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The objective of this study was to determine the serum pharmacokinetics of terbutaline in healthy cows. In the initial experiment, terbutaline was administered once as an intravenous (IV) bolus to 6 near-term pregnant beef cows within 24 h after parturition at a low but therapeutically relevant dose, 5 μg/kg. A 2nd experiment was conducted in the same cows with a higher dose, 0.5 mg/kg, but an otherwise similar experimental design. The serum concentration of terbutaline was determined by means of high-performance liquid chromatography with fluorescence detection in both experiments. After IV administration of 0.5 mg/kg, the mean peak serum concentration, residence time, and half-life were 708.22 (standard deviation 509.6) ng/mL, 6.75 (3.6) min, and 6.93 (2.4) min, respectively. The results indicate that terbutaline is rapidly eliminated from the bloodstream after IV administration in cattle, falling below the assay’s limit of detection 30 min after administration.
L’objectif de cette étude était de déterminer la pharmacocinétique sérique de la terbutaline chez des vaches en santé. Dans l’expérience initiale, de la terbutaline a été administrée en une fois sous forme de bolus intraveineux (IV) à six vaches d’embouche à moins de 24 h après la parturition à un dosage faible mais adéquat pour un usage thérapeutique, soit 5 μg/kg. Une deuxième expérience a été effectuée chez les mêmes vaches en utilisant un design expérimental similaire mais en administrant une dose plus élevée, soit 0,5 mg/kg. La concentration sérique de terbutaline a été déterminée par chromatographie en phase liquide à haute performance avec détection par fluorescence dans les deux expériences. Après l’administration IV de 0,5 mg/kg, le pic moyen de la concentration sérique, le temps de résidence et la demi-vie étaient, respectivement, 708,22 (écart type de 509,6) ng/mL, 6,75 (3,6) min et 6,93 (2,4) min. Ces résultats indiquent que la terbutaline est rapidement éliminée de la circulation sanguine chez les vaches après administration IV, se retrouvant sous la limite de détection de l’épreuve 30 min après l’administration.
(Traduit par Docteur Serge Messier)
Management of dystocia in cattle is sometimes complicated by high uterine tone, which makes manipulation of the fetus for correction of abnormal position or posture difficult. Exteriorization of the uterus during cesarean section is also impaired by high uterine tone. Previous clinical studies indicated that clenbuterol administration facilitated management of dystocia and performance of cesarean section (1). However, owing to reports of human intoxication with clenbuterol after its use as a repartitioning agent in livestock (2), clenbuterol was banned in Europe, Canada, and the United States in 1997, and its use in food-producing animals remains illegal.
Terbutaline is a phenethanolamine β2-adrenergic agonist that is used as a tocolytic agent in humans. Limited experimental data have demonstrated its tocolytic activity in rats, sheep, and buffalo (3–5). Terbutaline has been documented to have variable but moderate adverse effects, such as tachycardia, hypotension, and hyperglycemia (4,6). Our preliminary pharmacodynamic study confirmed that terbutaline has potential as a tocolytic drug for bovine obstetric use. However, the use of terbutaline in food-producing animals is problematic owing to a paucity of pharmacokinetic information that can be used to establish suitable dosage and to estimate appropriate tissue residues. Such information is particularly relevant to terbutaline because of the history of intoxication associated with clenbuterol, another β2-agonist. Although accurate withdrawal times can be calculated only by extensive sampling of milk and a variety of relevant tissues, preliminary estimates can be obtained by investigating the serum pharmacokinetics after intravenous (IV) administration of a single dose. Therefore, the objective of this study was to determine the serum pharmacokinetics of terbutaline in healthy cows.
Six near-term pregnant crossbred beef cows ranging in age from approximately 2 to 10 y, with an initial body weight (BW) of 477 to 638 kg, were enrolled in the 1st experiment after confirmation that they were free from medical or surgical conditions and were at least 8 mo pregnant. Parturition was induced in each cow by intramuscular administration of dexamethasone (Dexamethasone solution; Phoenix Scientific, St. Joseph, Missouri, USA), 20 mg/cow, and dinoprost tromethamine (Lutalyse; Pharmacia & Upjohn Company, Kalamazoo, Michigan, USA), 25 mg/cow. Each cow was cared for according to the guidelines of the Institutional Animal Care and Use Committee of Oklahoma State University, Stillwater, Oklahoma, USA, and following the Canadian Council on Animal Care Guidelines on Animal Use. The cows were maintained in individual barn stalls until parturition and throughout the study period. They were fed ad libitum grass hay and a quantity of commercial sweet feed sufficient to meet US National Research Council requirements for late-gestation cows.
The 1st pharmacokinetic experiment was initiated within 24 h after parturition. We placed a 16-gauge, 3-in and a 12-gauge, 5-in IV catheter (Mila International, Florence, Kentucky, USA) in the right caudal auricular and jugular veins, respectively, of each animal. Terbutaline sulfate (Brethine, Terbutaline sulfate for injection; Novartis Pharmaceuticals, Summit, New Jersey, USA) was administered as an IV bolus via the auricular catheter (AC) at a dose of 5 μg/kg BW over 30 s. Blood samples were collected from the jugular catheter (JC) before drug administration and at 5, 10, 20, 30, and 45 min and 1, 2, 3, 4, 6, 8, 12, 16, 18, 24, 48, and 72 h thereafter. After collection of each blood sample, the JC was flushed with 5 mL of heparinized saline solution (10 U heparin/mL of 0.9% saline). All samples were collected into evacuated glass tubes without additive (Vacutainer blood collection tube; Becton Dickinson, Franklin Lakes, New Jersey, USA). The blood samples were centrifuged (Centra GP8R; BJB Lab care, Milton Keynes, England) at 600 × g for 15 min, and the serum was harvested in polypropylene tubes (Fischer Scientific, Pittsburgh, Pennsylvania, USA) and stored at −70°C until analysis.
A 2nd experiment was performed with the same 6 cows approximately 8 mo after the initial experiment, when the cows were neither pregnant nor lactating. A higher dose of terbutaline sulfate (0.5 mg/kg) was administered once as an IV bolus. Each dose of terbutaline sulfate crystalline powder (Sigma Chemical Company, St. Louis, Missouri, USA) was measured precisely with the use of an electronic scale (Ohaus Analytical Plus; Aldinger Company, Pine Brook, New Jersey, USA) and reconstituted with sterile water; the final terbutaline concentration was 12.5 mg/mL. The solution was injected through a 0.2-μm syringe filter during IV administration via the AC over 30 s. Blood collection times were the same as in the 1st experiment. Heart rate, respiratory rate, and presence of cardiac arrhythmias or muscle tremors were monitored clinically at 2, 5, 10, 15, 20, 25, 30, 45, and 60 min after administration.
The concentrations of terbutaline in the serum samples were measured by high-performance liquid chromatography (HPLC) with fluorescence detection by a combination of 2 previously published methods (7,8) slightly modified to optimize the assay. The limit of detection was 25 ng/mL. Briefly, 100 μL of an internal standard solution of betaxolol (1 mg/mL) (Sigma) and 1 mL of acetonitrile (Sigma) were added to 1 mL of each serum sample and vortexed (Genie Scientific, Fountain Valley, California, USA) for 30 s. Each sample was centrifuged at 3000 × g for 15 min at 4°C and then extracted in 6-mL polypropylene reverse-phase columns packed with 1 g of carbon-18 (18C) (Varian; Palo Alto, California, USA) that had been preconditioned twice with 3 mL of ethanol (Sigma) and 3 mL of distilled water. After loading of each sample, the column was rinsed twice with 3 mL of distilled water. Drug and internal standard were eluted with 1 mL of ethanol containing 50 mM of ammonium chloride buffer (pH 8.5; 95:5 v/v) and placed in receiver tubes in a vacuum manifold. The solvent was removed by drying the samples with a Savant SpeedVac (TeleChem International, Sunnyvale, California, USA) at 45°C. The dried residue was reconstituted with 100 μL of mobile phase (25 mM sodium phosphate buffer, pH 7.4, and methanol; 77:23 v/v) (Sigma), vortexed, and centrifuged at 13 400 × g for 2 min. The supernatant was transferred into new tubes and stored at −20°C until analyzed. Extracted samples were subjected to HPLC with the use of 18C reverse-phase columns and fluorescence detection at excitation and emission wavelengths of 224 and 310 nm, respectively. Standard curves (for 0, 25, 50, 100, 250, 500, 1000, and 2000 ng of terbutaline/mL) were prepared by adding known amounts of terbutaline sulfate to drug-free bovine serum and measuring the terbutaline concentration as described above.
Concentration-versus-time data were subjected to pharmacokinetic analysis by use of noncompartmental methods based on statistical-moment theory and a commercially available software program, WinNonlin (version 4.1; Pharsight Corporation, Mountain View, California, USA). Pharmacokinetic parameters (area under the curve [AUC], residence time, volume of distribution [Vd], total body clearance, and half-life) were calculated with standard formulas for a noncompartmental model.
The concentrations of terbutaline in the initial experimental samples were not high enough to be measured, despite the use of a sensitive assay. Therefore, pharmacokinetic data could not be calculated. For the 2nd experiment, the mean serum concentration-versus-time curves obtained for the 6 cows after IV administration of 0.5 mg/kg of terbutaline are shown in Figure 1. Tachycardia (mean heart rate 174 [standard deviation 10.4] beats/min, compared with a baseline mean of 58 [7.5] beats/min) was observed approximately 2 min after the administration of terbutaline. Cardiac arrhythmias and muscle fasciculation, lasting approximately 45 min, were also noted in 2 cows. Their recovery was uneventful and did not necessitate therapy. Pharmacokinetic parameters describing terbutaline disposition in the 2nd experiment are presented in Table I.
The dose of terbutaline selected for the 1st experiment (5 μg/kg) was extrapolated from the most commonly reported dose used in women for tocolysis: 250 to 300 μg/min for an average 60-kg woman (9–11). Comparisons between the pharmacokinetics of the β2-agonist in humans and domestic animals cannot be made. However, in a pilot study (data not shown) involving 4 cows in mid- to late pregnancy that underwent cesarean section, relaxation of the uterus after a single IV bolus of terbutaline at a dose of 5 μg/kg was noted by observation and palpation.
In the 2nd experiment, the dose was increased to produce quantifiable serum concentrations. Using an estimated Vd of 0.5 L/kg, based on the reported values of 0.9 L/kg in horses (12) and 0.8 to 1.9 L/kg in humans (10,13,14), and a target initial serum concentration of 1 μg/mL, a new dose of 0.5 mg/kg was calculated (dose = concentration × Vd) for the 2nd experiment. This dose was higher than that used previously in humans and domestic animals (9,11,12,14). An initial pilot experiment was performed, with a 2-y-old healthy intact male alpine goat, to confirm that IV administration of this dose of terbutaline would be tolerated without life-threatening adverse effects. The goat’s heart rate increased from 90 to 230 beats/min and respiratory rate from 36 to 65 breaths/min within 2 min after administration; the rates returned to normal within 4 h. Although substantial, these cardiovascular effects were not considered life-threatening. This finding was consistent with the much greater median lethal dose of terbutaline reported for rats and mice (61.5 and 48.4 mg/kg, respectively) (15).
Pharmacokinetic analysis of the concentration-versus-time data was performed with a noncompartmental approach, which best suited the limited data obtained per cow. This model employs statistical-moment theory and is based on the assumption that at least a 1-compartment model (central) exists since the drug’s behavior in the body is being linked to the plasma-concentration profile (16,17). However, evidence of 2 phases (distribution and elimination) was found when the concentration-versus-time data were plotted semi-logarithmically (Figure 2), thus suggesting that the data may fit a multicompartmental model. Both noncompartmental and multicompartmental models have been used for pharmacokinetic analysis of terbutaline in humans and domestic animals (12,18,19).
The concentration-versus-time data obtained in the 2nd experiment indicated a variable serum peak concentration of terbutaline, detected approximately 5 min after administration and averaging 708.22 (standard deviation 509.6) ng/mL (Table I). Terbutaline was rapidly eliminated: the serum concentration in all 6 cows decreased below the quantification limit of the assay within approximately 30 min of administration (Figure 1), and the β2-agonist was undetectable 45 min to 72 h thereafter. A similarly rapid decline in serum concentration was reported for equine species after administration of the drug as a continuous infusion (12). Variable elimination half-lives for terbutaline have been reported in humans (3.7, 12.1, and 17 h in women, asthmatic children, and men, respectively) and animals (1.2 h in equine species) (10,12–14). Short terminal half-lives, such as those estimated in the present experiment (mean 6.93 [2.4] min), may actually represent distributional rather than elimination half-lives (17). The wide range of values reported for the elimination half-life of terbutaline is probably due to differences in duration of blood collection and assay sensitivity (14). The calculated mean residence time (MRT), 6.75 (3.6) min, was shorter than the MRT of 30 min reported for equine species by Torneke (12), who suggested that a short MRT can be attributed to a high or overestimated total body clearance (ClB) and a low or underestimated steady-state Vd (Vdss), was 43.29 since MRT = Vdss/ClB. In our experiment, the mean ClB (17.2) mL/min·kg (2.58 L/h·kg), which is high and similar to that previously reported for horses (1.9 L/h· kg) (12). The Vdss represents the sum of all compartmental volumes and provides an estimate of extravascular drug distribution that is independent of the elimination process (20). The mean Vdss for terbutaline in our experiment, 0.34 (0.28) L/kg, was lower than that reported for humans and other domestic animals (10,12–14), which suggests that terbutaline does not have an extensive extravascular distribution in the cow.
The mean AUC to infinity (AUCinf) calculated in the 2nd experiment was 13 692.8 (6869) ng·min/mL or 228.2 μg·h/L. In order to compare reported AUCs for terbutaline in humans (18) and domestic animals (9,12), we calculated a dose-normalized value by dividing by the total dose of terbutaline administered. The result the AUCinf in the 2nd experiment was 0.81 × 10−3 h/L, lower than the AUC reported for humans (69.71 × 10−3 h/L) (18) but similar to that calculated for equine species (1.18 × 10−3 h/L) (12). Differences in AUC may be due to failure to accurately define the terminal half-life of a drug, as the calculation of elimination half-life, AUC, and ClB depends on the extent to which the blood concentration-versus-time profile is described (20).
The pharmacokinetic data obtained in this experiment will be used as a basis for a future study, in which a loading dose and continuous IV infusion of terbutaline will be calculated to achieve a steady-state serum concentration within quantifiable limits. This may allow more complete characterization of the distribution and elimination phases in serum, as well as the determination of terbutaline concentrations in milk, both of which are necessary for the estimation of withdrawal times for beef and dairy cattle.
We thank Lisa Colclazier for her technical support and the Research Advisory Council of Oklahoma State University Center for Veterinary Health Sciences, College of Veterinary Medicine, Stillwater, Oklahoma, USA, for financial support.