifications for increasing the absorption of the tetracycline antibiotics (Harms andWaldroup, 1961;Panisset and Mathieu, 1967), and tetracycline residues in tissues and eggs (Meredith et al, 1965; Polujanski, 1968), but few studies have evaluated its pharmacokinetic properties (Pindell et al, 1959; Black, 1977). The purpose of this study was to determine the plasma drug levels, biliary excretion, and pharmacokinetic parameters of tetracycline in chickens after iv administration.

MATERIALS AND METHODS

Animals and Rearing Conditions Eighteen 40 to 60-day-old broiler chickens (Hubbard X Hubbard) weighing 1.5 to 2.0 kg, were used in the trials. These chickens, purchased at a poultry farm in the neighbourhood, were introduced into single cages in the animal house of this laboratory 2 weeks before the medication. No disease was observed. The animal house was maintained at a 25 ± 2 C room temperature and at 45 to 65% humidity. Food and water were supplied ad libitum.

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ABSTRACT Plasma levels of tetracycline in chickens were determined after intravenous (iv) administration of a 65-mg/kg dose. The disposition kinetics of tetracycline in chickens were fitted to a two-compartment open model. Pharmacokinetic parameters were found to be: A (Mg/ml) = 2000 ± 450, a (hr - 1 ) = 4.3 ± .5, B (Mg/ml) = 82 ± 6, (3 ( h f 1 ) = .252 ± .009, K 12 (hr - 1 ) = 1.515, K21 (hr" 1 ) = .049, and K10 ( h f 1 ) = 2.652. Biliary excretion of tetracycline was also studied in chickens fitted with cannulae inserted into both bile ducts. The maximum values for tetracycline biliary excretion rate (407 and 606 Mg/hr) were reached at about 1 hr after iv administration of 10and 15-mg/kg doses. First-order rate constants for the biliary excretion, K[,; (hr - 1 ), were .834 and .665, respectively. The cumulative biliary excretion study showed that about 7% of both administered doses was recovered from the bile within the first 6 hr. In contrast, there was a low recovery of antibiotic in the bile after oral administration of 100 and 200 mg/kg doses. (Key words: pharmacokinetics, tetracycline, chickens)

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Experimental

These chickens were divided into four groups of three animals each. Group 1 chickens were given a single iv injection of tetracycline at a dose level of 10 mg/kg. Group 2 chickens were given a single iv injection of tetracycline at a dose level of 15 mg/kg. Group 3 chickens received a single oral dose of 100 mg/kg of tetracycline, and Group 4 chickens received a single oral dose of 200 mg/kg of tetracycline. Tetracycline hydrochloride (Sigma Co.) was diluted with sterile saline prior to administration. According to groups, tetracycline was injected iv into the brachial vein or was administered orally directly into the crop using a thin plastic tube attached to a syringe. Because

Analytic Method and Data Analysis Plasma and bile samples were analyzed for tetracycline concentration using the high performance liquid chromatography (HPLC) method of Sarma and Bevill (1978), (Varian liquid chromatograph, Model 5000, equipped with variable wavelength detector UV-50, CDS 111L chromatography data system, and recorder). Samples were extracted with ethyl acetate and re-extracted into a phosphoric acid solution. Separations were performed on a reverse phase 10-/X MicroPak MCH-10 column using a phosphate-buffered, acetonitrile-water (40:60, v/v) mobile phase. Absorbance was measured at 355 nm and tetracycline concentration was automatically determined by peak area measurements relative to a tetracycline standard. The average plasma data were fitted by an HP-85 computer using the nonlinearleast squares program-ELSFIT (Sheiner, 1981). The curves of tetracycline concentrations in plasma vs. time were fitted to a two-compartment open model for kinetic analysis; such curves could be described by the following biexponential equation:

Cp = A ' e ~ a t + B*e-/ft TABLE 1. Plasma concentrations (ng/ml) of tetracycline (mean ± SEM) in chickens receiving 65 mg/kg tetracycline hydrochloride intravenously* Time after administration 15 min 30 min 1 hr 2 hr 4hr 8hr 12 hr ' N = 6.

690 ± 17 359 ±12 81 ± 5 52 ± 6 29 ± 4 12 ± 2.3 3.8+ .9

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Experiment 1. Tetracycline was injected iv into the left brachial vein of each of a group of six chickens at a single dose of 65 mg/kg. Tetracycline hydrochloride (Sigma Co.) was diluted with sterile saline prior to administration. Blood samples were collected in heparinized syringes from the brachial vein (opposite the one used for iv treatment) through a special permanent plastic cannula at each of the following times after administration of the drug: 15 min, 30 min, 1, 2, 4, 8, and 12 hr. Blood samples were centrifugated at 3000 rpm for 7 min, and the separated plasma samples were stored frozen until analyzed for tetracycline concentration. Experiment 2. Twelve chickens were fasted the night before and the day of the study, approximately 18 to 20 hr before surgery. The chickens were anesthetized by means of a single intramuscular (im) injection of ketamine (10 mg/kg) (Ketolar, Substancia Parke-Davis) into the pectoral muscle. The right leg was doubled back to facilitate access to the site of the incision and to minimize tension on the abdominal muscles. A 4-cm incision was made in the right abdominal wall, starting slightly posterior to the juncture of the last rib and the sternal member of that rib and proceeding in a straight line to a point slightly dorsal to the caudal end of the sternum. The hepatic and cystic ducts were carefully exposed by blunt dissection to minimize bleeding and injury to the surrounding connective and pancreatic tissues. The bile ducts were cannulated with polyethylene tubing. Body temperatures were maintained at 37 ± .5 C by placing a heating lamp over the animals during the experiment.

some of the chickens vomited shortly after the oral administration, more chickens were given the dose. Only chickens that did not vomit were used in this experiment. Bile samples were collected at 30 min and after every hr for a total of 8 hr after administration of the drug. The volume of bile samples was measured and an aliquot was immediately frozen until the time of assay.

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TABLE 2. Pharmacokinetic parameters for tetracycline administered intravenously (65 mg/kg) to chickens Values

Parameters

±450 x ±i 6 4.274 d .489 .252 i .009 .162 2.772 .037 .137 .174 1.497 .408 2.614 796.38 .0979 3.667 .573 .156

A, Mg/ml B, Mg/ml a, hr 0, hr"1 ty2a, hr ty2(3, hr Vc = Central compartment, 1/kg Peripheral compartment, 1/kg Total volume of distribution, 1/kg Kn,hr_1 K „ , hr"1 K.o.hr" 1 Concentration time curve, mg/hr/1 Total body clearance, 1/hr/kg K 1 2 /K 2 l K l2 /K 1( , K 2 1 /K 1 0 Mean value ± standard error for six chickens.

T h e p h a r m a c o k i n e t i c parameters investigated w e r e : e x t r a p o l a t e d zero-time drug concent r a t i o n s (A and B), hybrid rate c o n s t a n t s a and j3, t h e rate constants for drug transfer ( K i 2 and K 2 i ) , t h e rate c o n s t a n t for drug elimination ( K i o ) , t h e half-lives in t h e a and j3-phases (ty2ot, ti/2/3), t h e a p p a r e n t volume of drug distribution in t h e central c o m p a r t m e n t (Vc), peripheral c o m p a r t m e n t (Vp) and t h e t o t a l volume of distribution (Vdss), t h e t o t a l b o d y clearance ( C I B ) , and the area u n d e r t h e c o n c e n t r a t i o n t i m e curve (AUC). Biliary excretion p a r a m e t e r s were evaluated b y linear least-squares regression analysis using an HP-85 c o m p u t e r . Biliary excretion rates (/ng/hr) of tetracycline vs. t i m e were fitted t o a o n e - c o m p a r t m e n t m o d e l . T h e first-order rate c o n s t a n t (Kb;) for biliary excretion and t h e corresponding half-life (ti^bi) were investigated. RESULTS Plasma Levels. Plasma c o n c e n t r a t i o n s of tetracycline after 65 mg/kg iv administration are s h o w n in Table 1. A rapid decline in drug c o n c e n t r a t i o n s occurred. T h e corresponding curve was: Cp = 2 0 0 0 e - 4 . 2 7 4 t + 82 e ~ - 2 5 2 t T h e p h a r m a c o k i n e t i c parameters which describe t h e distribution and elimination phases

of tetracycline after a 65 mg/kg iv dosage are given in Table 2. A semilogarithmic plot of average plasma c o n c e n t r a t i o n vs. t i m e is presented in Figure 1. Biliary Excretion. Tetracycline was excreted into t h e bile following b o t h iv and oral administration. T h e biliary excretion rates after iv administration are shown in Table 3. T h e m a x i m u m values for t h e biliary excretion rate of tetracycline, 4 0 7 and 606 jUg/hr, were reached at a b o u t 1 h r after iv administration of 10 and 15 mg/kg, respectively. Semilogarithmic

TABLE 3. Biliary excretion rates (fig/hr) of tetracycline (mean ± SEM) after intravenous administration in chickens1 Time after administration

Dose 15 mg/kg

10 mg/kg „

30 min 1 hr 2hr 3 hr 4hr 5 hr 6hr

225.0 407.0 308.6 44.8 24.0 15.7 8.0

± ± ± ± + ± ±

21.0 10.8 36.2 5.6 3.8 1.9 1.7

s

(mg/kg) 329.0 606.0 275.3 118.0 70.4 39.1 20.5

± ± ± ± ± ± ±

17.3 36.2 13.8 13.1 8.5 2.9 4.9

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1

2000 82

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12

Time (h)

FIG. 1. Semilogarithmic plot of average plasma tetracycline concentration vs. time following intravenous administration of 65 mg/kg tetracycline to chickens. Each point represents the mean ± SEM from six chickens.

plots of the terminal portion of these curves are illustrated in Figure 2. The lines drawn are calculated regression lines. The correlation coefficients found were .9695 and .9964, respectively. With both of the doses there was a log-linear decline of bile concentration indicative of a one compartment model. The results reveal that with the iv doses used (10 and 15 mg/kg), the median rate constants (Kbi) for drug biliary excretion were .834 and .665

hr 1, respectively. The corresponding median half-lives were calculated to be .831 and 1.05 hr. Figure 3 illustrates the average cumulative amount of tetracycline recovered into the bile in 6 hr following iv administration. During this period, 7% of the total doses was excreted into the bile. After oral administration, the percentages of the total doses recovered from the bile were

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FIG. 2. Semilogarkhmic plots of biliary tetracycline excretion rate vs. time in chickens after the intravenous administration of 10 mg/kg (o) and after the intravenous administration of 15 mg/kg (•). Each point represents the mean + SEM obtained from 3 chickens for every dose.

much lower than following iv administration, a phenomenon probably related to the absorption phase. The average maximum cumulative amount of tetracycline excreted into the bile within 8 hr was 260 and 1480 /Ug after oral administration of 100 and 200 mg/kg, respectively, which corresponds to .2 and .5% of the total doses.

DISCUSSION The plasma concentrations of the antibiotic measured in chickens were proportionally in accordance with those obtained in man (Kunin and Finland, 1961), laboratory animals (Barber, 1964; Schach Von Wittenau and Yeary, 1963), and cows (Ziv and Sulman, 1974; Schipper, 1965). In addition, the pharmacokinetic behaviour for tetracycline, administered iv to chickens, agree with the two compartment open model previously described in cows (Ziv and Sulman, 1974) and for oxytetracycline in cows, buffalo, and horses (Pilloud, 1973; Ziv and Sulman, 1974; Varma et al, 1983; Trevor

et al, 1983). However, marked differences were found among the pharmacokinetic parameters for tetracycline in chickens vs. other domestic animals. The elimination ty2|3 of 2.772 hr obtained in the present study is lower than the value derived from the report by Ziv and Sulman (1974) in cows. Magnitudes of the rate constants found in this work indicate that tetracycline was rapidly distributed in the body but was eliminated rather slowly. Likewise, the small value of apparent volume of drug distribution in the central compartment, .037 1/kg, indicates a deep tissue sequestration of the drug and suggests a slow elimination process. This study also clearly shows that tetracycline is excreted into the bile even after iv administration. Biliary elimination within 6 hr reached 7% of both doses given (10 and 15 mg/kg), a value greater than the one obtained in dogs by Schach Von Wittenau and Yeary (1963) who reported a biliary excretion lesser than 5% within 24 hr when various tetracyclines were administered iv. Such a difference is important because the dog is an animal model

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Time (h)

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15 mg/kg 10 mg/kg 1.500

=

1.000 -

Time (h) FIG. 3. Plot of cumulative amount of tetracycline excreted into bile vs. time following intravenous administration to chickens.

usually considered as a " g o o d " biliary excreter (Smith, 1 9 7 3 ) . T h e finding of tetracycline in the bile after oral administration suggests the existence of at least a minimal enterohepatic circulation. Few studies have questioned t h e existence of an e n t e r o h e p a t i c circulation for tetracycline in domestic animals. In chickens, t h e m o d e r a t e recovery of t h e oral doses administered, a r o u n d . 5 % within 8 hr, indicated a low biliary excretion rate. Nevertheless, e n t e r o h e p a t i c circulation, which leads t o a longer persistence of tetracycline in t h e b o d y , w o u l d increase t h e availability of t h e c o m p o u n d . This has t o be t a k e n into consideration w h e n estimating t h e magnitude of t h e first-pass elimination, which otherwise tends t o be u n d e r e s t i m a t e d . In conclusion, t h e discrepancies f o u n d between p h a r m a c o k i n e t i c properties of this antibiotic in chickens and in o t h e r d o m e s t i c animals illustrate t h e i m p o r t a n c e of t h e pharm a c o k i n e t i c studies for establishing a correct dosage regimen or an optimal drug t h e r a p y .

ACKNOWLEDGMENTS We would like t o express our grateful a c k n o w l e d g m e n t s t o t h e Spanish C o m m i t t e e for t h e Science and T e c h n o l o g y for t h e financial s u p p o r t granted (Ref. 0 4 9 7 / 8 1 , CAICYT).

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Meredith, W. E., H. H. Weiser, and A. R. Winter, 1965. Chlortetracycline and oxytetracycline residues in poultry tissues and eggs. Appl. Microbiol. 13(1): 86-88. Panisset, J. C , and L. G. Mathieu, 1967. The effect of sodium sulfate on blood levels of oxytetracycline in birds. Poultry Sci. 46:560-563. Pilloud, M., 1973. Pharmacokinetics, plasma protein binding and dosage of oxytetracycline in cattle and horses. Res. Vet. Sci. 15:224-230. Pindell, M. H., K. M. Cull, K. M. Doran, and H. L. Dickison, 1959. Absorption and excretion studies of tetracycline. J. Pharmacol. Exp. Ther. 125: 287-294. Polujanski, P., 1968. Tissue fluorescence after feeding fowls with feed mixtures containing oxytetracycline. Medycyna Weterynaryjan 24:267—277. Schach, Von Wittenau, M. and R. Yeary, 1963. The excretion and distribution in body fluids of tetracycline after intravenous administration to dogs. J. Pharmacol. Exp. Ther. 140:258-266. Schifferli, D., R. L. Galeazzi, J. Nicolet, and M. Wanner, 1982. Pharmacokinetics of oxytetracycline and therapeutic implications in veal calves. J. Vet. Pharmacol. Ther. 5:247-257. Schipper, I. A., 1965. Milk and blood levels of chemotherapeutic agents in cattle. J. Am. Vet. Med. Assoc. 147:1403-1407. Sharma, L. P., and R. F. Bevill, 1978. Improved high-performance liquid chromatographic procedure for the determination of tetracyclines in

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