Pharmacokinetics of tepoxalin and its active metabolite in broiler chickens

Tepoxalin, [5(4-chlorophenyl)-N-hydroxy-(4-methoxyphenyl)-N-methyl-1H-pyrazole-3-propanamide], is an orally active dual cyclooxygenase/lipoxygenase inhibitor, with a favourable gastrointestinal profile with respect to gastric mucosal injury (Wallace et al., 1991, 1993; Argentieri et al., 1994; Knight et al., 1996; Kirchner et al., 1997). Tepoxalin is indicated for the control of pain and inflammation associated with osteoarthritis in dogs. Besides its anti-inflammatory and analgesic properties, tepoxalin has been reported to inhibit T cell proliferation and to have cytokine modifying activity (Rainsford et al., 1993, 1996; Zhou et al., 1994; Kazmi et al., 1995; Ritchie et al., 1995; Willburger et al., 1998; Fiebich et al., 1999). After oral administration in humans and dogs, tepoxalin is rapidly converted to its active, carboxylic acid metabolite RWJ-20142, which inhibits cyclooxygenase but not lipoxygenase (Waldman et al., 1996; Homer et al., 2005). The objective of the present experiment was to study the pharmacokinetics of tepoxalin and its active metabolite in broiler chickens, in order to use it in future experiments as an anti-inflammatory and a potential cytokine inhibiting drug in an inflammation model in chickens developed in our laboratory (Baert et al., 2005a,b; De Boever et al., 2008). Experiments were carried out on six healthy broiler chickens (Ross, mean body weight 1.16 ± 0.108 kg), obtained from a local commercial poultry farm. The study was approved by the Ethics Committee of the Faculty of Veterinary Medicine (2004/077). The animals were housed according to the requirements of the EU (Anonymous, 2004). The study was designed as a two-way cross-over study using two groups (n = 6) of broilers. A drug free period of 1 week was allowed between the two treatments. Before oral administration, chickens were fasted for 14 h. Intravenous tepoxalin was prepared by dissolving the tepoxalin standard (Schering-Plough Co., Wicklow, Ireland) in sterile polyethylene glycol at a concentration of 60 mg/mL. The drug was slowly injected at a dose of 30 mg/kg via a 25 G needle in the wing vein. For the oral administration, tepoxalin lyophilisates tablets [Zubrin Oral Lyophilisates, 50 mg; Schering-Plough, Brussels, Belgium; Schering-Plough Animal Health (2003)] were weighed and the amount corresponding to 30 mg/kg was given directly into the beak of the animal. Blood samples were collected from the leg vein (vena metatarsea plantaris) before (0) and at 15, 30, 45 min, 1, 1.5, 2, 2.5, 3, 4, 6, 7, 8 and 10 h after administration. Plasma was collected after centrifugation of the blood sample for 10 min at 2400 g and stored at −20 °C until assayed. Plasma concentrations of tepoxalin and its active, acid metabolite were determined by a HPLC method with fluorescence detection. Briefly; 225 μL of plasma were pipetted into a 1.5 mL Eppendorf tube, followed by the addition of 25 μL of internal standard (RWJ-20294, Schering-Plough Co.) (25 or 250 μg/mL) and 500 μL of acetonitrile. After vortexing briefly and centrifugation (10 000 g for 10 min at room temperature), 50 μL of the supernatant were injected in the HPLC system (TSP, Fremont, CA, USA) (λex = 290 nm and λem = 440 nm). A PLRP-S column (Polymer Laboratories, Shropshire, UK) attached to an appropriate guard column was used. The mobile phase (MF) A consisted of 0.01 m 1-octane-sulfonic acid in 0.01 m acetic acid in water, mobile phase B consisted of tetrahydrofuran. The retention time was 6.5, 8.8 and 12.6 min. for tepoxalin, its acid metabolite and the IS, respectively, using an isocratic elution at 58% MF A and 42% MF B at 0.7 mL/min. The calibration curves for tepoxalin and acid metabolite were linear between 0.025 and 1 μg/mL and 2.5 and 100 μg/mL (r > 0.99). The precision fell between the ranges specified by EU at for different concentration levels and the accuracy fell within ranges of −20% to +10% at the same concentration levels (Anonymous, 2005). The LOQ was set at 25 ng/mL for both compounds and the LOD was 5.9 ng/mL and 6.8 ng/mL for tepoxalin and its acid metabolite, respectively (Anonymous, 2005). The extraction recovery was 98% and 95% for tepoxalin and the acid metabolite, respectively. Stock solutions were stable for at least 3 months. where C is the plasma concentration, C0 is the plasma concentration at time 0 h, kel is the first-order elimination rate constant for the elimination phase and t is the time. For the acid metabolite the area under the curve (AUC), the half-life of elimination (t½), the maximum observed concentration (Cmax) and the time to maximum concentration (Tmax) were calculated by the non compartmental PK functions for Microsoft Excel. The mean plasma concentrations of tepoxalin and its acid metabolite vs. time curves are shown in Fig. 1. For both administration routes, the mean pharmacokinetic parameters of both substances are reported in Table 1. After oral administration of tepoxalin, plasma concentrations of the parent compound did not rise above the LOQ of 25 ng/mL. Mean (+SD) plasma concentrations vs. time curve of tepoxalin and its acid metabolite after oral and intravenous administration of tepoxalin to broiler chickens at a dose of 30 mg/kg (n=6). The Student's t-test (α = 0.05) was used to demonstrate possible differences in the AUC and half-life of elimination of the acid metabolite after oral and intravenous administration of tepoxalin. Because we expected a low oral bioavailability of tepoxalin in chickens, we selected a dose of 30 mg/kg. Nevertheless, plasma concentrations of tepoxalin after oral administration of the drug did not rise above the LOQ of 25 ng/mL. Comparison of the pharmacokinetic parameters of tepoxalin after intravenous administration is only possible in dogs at this moment (Homer et al., 2005). After intravenous tepoxalin administration in chickens, a mean half-life of elimination of 0.52 h was calculated. In dogs, tepoxalin was eliminated five times slower. Differences in elimination half-life could be attributed to dissimilarities between animal species in the clearance or the volume of distribution of the drug. A difference in clearance of tepoxalin has been described for dogs and mice. In dogs, the excretion route was almost exclusively via faeces and only 1% via urine. In mice, on the contrary, about 30% of tepoxalin was eliminated via urine (EMEA, 2003). Another explanation could be a quantitative difference in biotransformation rate of tepoxalin between dogs and chickens. In mammals, including rats and dogs, tepoxalin undergoes both rapid and extensive in vivo conversion to its carboxylic acid hydrolysis product as shown in Fig. 2. Plasma concentrations of the acid metabolite were substantially higher and remained in the systemic circulation for a longer period than the parent compound (Knight et al., 1996). These data were confirmed in chickens in the present experiment. Despite the levels of the parent compound in plasma after oral administration being below the LOQ, the metabolite was abundantly present indicating the existence of a substantial presystemic first-pass metabolism. In this way, tepoxalin could be considered as a pro-drug. Tepoxalin may reach substantial higher concentrations at sites of inflammation than can be expected from plasma levels, since a high Vd of 5.80 L/kg was calculated after intravenous administration. Chemical structures of (a) tepoxalin, (b) its acid metabolite (RWJ-20142) and (c) the internal standard RWJ-20294 (IS). In contrast to humans and dogs, where the half-life of elimination of the metabolite was about 14 h after oral tepoxalin administration, chickens eliminated the acid metabolite much faster with a mean half-life of elimination of 2.80 and 1.07 h after oral and intravenous administration, respectively (Waldman et al., 1996; Homer et al., 2005). Similarly in dogs and in chickens, the half-life of elimination of the metabolite was faster after intravenous than oral administration of the parent drug (Homer et al., 2005). This could be due to the presence of a flip-flop phenomenon for the parent drug, suggesting that the rate of absorption is slower than the rate of elimination. A slower absorption of tepoxalin would also result in a slower formation of the metabolite and a longer persistence of the metabolite in plasma after oral administration of the parent drug. Besides the acid metabolite, an unknown metabolite was detected in plasma of dogs after tepoxalin administration (EMEA, 2003). Also in this study in chickens the presence of an unknown metabolite in plasma was seen as a peak eluting between the acid metabolite and tepoxalin, both after oral and intravenous tepoxalin administration (data not shown). The unknown metabolite represented approximately 10% in peak area of the active metabolite. Knowing the pharmacokinetic properties of tepoxalin in chickens, we intend to study the anti-inflammatory and cytokine inhibiting potentials of the drug in an intravenous LPS inflammation model in chickens whereby the inflammatory pathways could be elucidated. The assistance of P. Wassink and K. Jonckheere for sample analysis and the assistance of D. De Clercq in the animal experiment was greatly appreciated.