Pharmacokinetics of florfenicol in turkey plasma, lung tissue, and pulmonary epithelial lining fluid after single oral bolus or continuous administration in the drinking water

Pharmacokinetics of florfenicol in turkey plasma, lung tissue, and pulmonary epithelial lining... Abstract Florfenicol (FF) is registered for treatment of bovine and swine respiratory diseases. Although, turkeys often suffer from bacterial respiratory tract infections, there is no registered formulation based on FF for poultry available in Europe. The aim of this study was to evaluate the pharmacokinetic behavior of FF in turkeys in plasma, lung tissue, and pulmonary epithelial lining fluid (PELF). The concentration and pharmacokinetic characteristics of FF in plasma, lung tissue, and PELF in turkeys were determined, either after a single oral bolus (30 mg/kg body weight, BW) or during and after continuous drinking water medication (30 mg/kg BW/d for 5 d). Plasma, lung tissue, and PELF samples were collected at different intervals after administration, and FF was quantified by liquid chromatography-tandem mass spectrometry. After single bolus administration, FF was rapidly absorbed in plasma (the time to maximum concentration, tmax, was 1.02 h) and distributed to the respiratory tract (mean tmax = 1.00 h). The mean t1/2el in plasma and lung tissue was similar, around 6 h, whereas it was slightly higher in PELF, namely, 8.7 hours. After oral bolus dosing, the mean maximum concentration in plasma was twice as high as in the lung tissue, 4.26 μg/mL and 2.64 μg/g, respectively, while in PELF it was much lower, 0.39 μg/mL. During continuous drinking water medication, lung FF concentrations were slightly higher than plasma concentrations, with lung/plasma ratios of 2.01 and 1.27 after 24 h and 72 h, respectively. FF was not detected in PELF during continuous drinking water medication. INTRODUCTION Florfenicol (FF) has a broad antibacterial action against several pathogens responsible for infections of the respiratory, urinary, and gastrointestinal tract. An excellent clinical response to FF in bovine and swine respiratory diseases can be attributed to the remarkable pharmacokinetic (PK) characteristics, such as high absorption and rapid penetration in the respiratory tract, and the low resistance of cattle and swine pathogens, such as Mannheimia haemolytica, Pasteurella multocida, and Actinobacillus pleuropneumoniae, which all have a minimum inhibitory concentration (MIC90) below 1 μg/mL (Shin et al., 2005). In turkeys, FF has been proven to be effective against Ornithobacterium rhinotracheale infection, using drinking water medication during 5 d at a dose of 30 mg/kg body weight (BW) per d (Marien et al., 2007; Watteyn et al., 2013). However, only few data regarding the PK characteristics of FF in respiratory tissues are available, which is of primary interest given the localization of O. rhinotracheale (Afifi & Abo el-Sooud, 1997; Liu et al., 2002). Due to species-dependent differences in anatomy and physiology, PK studies have to be performed in the species of interest and with the pathogen of interest. To the authors knowledge, only one PK study of FF has been performed in turkeys after single bolus administration of 30 mg/kg BW either per os (orally, PO) or intravenously (IV) (Switala et al., 2007), but concentrations in lung tissue or pulmonary epithelial lining fluid (PELF), the sites of action, have not been reported yet. The first aim of this research was to determine the concentrations and PK characteristics of FF in plasma, lung tissue, and PELF after single oral bolus administration (30 mg FF/kg BW). In order to simulate field conditions, plasma, lung tissue, and PELF concentrations and PK characteristics of FF also were studied during and after continuous drinking water medication for 5 d at the same recommended dose of 30 mg/kg BW/d. MATERIALS AND METHODS Veterinary Drug FF, 2,2-dichloro-N-[1S,2R]-1-(fluoromethyl)-2-hydro-xy-2-[4-(methylsulfonyl)-phenyl]ethyl]-acetamide, used for the animal experiments was obtained from Zhejiang Hisoar Pharmaceutical Co., LTD (Zhejiang, China). The antibiotic was used solely as an active ingredient, without formulation. Because of the low aqueous solubility (1 mg/mL), the FF bolus for the single oral bolus PK study was given as a suspension of FF in tap water at a concentration of 6 mg/mL. For the continuous drinking water PK study, the medicated drinking water was prepared daily by mixing FF in tap water, stirring for 30 min, followed by sonication for 20 min to dissolve the FF. The mean (± SD) concentration was 74.3 (± 3.4) mg FF/L tap water. PK-experiment—single Oral Bolus Administration This study was performed using 54 6-week-old female turkey poults (Hybrid Converter, local commercial turkey farm) with a mean (± SD) BW of 2.063 (± 0.195) kg that were housed according to the requirements of the European Union (Anonymous, 2010). During the whole experiment, the turkeys were housed together, and the light scheme was set at 16 h light and 8 h dark. After a fasting period of 12 h, the birds received a FF bolus of 30 mg/kg BW by gavage in the crop, followed by rinsing with tap water. Four h after the bolus administration, the birds received feed again. Blood (1 mL) was collected from 6 turkeys by venipuncture from the medial metatarsal vein into heparinized tubes (Vacutest Kima, Novolab, Geraardsbergen, Belgium) at different intervals, pre (time 0) and post administration (p.a.; 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 10, and 24 h). Plasma was separated by centrifugation and stored at ≤ −15°C, pending analysis. Furthermore, animals (n = 6 at each interval) were euthanized at different intervals post FF administration to collect plasma, lung tissue, and PELF. Euthanasia was performed at 1, 2, 4, 6, 8, 12, 24, 36, and 48 h after the oral bolus administration. Birds were anesthetized by an intramuscular injection of a combination of xylazine (Xyl-M 2%, VMD, Arendonk, Belgium), zolazepam, and tiletamine (Zoletil 100, Virbac, Wavre, Belgium), followed by exsanguination. The whole right lung was removed for FF analysis. The complete left lung was used to collect PELF, as described by Bottje et al. (1999). In brief, after weighing the lung, it was lavaged with heparin-saline (200 units heparin per mL of 0.9% saline) at a volume of 2 mL/g lung through a cannula in the first bronchus. The PELF/saline solution was collected in a petri dish, and the amount of fluid was measured to determine the recovery, which ranged from 80.0 to 100%. The fluid was centrifuged (5,250 x g for 3 min) to remove red blood cells. Both the lung tissue and PELF were stored at ≤ −15°C until analysis. The animal experiment was approved by the Ethical Committee of the Faculty of Veterinary Medicine and Bioscience Engineering, Ghent University (EC 2014/68). PK-experiment—continuous Drinking Water Administration During an acclimatization period of 5 d, water consumption (non-medicated tap water) of 20 3-week-old female turkey poults (Hybrid Converter, local commercial turkey farm) with a mean (± SD) BW of 0.812 (± 0.074) kg was measured to calculate a correct dose of the medicated drinking water. Thereafter, the turkeys received FF via the drinking water during a 5-day period (target dose: 30 mg/kg BW/d), supplied in 3 plastic drinking troughs. During the whole experiment, the turkeys were housed together, and the light scheme was set at 16 h light and 8 h dark. Blood (1 mL) of 6 turkeys was collected and stored in the same way as the single oral bolus study, except for sampling points. Blood was taken immediately pre (time 0), and at 10, 24, 34, 48, 58, 72, 82, 96, 106, and 120 h after the start of the medicated water administration. Also, the collection of lung tissue and PELF was similar as for the oral bolus experiment, although the euthanasia of 4 birds at each interval took place at d 2 (24 h), 4 (72 h), 6 (120 h), 8 (168 h), and 10 (216 h) after the start of the continuous drinking water medication. The recovery of the PELF/saline solution ranged between 71.4 and 96.0%. The animal experiment was approved by the Ethical Committee of the Faculties of Veterinary Medicine and Bioscience Engineering, Ghent University (EC 2013/108). Florfenicol Analyses in Plasma, Lung Tissue, and PELF Quantification of FF in the plasma samples was performed using an in-house developed and validated LC-MS/MS method, described by Watteyn et al. (2013). Sample preparation for lung tissue consisted of homogenization with an equal weight of water, using an Ultra Turrax mixer (Ika, Staufen, Germany). A 0.5 g aliquot of this lung tissue homogenate (corresponding with 0.25 g of lung tissue) was used for FF analysis. The further sample preparation procedure of lung tissue and the preparation of the PELF samples were similar to that for plasma samples. The method for quantification of FF in lung tissue and PELF was validated in-house by a set of parameters (linearity, within-run and between-run accuracy and precision, limit of quantification [LOQ], limit of detection [LOD], and selectivity) that were in compliance with the recommendations as defined by the European Community (Anonymous, 2002) and with international reference guidelines (Knecht and Stork, 1974; Heitzman, 1994; VICH GL 49, 2015). Quadratic calibration curves were constructed using matrix-matched calibrator samples (concentration range: 10 to 5,000 ng/mL or ng/g) and the correlation coefficients (r = 0.9998 and 0.9981 for lung tissue and PELF, respectively) and goodness-of-fit coefficients (g = 7.54 and 9.17% for lung tissue and PELF, respectively) fell within the accepted ranges, i.e., r ≥ 0.99 and g < 20%, respectively. Within-run precision (repeatability) and accuracy were determined by analyzing blank samples that were spiked on the same day. The samples were spiked at 25, 250, and 2,500 ng/g; 25, 100, and 1,000 ng/mL for lung tissue and PELF samples, respectively. The between-run precision and accuracy were determined by analyzing quality control samples together with each analytical batch of samples, run on different days. The concentration levels for lung tissue and PELF were 25, 250, and 2,500 ng/g; 100 and 1,000 ng/mL, respectively. The LOQ was 25 ng/mL for plasma, 25 ng/g for lung tissue, and 20 ng/mL for PELF. Values below the LOQ were not included in the plasma concentration-time curves and the PK analysis. Pharmacokinetic Analyses The following plasma PK parameters were determined by one-compartmental analysis (WinNonlin 6.3, Pharsight, Princeton, NJ, USA): area under the plasma concentration-time curve from time 0 to infinity (AUCinf); absorption rate constant (kabs); elimination rate constant (kel); absorption half-life (t1/2abs); elimination half-life (t1/2el), expressed as the harmonic mean; volume of distribution, scaled by absolute oral bioavailability (Vd/Fabs); total body clearance, scaled by absolute oral bioavailability (Cl/Fabs); maximum plasma concentration (Cmax); and time to Cmax (tmax). For lung tissue, AUCinf, kel, t1/2el, Cmax, and tmax were calculated in a similar way. The PK data are expressed as mean ± SD for plasma. For lung and PELF, a sparse sampling protocol was applied, and values are expressed as mean. RESULTS Validation of FF in Lung Tissue and PELF The results of the validation are shown in Table 1. As can be seen, these results fell within the accepted ranges for accuracy (−20% to +10% of the theoretical concentration) and precision (within-run precision: relative standard deviation (RSD) ≤ RSDmax with a RSDmax of 15% for concentration levels ≥ 10 and < 100 ng/mL or ng/g, and a RSDmax of 10% for concentration levels ≥ 100 ng/mL or ng/g; between-run precision: RSD ≤ RSDmax with RSDmax = 2(1−0.5logConc), i.e., 27.9, 22.6, 19.7, 16.0, and 13.9% at 25, 100, 250, 1,000, and 2,500 ng/mL or ng/g, respectively). Table 1. Results for the within-run and between-run accuracy and precision experiments for the analyses of FF in lung tissue and pulmonary epithelial lining fluid (PELF).   Lung tissue ng/g  PELF ng/mL    25  250  2,500  25  100  1,000  Within-run  Accuracy (%)  5.5  −19.8  4.3  −13.7  −2.2  −10.9  Precision (RSD) (%)  6.4  4.2  8.6  11.0  5.2  5.2  Between-run  Accuracy (%)  0.8  3.2  −6.1  ND  −1.7  −2.6  Precision (RSD) (%)  15.2  14.0  12.0  ND  9.6  9.4    Lung tissue ng/g  PELF ng/mL    25  250  2,500  25  100  1,000  Within-run  Accuracy (%)  5.5  −19.8  4.3  −13.7  −2.2  −10.9  Precision (RSD) (%)  6.4  4.2  8.6  11.0  5.2  5.2  Between-run  Accuracy (%)  0.8  3.2  −6.1  ND  −1.7  −2.6  Precision (RSD) (%)  15.2  14.0  12.0  ND  9.6  9.4  ND, not determined; RSD, relative standard deviation. Acceptance criteria for accuracy: −20% to +10% of the theoretical concentration. Acceptance criteria for precision: Within-run precision: relative standard deviation (RSD) ≤ RSDmax with RSDmax of 15% for concentration levels ≥ 10 and < 100 ng/mL or ng/g, and RSDmax of 10% for concentration levels ≥ 100 ng/mL or ng/g; between-run precision: RSD ≤ RSDmax with RSDmax = 2(1−0.5logConc), i.e., 27.9, 22.6, 19.7, 16.0, and 13.9% at 25, 100, 250, 1,000, and 2,500 ng/mL or ng/g, respectively. View Large PK—single Oral Bolus vs. Continuous Drinking Water Administration In the experiment with continuous drinking water administration, a reduction in the mean water consumption during the treatment period was observed (5.35 ± 0.21 L/kg), in comparison with the acclimatization period (8.12 ± 0.37 L/kg). Consequently, the target dosage of FF of 30 mg/kg BW/d was not reached, but the mean real dose was 26.3 ± 3.12 mg/kg BW/d. The plasma concentration-time profiles of both the oral bolus and continuous drinking water experiment are depicted in Figure 1. The concentrations during continuous medicated drinking water administration were nearly constant during 5 d, followed by a rapid elimination when drinking water medication was ceased (at 110 h). During the elimination phase, at the interval of 8 h, a slight rise in plasma concentration can be observed for the single bolus treatment. After 24 h, all plasma concentrations were below the LOQ. Figure 1. View largeDownload slide Mean (+ SD) plasma concentration (log scale) vs. time curve of florfenicol (FF) in turkeys, during and after either a 5-day continuous oral administration of FF via medicated drinking water at a target dose of 30 mg/kg BW/d (n = 6, continuous experiment, ▴) or after a single oral bolus administration of FF at a dose of 30 mg/kg BW (n = 6, oral bolus experiment, •). Figure 1. View largeDownload slide Mean (+ SD) plasma concentration (log scale) vs. time curve of florfenicol (FF) in turkeys, during and after either a 5-day continuous oral administration of FF via medicated drinking water at a target dose of 30 mg/kg BW/d (n = 6, continuous experiment, ▴) or after a single oral bolus administration of FF at a dose of 30 mg/kg BW (n = 6, oral bolus experiment, •). Table 2 presents the mean (± SD) PK characteristics of FF in plasma, lung tissue, and PELF after bolus administration. FF was rapidly absorbed in plasma and distributed to the respiratory tract (mean kabs is 4.64 h−1 in plasma; mean tmax is 1 h in plasma, lung tissue, as well as in PELF). The mean t1/2el in plasma and lung tissue was similar, 6.27 h and 5.96 h respectively, whereas it was slightly higher in PELF, 8.70 h. In plasma, the mean Cmax was twice as high as in the lung tissue, 4.26 μg/mL and 2.64 μg/g, respectively. The mean concentration in PELF was much lower, i.e., at 0.39 μg/mL. Table 2. Pharmacokinetic properties of florfenicol in turkey poults after oral (PO) bolus administration of 30 mg/kg body weight, in plasma (n = 6), lung tissue, and PELF (both n = 6 at each interval). Results are presented as mean ± SD (plasma) or mean (lung and PELF). Parameter  Units  Plasma  Lung  PELF  AUCinf  h.μg/mL or h.μg/g  48.56 ± 18.76  32.63  2.97  kabs  h−1  4.64 ± 3.24  −  −  kel  h−1  0.011 ± 0.05  0.12  0.08  t1/2 abs  h  0.15A  −  −  t1/2 el  h  6.27A  5.96  8.70  Vd/Fabs  L/kg  6.75 ± 1.56  −  −  Cl/Fabs  L/kg/h  0.74 ± 0.42  −  −  tmax  h  1.02 ± 0.39  1.00  1.00  Cmax  μg/mL or μg/g  4.26 ± 1.30  2.64  0.39  Parameter  Units  Plasma  Lung  PELF  AUCinf  h.μg/mL or h.μg/g  48.56 ± 18.76  32.63  2.97  kabs  h−1  4.64 ± 3.24  −  −  kel  h−1  0.011 ± 0.05  0.12  0.08  t1/2 abs  h  0.15A  −  −  t1/2 el  h  6.27A  5.96  8.70  Vd/Fabs  L/kg  6.75 ± 1.56  −  −  Cl/Fabs  L/kg/h  0.74 ± 0.42  −  −  tmax  h  1.02 ± 0.39  1.00  1.00  Cmax  μg/mL or μg/g  4.26 ± 1.30  2.64  0.39  AUCinf, the area under the plasma concentration-time curve from time 0 to infinity; kabs, absorption rate constant; kel, elimination rate constant; t1/2abs, half-life of absorption; t1/2el, half-life of elimination; Vd/Fabs, volume of distribution (scaled by absolute oral bioavailability); Cl/Fabs, clearance (scaled by absolute oral bioavailability); tmax, time to maximum plasma concentration; Cmax, maximum plasma concentration. AHarmonic mean. View Large An overview of the mean FF concentrations in plasma, lung tissue, and PELF at different intervals during and after a 5-day continuous oral administration of FF via medicated drinking water (target dose: 30 mg FF/kg BW/d) or after a single oral bolus administration of FF (dose: 30 mg/kg BW) is shown in Figure 2A and 2B, respectively. During the 5-day continuous drinking water medication, the FF concentrations in plasma and lung tissue increased up to the 72 h sampling point, with lung/plasma ratios above 1 (Table 3). After treatment, from d 6 onwards, no FF concentrations could be detected in plasma and only very low concentrations in lung tissue. The concentrations in PELF were at all intervals below the LOQ. After oral bolus administration, the concentrations in plasma, lung tissue, and PELF were higher compared to the continuous drinking water medication. The FF concentrations after oral bolus administration were higher in lung tissue compared to plasma only at 6 and 24 h (Table 3). Figure 2. View largeDownload slide Mean (+SD) plasma, lung tissue, and PELF concentrations of florfenicol (FF) in turkeys, during and after either a 5-day continuous oral administration of FF via medicated drinking water at a target dose of 30 mg/kg BW/d (panel A) or after a single oral bolus administration of FF at a dose of 30 mg/kg BW (panel B). At each interval, 4 (continuous drinking water) or 6 (oral bolus) turkeys were taken into account. Values below the LOQ are indicated by ◊. At 36 and 48 h after oral bolus, all concentrations were below the LOQ and are not presented. Figure 2. View largeDownload slide Mean (+SD) plasma, lung tissue, and PELF concentrations of florfenicol (FF) in turkeys, during and after either a 5-day continuous oral administration of FF via medicated drinking water at a target dose of 30 mg/kg BW/d (panel A) or after a single oral bolus administration of FF at a dose of 30 mg/kg BW (panel B). At each interval, 4 (continuous drinking water) or 6 (oral bolus) turkeys were taken into account. Values below the LOQ are indicated by ◊. At 36 and 48 h after oral bolus, all concentrations were below the LOQ and are not presented. Table 3. Mean concentration ratios for FF in lung/plasma and pulmonary epithelial lining fluid (PELF)/plasma after an oral bolus administration of FF at a dose of 30 mg/kg BW or a continuous medicated drinking water administration for 5 d of FF at a target dose of 30 mg/kg BW/d. Oral bolus  Continuous drinking water  Time p.a.  Lung/Plasma  PELF/Plasma  Time  Lung/Plasma  PELF/Plasma  1 h  0.64 ± 0.47  0.12 ± 0.10  24 h (d 2)  2.01 ± 1.01  ND  2 h  0.27 ± 0.21  0.24 ± 0.33  72 h (d 4)  1.27 ± 0.42  ND  4 h  0.79 ± 0.53  0.55 ± 1.03  120 h (d 6)  ND  ND  6 h  1.16 ± 0.24  0.15 ± 0.05  168 h (d 8)  ND  ND  8 h  0.91 ± 0.35  0.12 ± 0.04  216 h (d 10)  ND  ND  12 h  0.98 ± 0.45  0.08 ± 0.03        24 h  1.09 ± 0.31  ND        Oral bolus  Continuous drinking water  Time p.a.  Lung/Plasma  PELF/Plasma  Time  Lung/Plasma  PELF/Plasma  1 h  0.64 ± 0.47  0.12 ± 0.10  24 h (d 2)  2.01 ± 1.01  ND  2 h  0.27 ± 0.21  0.24 ± 0.33  72 h (d 4)  1.27 ± 0.42  ND  4 h  0.79 ± 0.53  0.55 ± 1.03  120 h (d 6)  ND  ND  6 h  1.16 ± 0.24  0.15 ± 0.05  168 h (d 8)  ND  ND  8 h  0.91 ± 0.35  0.12 ± 0.04  216 h (d 10)  ND  ND  12 h  0.98 ± 0.45  0.08 ± 0.03        24 h  1.09 ± 0.31  ND        p.a., post administration; ND, not determined. View Large DISCUSSION Notwithstanding that FF may be used to treat turkeys from bacterial respiratory infections and PK studies in the species of interest are essential, no data on PK characteristics of FF in respiratory tissue of turkeys have been published before. An important drawback of FF when used as an active pharmaceutical ingredient (not formulated) in medicated drinking water is the low aqueous solubility (1 g/L). Only after stirring and sonication could the drug be dissolved in the drinking water. Plasma Pharmacokinetics After a single oral bolus administration of 30 mg/kg BW, FF showed a rapid absorption (tmax of 1.02 h). This is in accordance with other studies in avian species after an oral bolus dosing of the same dose, where the mean reported plasma tmax values varied from 0.30 to 2.00 h (Afifi & Abo El-Sooud, 1997; Shen et al., 2003; Abu-Basha et al., 2007; Switala et al., 2007; Chang et al., 2010). Also in pigs, the tmax was similar, namely, 1.50 h, after a single oral bolus of 20 mg FF/kg BW (Jiang et al., 2006). The mean Cmax in plasma determined in this study (4.26 μg/mL) was comparable to that in broiler chickens (range from 3.20 to 6.79 μg/mL, Afifi & Abo El-Sooud, 1997; Shen et al., 2003), but lower compared to Switala et al. (2007) in turkeys using the same dose (12.25 μg/mL). Also, the AUC in the present study was lower compared to the other study in turkeys, 48.56 and 77.62 μg.h/mL, respectively (Switala et al., 2007). The results of both parameters suggest a lower oral bioavailability of the active substance used in the present study, whereas Switala et al. (2007) used a commercially available FF formulation. In Leghorn and Taiwan native chickens, Chang et al. (2010) also used a commercial oral formulation of FF, and the plasma concentrations were similar to the levels in turkeys as reported by Switala et al. (2007). With a Vd above 1 L/kg BW, FF is moderately distributed. The Vd of FF varies among different bird species, with a Vd ranging from 1.06 L/kg BW in turkeys, over around 5 L/kg BW in quails, pigeons, ducks, and broiler chickens, and up to 8.70 L/kg BW in Japanese quails (Afifi & Abo El-Sooud, 1997; El-Banna, 1998; Switala et al., 2007; Ismail & El-Kattan, 2009; Koc et al., 2009). Although plasma protein binding was not determined in this study, many others reported a low binding for FF in different animal species, such as calves, rabbits, and chickens, < 25% (Adams et al., 1987; Afifi & Abo El-Sooud, 1997; Abd El-Aty et al., 2004). This low extent is consistent with the moderate Vd. Since no IV bolus was administered, Cl was not corrected for the absolute bioavailability (Fabs). Therefore, the mean Cl found in our study (0.74 L/kg/h) could be even lower. There is a wide range in Cl values reported among avian species, from 0.3 to 0.6 L/kg/h in larger birds (turkey and Muscovy ducks) to 1.6 L/kg/h in broiler chickens, 3.9 L/kg/h in pigeons, and 5.3 L/kg/h in quails (Afifi & Abo El-Sooud, 1997; El-Banna, 1998; Switala et al., 2007; Ismail & El-Kattan, 2009). A mean plasma t1/2el of 6.27 h in turkeys was comparable to that reported for Muscovy ducks (El-Banna, 1998), whereas it was twice as long as reported in chickens (Afifi & Abo El-Sooud, 1997; Shen et al., 2003; Ismail & El-Kattan, 2009). Also Switala et al. (2007) found a lower t1/2el value, i.e., 3.76 h in turkeys. In the present study, almost all FF was eliminated from the body at 24 h p.a. Regarding the plasma concentration-time profile of the continuous drinking water medication, the plasma concentrations balanced around 1 μg/mL during the whole drug administration period (5 d). After stopping the medicated drinking water administration, FF was rapidly eliminated from plasma. Lung and PELF Pharmacokinetics Despite that concentrations of FF in tissues have been reported in avian (Anadón et al., 2008; Chang et al., 2010) as well as in mammalian species (Lane et al., 2008), only one study described the PK characteristics of FF in lung tissue. After intramuscular administration of 20 mg FF/kg BW to pigs (Liu et al., 2002), the mean Cmax and tmax of FF in lung tissue were comparable to turkeys, 2.64 μg/g and 1 h compared to 2.46 μg/g and 2 h for turkeys and pigs, respectively. This confirms the rapid distribution of FF from plasma to lung tissue. However, a great discrepancy can be observed between turkeys and pigs concerning the elimination of FF from lung tissue. In pigs, the mean t1/2el of FF from lung tissue was 38.5 h, whereas in turkeys a mean t1/2el of 5.96 h was observed. This could be attributed to the pharmaceutical formulation of the used intramuscular injection of FF in the study of Liu et al. (2002). They used a long-acting formulation, which resulted in a delayed absorption period and a prolonged elimination time. In order to compare the concentrations in either lung tissue or PELF to the plasma concentrations, lung/plasma and PELF/plasma ratios were calculated. For the continuous drinking water administration experiment, the lung/plasma concentration ratio was above 1 during the treatment, confirming the high affinity of FF for the respiratory tract. Throughout the administration period, the FF lung concentrations increased. After stopping the treatment, only low FF lung concentrations and no plasma concentrations could be detected. In all PELF samples, the FF concentrations were below the LOQ. It is likely that these low concentrations in PELF are related to the low concentrations in plasma and lung tissue. After the oral bolus administration, the lung/plasma concentration ratio was above 1 only at 6 h and 24 h p.a. This would imply a more rapid elimination from plasma compared to lung tissue, although the t1/2el of FF in plasma and lung are comparable. After 8 h, the plasma concentrations increased with 0.7 μg/mL, and this is also reflected in a higher lung FF concentration at 12 h (increase of 0.9 μg/g). This phenomenon could be explained by enterohepatic circulation of FF (Pasmans et al., 2008). The high concentration of FF in bile from chickens confirms this suggestion (Afifi & Abo El-Sooud, 1997). The same authors found after multiple oral doses of 30 mg/kg BW/d to chickens for 5 successive d detectable lung concentrations (20 μg/g) until 48 h after the last dose. The Cmax reported in that study was similar to the mean Cmax in the present study, i.e., 2.80 μg/g compared to 2.64 μg/g, respectively. In contrast to continuous drinking water medication, FF was detectable in PELF after a single bolus administration. However, the FF PELF/plasma concentration ratios were very low (range between 0.55 and 0.08). The low FF concentrations in PELF could be due to the collection method of PELF and/or to the anatomy and physiology of the respiratory tract of birds (Watteyn et al., 2015). Birds have a typical arrangement of the respiratory system, with flow-through lungs and bronchi that are connected with the air sacs via ostia. In contrast with a bronchoalveolar lavage in mammals, the used technique in this study was based on flushing the ex vivo lungs. The flush solution, heparin-saline, was distributed in and immediately flushed out of the lung through the ostia. These differences with the mammalian respiratory tract have an influence on the collection method for PELF. CONCLUSION FF was very rapidly absorbed and distributed to the lung tissue after a single oral bolus administration in turkeys. However, FF as such is not applicable for drinking water medication, as the water solubility is very low (only 1 mg/mL), which implies an elaborate preparation, including stirring and sonication, of the medicated drinking water. Therefore, efforts should be made to develop and register a soluble pharmaceutical FF formulation for poultry. Acknowledgements The authors wish to thank Jelle Lambrecht* and Matteo Stecca‡ for their excellent laboratory assistance. For the aid in the animal experiment, we thank Gunther Antonissen*†, Nathan Broekaert*, Thomas De Mil*, Sophie Fraeyman*, Elke Gasthuys*, Elke Plessers*, Charlotte Watteyn#, and Heidi Wyns*. *Department of Pharmacology, Toxicology and Biochemistry, †Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium. ‡Department of Comparative Biomedicine and Food Science, University of Padova, Padova, Italy. #Department of Forest Ecology and Forest Management, Wageningen University, Wageningen, The Netherlands. REFERENCES Abd El-Aty A. M., Goudah A., Abo El-Sooud K., El-Zorba H. Y., Shimoda M., Zhou H. H.. 2004. Pharmacokinetics and bioavailability of florfenicol following intravenous, intramuscular and oral administrations in rabbits. Vet. Res. Comm.  28: 515– 524. Google Scholar CrossRef Search ADS   Abu-Basha E. A., Idkaidek N. M, Al-Shunnaq A. F.. 2007. 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Pharmacokinetics of florfenicol in turkey plasma, lung tissue, and pulmonary epithelial lining fluid after single oral bolus or continuous administration in the drinking water

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Abstract

Abstract Florfenicol (FF) is registered for treatment of bovine and swine respiratory diseases. Although, turkeys often suffer from bacterial respiratory tract infections, there is no registered formulation based on FF for poultry available in Europe. The aim of this study was to evaluate the pharmacokinetic behavior of FF in turkeys in plasma, lung tissue, and pulmonary epithelial lining fluid (PELF). The concentration and pharmacokinetic characteristics of FF in plasma, lung tissue, and PELF in turkeys were determined, either after a single oral bolus (30 mg/kg body weight, BW) or during and after continuous drinking water medication (30 mg/kg BW/d for 5 d). Plasma, lung tissue, and PELF samples were collected at different intervals after administration, and FF was quantified by liquid chromatography-tandem mass spectrometry. After single bolus administration, FF was rapidly absorbed in plasma (the time to maximum concentration, tmax, was 1.02 h) and distributed to the respiratory tract (mean tmax = 1.00 h). The mean t1/2el in plasma and lung tissue was similar, around 6 h, whereas it was slightly higher in PELF, namely, 8.7 hours. After oral bolus dosing, the mean maximum concentration in plasma was twice as high as in the lung tissue, 4.26 μg/mL and 2.64 μg/g, respectively, while in PELF it was much lower, 0.39 μg/mL. During continuous drinking water medication, lung FF concentrations were slightly higher than plasma concentrations, with lung/plasma ratios of 2.01 and 1.27 after 24 h and 72 h, respectively. FF was not detected in PELF during continuous drinking water medication. INTRODUCTION Florfenicol (FF) has a broad antibacterial action against several pathogens responsible for infections of the respiratory, urinary, and gastrointestinal tract. An excellent clinical response to FF in bovine and swine respiratory diseases can be attributed to the remarkable pharmacokinetic (PK) characteristics, such as high absorption and rapid penetration in the respiratory tract, and the low resistance of cattle and swine pathogens, such as Mannheimia haemolytica, Pasteurella multocida, and Actinobacillus pleuropneumoniae, which all have a minimum inhibitory concentration (MIC90) below 1 μg/mL (Shin et al., 2005). In turkeys, FF has been proven to be effective against Ornithobacterium rhinotracheale infection, using drinking water medication during 5 d at a dose of 30 mg/kg body weight (BW) per d (Marien et al., 2007; Watteyn et al., 2013). However, only few data regarding the PK characteristics of FF in respiratory tissues are available, which is of primary interest given the localization of O. rhinotracheale (Afifi & Abo el-Sooud, 1997; Liu et al., 2002). Due to species-dependent differences in anatomy and physiology, PK studies have to be performed in the species of interest and with the pathogen of interest. To the authors knowledge, only one PK study of FF has been performed in turkeys after single bolus administration of 30 mg/kg BW either per os (orally, PO) or intravenously (IV) (Switala et al., 2007), but concentrations in lung tissue or pulmonary epithelial lining fluid (PELF), the sites of action, have not been reported yet. The first aim of this research was to determine the concentrations and PK characteristics of FF in plasma, lung tissue, and PELF after single oral bolus administration (30 mg FF/kg BW). In order to simulate field conditions, plasma, lung tissue, and PELF concentrations and PK characteristics of FF also were studied during and after continuous drinking water medication for 5 d at the same recommended dose of 30 mg/kg BW/d. MATERIALS AND METHODS Veterinary Drug FF, 2,2-dichloro-N-[1S,2R]-1-(fluoromethyl)-2-hydro-xy-2-[4-(methylsulfonyl)-phenyl]ethyl]-acetamide, used for the animal experiments was obtained from Zhejiang Hisoar Pharmaceutical Co., LTD (Zhejiang, China). The antibiotic was used solely as an active ingredient, without formulation. Because of the low aqueous solubility (1 mg/mL), the FF bolus for the single oral bolus PK study was given as a suspension of FF in tap water at a concentration of 6 mg/mL. For the continuous drinking water PK study, the medicated drinking water was prepared daily by mixing FF in tap water, stirring for 30 min, followed by sonication for 20 min to dissolve the FF. The mean (± SD) concentration was 74.3 (± 3.4) mg FF/L tap water. PK-experiment—single Oral Bolus Administration This study was performed using 54 6-week-old female turkey poults (Hybrid Converter, local commercial turkey farm) with a mean (± SD) BW of 2.063 (± 0.195) kg that were housed according to the requirements of the European Union (Anonymous, 2010). During the whole experiment, the turkeys were housed together, and the light scheme was set at 16 h light and 8 h dark. After a fasting period of 12 h, the birds received a FF bolus of 30 mg/kg BW by gavage in the crop, followed by rinsing with tap water. Four h after the bolus administration, the birds received feed again. Blood (1 mL) was collected from 6 turkeys by venipuncture from the medial metatarsal vein into heparinized tubes (Vacutest Kima, Novolab, Geraardsbergen, Belgium) at different intervals, pre (time 0) and post administration (p.a.; 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 10, and 24 h). Plasma was separated by centrifugation and stored at ≤ −15°C, pending analysis. Furthermore, animals (n = 6 at each interval) were euthanized at different intervals post FF administration to collect plasma, lung tissue, and PELF. Euthanasia was performed at 1, 2, 4, 6, 8, 12, 24, 36, and 48 h after the oral bolus administration. Birds were anesthetized by an intramuscular injection of a combination of xylazine (Xyl-M 2%, VMD, Arendonk, Belgium), zolazepam, and tiletamine (Zoletil 100, Virbac, Wavre, Belgium), followed by exsanguination. The whole right lung was removed for FF analysis. The complete left lung was used to collect PELF, as described by Bottje et al. (1999). In brief, after weighing the lung, it was lavaged with heparin-saline (200 units heparin per mL of 0.9% saline) at a volume of 2 mL/g lung through a cannula in the first bronchus. The PELF/saline solution was collected in a petri dish, and the amount of fluid was measured to determine the recovery, which ranged from 80.0 to 100%. The fluid was centrifuged (5,250 x g for 3 min) to remove red blood cells. Both the lung tissue and PELF were stored at ≤ −15°C until analysis. The animal experiment was approved by the Ethical Committee of the Faculty of Veterinary Medicine and Bioscience Engineering, Ghent University (EC 2014/68). PK-experiment—continuous Drinking Water Administration During an acclimatization period of 5 d, water consumption (non-medicated tap water) of 20 3-week-old female turkey poults (Hybrid Converter, local commercial turkey farm) with a mean (± SD) BW of 0.812 (± 0.074) kg was measured to calculate a correct dose of the medicated drinking water. Thereafter, the turkeys received FF via the drinking water during a 5-day period (target dose: 30 mg/kg BW/d), supplied in 3 plastic drinking troughs. During the whole experiment, the turkeys were housed together, and the light scheme was set at 16 h light and 8 h dark. Blood (1 mL) of 6 turkeys was collected and stored in the same way as the single oral bolus study, except for sampling points. Blood was taken immediately pre (time 0), and at 10, 24, 34, 48, 58, 72, 82, 96, 106, and 120 h after the start of the medicated water administration. Also, the collection of lung tissue and PELF was similar as for the oral bolus experiment, although the euthanasia of 4 birds at each interval took place at d 2 (24 h), 4 (72 h), 6 (120 h), 8 (168 h), and 10 (216 h) after the start of the continuous drinking water medication. The recovery of the PELF/saline solution ranged between 71.4 and 96.0%. The animal experiment was approved by the Ethical Committee of the Faculties of Veterinary Medicine and Bioscience Engineering, Ghent University (EC 2013/108). Florfenicol Analyses in Plasma, Lung Tissue, and PELF Quantification of FF in the plasma samples was performed using an in-house developed and validated LC-MS/MS method, described by Watteyn et al. (2013). Sample preparation for lung tissue consisted of homogenization with an equal weight of water, using an Ultra Turrax mixer (Ika, Staufen, Germany). A 0.5 g aliquot of this lung tissue homogenate (corresponding with 0.25 g of lung tissue) was used for FF analysis. The further sample preparation procedure of lung tissue and the preparation of the PELF samples were similar to that for plasma samples. The method for quantification of FF in lung tissue and PELF was validated in-house by a set of parameters (linearity, within-run and between-run accuracy and precision, limit of quantification [LOQ], limit of detection [LOD], and selectivity) that were in compliance with the recommendations as defined by the European Community (Anonymous, 2002) and with international reference guidelines (Knecht and Stork, 1974; Heitzman, 1994; VICH GL 49, 2015). Quadratic calibration curves were constructed using matrix-matched calibrator samples (concentration range: 10 to 5,000 ng/mL or ng/g) and the correlation coefficients (r = 0.9998 and 0.9981 for lung tissue and PELF, respectively) and goodness-of-fit coefficients (g = 7.54 and 9.17% for lung tissue and PELF, respectively) fell within the accepted ranges, i.e., r ≥ 0.99 and g < 20%, respectively. Within-run precision (repeatability) and accuracy were determined by analyzing blank samples that were spiked on the same day. The samples were spiked at 25, 250, and 2,500 ng/g; 25, 100, and 1,000 ng/mL for lung tissue and PELF samples, respectively. The between-run precision and accuracy were determined by analyzing quality control samples together with each analytical batch of samples, run on different days. The concentration levels for lung tissue and PELF were 25, 250, and 2,500 ng/g; 100 and 1,000 ng/mL, respectively. The LOQ was 25 ng/mL for plasma, 25 ng/g for lung tissue, and 20 ng/mL for PELF. Values below the LOQ were not included in the plasma concentration-time curves and the PK analysis. Pharmacokinetic Analyses The following plasma PK parameters were determined by one-compartmental analysis (WinNonlin 6.3, Pharsight, Princeton, NJ, USA): area under the plasma concentration-time curve from time 0 to infinity (AUCinf); absorption rate constant (kabs); elimination rate constant (kel); absorption half-life (t1/2abs); elimination half-life (t1/2el), expressed as the harmonic mean; volume of distribution, scaled by absolute oral bioavailability (Vd/Fabs); total body clearance, scaled by absolute oral bioavailability (Cl/Fabs); maximum plasma concentration (Cmax); and time to Cmax (tmax). For lung tissue, AUCinf, kel, t1/2el, Cmax, and tmax were calculated in a similar way. The PK data are expressed as mean ± SD for plasma. For lung and PELF, a sparse sampling protocol was applied, and values are expressed as mean. RESULTS Validation of FF in Lung Tissue and PELF The results of the validation are shown in Table 1. As can be seen, these results fell within the accepted ranges for accuracy (−20% to +10% of the theoretical concentration) and precision (within-run precision: relative standard deviation (RSD) ≤ RSDmax with a RSDmax of 15% for concentration levels ≥ 10 and < 100 ng/mL or ng/g, and a RSDmax of 10% for concentration levels ≥ 100 ng/mL or ng/g; between-run precision: RSD ≤ RSDmax with RSDmax = 2(1−0.5logConc), i.e., 27.9, 22.6, 19.7, 16.0, and 13.9% at 25, 100, 250, 1,000, and 2,500 ng/mL or ng/g, respectively). Table 1. Results for the within-run and between-run accuracy and precision experiments for the analyses of FF in lung tissue and pulmonary epithelial lining fluid (PELF).   Lung tissue ng/g  PELF ng/mL    25  250  2,500  25  100  1,000  Within-run  Accuracy (%)  5.5  −19.8  4.3  −13.7  −2.2  −10.9  Precision (RSD) (%)  6.4  4.2  8.6  11.0  5.2  5.2  Between-run  Accuracy (%)  0.8  3.2  −6.1  ND  −1.7  −2.6  Precision (RSD) (%)  15.2  14.0  12.0  ND  9.6  9.4    Lung tissue ng/g  PELF ng/mL    25  250  2,500  25  100  1,000  Within-run  Accuracy (%)  5.5  −19.8  4.3  −13.7  −2.2  −10.9  Precision (RSD) (%)  6.4  4.2  8.6  11.0  5.2  5.2  Between-run  Accuracy (%)  0.8  3.2  −6.1  ND  −1.7  −2.6  Precision (RSD) (%)  15.2  14.0  12.0  ND  9.6  9.4  ND, not determined; RSD, relative standard deviation. Acceptance criteria for accuracy: −20% to +10% of the theoretical concentration. Acceptance criteria for precision: Within-run precision: relative standard deviation (RSD) ≤ RSDmax with RSDmax of 15% for concentration levels ≥ 10 and < 100 ng/mL or ng/g, and RSDmax of 10% for concentration levels ≥ 100 ng/mL or ng/g; between-run precision: RSD ≤ RSDmax with RSDmax = 2(1−0.5logConc), i.e., 27.9, 22.6, 19.7, 16.0, and 13.9% at 25, 100, 250, 1,000, and 2,500 ng/mL or ng/g, respectively. View Large PK—single Oral Bolus vs. Continuous Drinking Water Administration In the experiment with continuous drinking water administration, a reduction in the mean water consumption during the treatment period was observed (5.35 ± 0.21 L/kg), in comparison with the acclimatization period (8.12 ± 0.37 L/kg). Consequently, the target dosage of FF of 30 mg/kg BW/d was not reached, but the mean real dose was 26.3 ± 3.12 mg/kg BW/d. The plasma concentration-time profiles of both the oral bolus and continuous drinking water experiment are depicted in Figure 1. The concentrations during continuous medicated drinking water administration were nearly constant during 5 d, followed by a rapid elimination when drinking water medication was ceased (at 110 h). During the elimination phase, at the interval of 8 h, a slight rise in plasma concentration can be observed for the single bolus treatment. After 24 h, all plasma concentrations were below the LOQ. Figure 1. View largeDownload slide Mean (+ SD) plasma concentration (log scale) vs. time curve of florfenicol (FF) in turkeys, during and after either a 5-day continuous oral administration of FF via medicated drinking water at a target dose of 30 mg/kg BW/d (n = 6, continuous experiment, ▴) or after a single oral bolus administration of FF at a dose of 30 mg/kg BW (n = 6, oral bolus experiment, •). Figure 1. View largeDownload slide Mean (+ SD) plasma concentration (log scale) vs. time curve of florfenicol (FF) in turkeys, during and after either a 5-day continuous oral administration of FF via medicated drinking water at a target dose of 30 mg/kg BW/d (n = 6, continuous experiment, ▴) or after a single oral bolus administration of FF at a dose of 30 mg/kg BW (n = 6, oral bolus experiment, •). Table 2 presents the mean (± SD) PK characteristics of FF in plasma, lung tissue, and PELF after bolus administration. FF was rapidly absorbed in plasma and distributed to the respiratory tract (mean kabs is 4.64 h−1 in plasma; mean tmax is 1 h in plasma, lung tissue, as well as in PELF). The mean t1/2el in plasma and lung tissue was similar, 6.27 h and 5.96 h respectively, whereas it was slightly higher in PELF, 8.70 h. In plasma, the mean Cmax was twice as high as in the lung tissue, 4.26 μg/mL and 2.64 μg/g, respectively. The mean concentration in PELF was much lower, i.e., at 0.39 μg/mL. Table 2. Pharmacokinetic properties of florfenicol in turkey poults after oral (PO) bolus administration of 30 mg/kg body weight, in plasma (n = 6), lung tissue, and PELF (both n = 6 at each interval). Results are presented as mean ± SD (plasma) or mean (lung and PELF). Parameter  Units  Plasma  Lung  PELF  AUCinf  h.μg/mL or h.μg/g  48.56 ± 18.76  32.63  2.97  kabs  h−1  4.64 ± 3.24  −  −  kel  h−1  0.011 ± 0.05  0.12  0.08  t1/2 abs  h  0.15A  −  −  t1/2 el  h  6.27A  5.96  8.70  Vd/Fabs  L/kg  6.75 ± 1.56  −  −  Cl/Fabs  L/kg/h  0.74 ± 0.42  −  −  tmax  h  1.02 ± 0.39  1.00  1.00  Cmax  μg/mL or μg/g  4.26 ± 1.30  2.64  0.39  Parameter  Units  Plasma  Lung  PELF  AUCinf  h.μg/mL or h.μg/g  48.56 ± 18.76  32.63  2.97  kabs  h−1  4.64 ± 3.24  −  −  kel  h−1  0.011 ± 0.05  0.12  0.08  t1/2 abs  h  0.15A  −  −  t1/2 el  h  6.27A  5.96  8.70  Vd/Fabs  L/kg  6.75 ± 1.56  −  −  Cl/Fabs  L/kg/h  0.74 ± 0.42  −  −  tmax  h  1.02 ± 0.39  1.00  1.00  Cmax  μg/mL or μg/g  4.26 ± 1.30  2.64  0.39  AUCinf, the area under the plasma concentration-time curve from time 0 to infinity; kabs, absorption rate constant; kel, elimination rate constant; t1/2abs, half-life of absorption; t1/2el, half-life of elimination; Vd/Fabs, volume of distribution (scaled by absolute oral bioavailability); Cl/Fabs, clearance (scaled by absolute oral bioavailability); tmax, time to maximum plasma concentration; Cmax, maximum plasma concentration. AHarmonic mean. View Large An overview of the mean FF concentrations in plasma, lung tissue, and PELF at different intervals during and after a 5-day continuous oral administration of FF via medicated drinking water (target dose: 30 mg FF/kg BW/d) or after a single oral bolus administration of FF (dose: 30 mg/kg BW) is shown in Figure 2A and 2B, respectively. During the 5-day continuous drinking water medication, the FF concentrations in plasma and lung tissue increased up to the 72 h sampling point, with lung/plasma ratios above 1 (Table 3). After treatment, from d 6 onwards, no FF concentrations could be detected in plasma and only very low concentrations in lung tissue. The concentrations in PELF were at all intervals below the LOQ. After oral bolus administration, the concentrations in plasma, lung tissue, and PELF were higher compared to the continuous drinking water medication. The FF concentrations after oral bolus administration were higher in lung tissue compared to plasma only at 6 and 24 h (Table 3). Figure 2. View largeDownload slide Mean (+SD) plasma, lung tissue, and PELF concentrations of florfenicol (FF) in turkeys, during and after either a 5-day continuous oral administration of FF via medicated drinking water at a target dose of 30 mg/kg BW/d (panel A) or after a single oral bolus administration of FF at a dose of 30 mg/kg BW (panel B). At each interval, 4 (continuous drinking water) or 6 (oral bolus) turkeys were taken into account. Values below the LOQ are indicated by ◊. At 36 and 48 h after oral bolus, all concentrations were below the LOQ and are not presented. Figure 2. View largeDownload slide Mean (+SD) plasma, lung tissue, and PELF concentrations of florfenicol (FF) in turkeys, during and after either a 5-day continuous oral administration of FF via medicated drinking water at a target dose of 30 mg/kg BW/d (panel A) or after a single oral bolus administration of FF at a dose of 30 mg/kg BW (panel B). At each interval, 4 (continuous drinking water) or 6 (oral bolus) turkeys were taken into account. Values below the LOQ are indicated by ◊. At 36 and 48 h after oral bolus, all concentrations were below the LOQ and are not presented. Table 3. Mean concentration ratios for FF in lung/plasma and pulmonary epithelial lining fluid (PELF)/plasma after an oral bolus administration of FF at a dose of 30 mg/kg BW or a continuous medicated drinking water administration for 5 d of FF at a target dose of 30 mg/kg BW/d. Oral bolus  Continuous drinking water  Time p.a.  Lung/Plasma  PELF/Plasma  Time  Lung/Plasma  PELF/Plasma  1 h  0.64 ± 0.47  0.12 ± 0.10  24 h (d 2)  2.01 ± 1.01  ND  2 h  0.27 ± 0.21  0.24 ± 0.33  72 h (d 4)  1.27 ± 0.42  ND  4 h  0.79 ± 0.53  0.55 ± 1.03  120 h (d 6)  ND  ND  6 h  1.16 ± 0.24  0.15 ± 0.05  168 h (d 8)  ND  ND  8 h  0.91 ± 0.35  0.12 ± 0.04  216 h (d 10)  ND  ND  12 h  0.98 ± 0.45  0.08 ± 0.03        24 h  1.09 ± 0.31  ND        Oral bolus  Continuous drinking water  Time p.a.  Lung/Plasma  PELF/Plasma  Time  Lung/Plasma  PELF/Plasma  1 h  0.64 ± 0.47  0.12 ± 0.10  24 h (d 2)  2.01 ± 1.01  ND  2 h  0.27 ± 0.21  0.24 ± 0.33  72 h (d 4)  1.27 ± 0.42  ND  4 h  0.79 ± 0.53  0.55 ± 1.03  120 h (d 6)  ND  ND  6 h  1.16 ± 0.24  0.15 ± 0.05  168 h (d 8)  ND  ND  8 h  0.91 ± 0.35  0.12 ± 0.04  216 h (d 10)  ND  ND  12 h  0.98 ± 0.45  0.08 ± 0.03        24 h  1.09 ± 0.31  ND        p.a., post administration; ND, not determined. View Large DISCUSSION Notwithstanding that FF may be used to treat turkeys from bacterial respiratory infections and PK studies in the species of interest are essential, no data on PK characteristics of FF in respiratory tissue of turkeys have been published before. An important drawback of FF when used as an active pharmaceutical ingredient (not formulated) in medicated drinking water is the low aqueous solubility (1 g/L). Only after stirring and sonication could the drug be dissolved in the drinking water. Plasma Pharmacokinetics After a single oral bolus administration of 30 mg/kg BW, FF showed a rapid absorption (tmax of 1.02 h). This is in accordance with other studies in avian species after an oral bolus dosing of the same dose, where the mean reported plasma tmax values varied from 0.30 to 2.00 h (Afifi & Abo El-Sooud, 1997; Shen et al., 2003; Abu-Basha et al., 2007; Switala et al., 2007; Chang et al., 2010). Also in pigs, the tmax was similar, namely, 1.50 h, after a single oral bolus of 20 mg FF/kg BW (Jiang et al., 2006). The mean Cmax in plasma determined in this study (4.26 μg/mL) was comparable to that in broiler chickens (range from 3.20 to 6.79 μg/mL, Afifi & Abo El-Sooud, 1997; Shen et al., 2003), but lower compared to Switala et al. (2007) in turkeys using the same dose (12.25 μg/mL). Also, the AUC in the present study was lower compared to the other study in turkeys, 48.56 and 77.62 μg.h/mL, respectively (Switala et al., 2007). The results of both parameters suggest a lower oral bioavailability of the active substance used in the present study, whereas Switala et al. (2007) used a commercially available FF formulation. In Leghorn and Taiwan native chickens, Chang et al. (2010) also used a commercial oral formulation of FF, and the plasma concentrations were similar to the levels in turkeys as reported by Switala et al. (2007). With a Vd above 1 L/kg BW, FF is moderately distributed. The Vd of FF varies among different bird species, with a Vd ranging from 1.06 L/kg BW in turkeys, over around 5 L/kg BW in quails, pigeons, ducks, and broiler chickens, and up to 8.70 L/kg BW in Japanese quails (Afifi & Abo El-Sooud, 1997; El-Banna, 1998; Switala et al., 2007; Ismail & El-Kattan, 2009; Koc et al., 2009). Although plasma protein binding was not determined in this study, many others reported a low binding for FF in different animal species, such as calves, rabbits, and chickens, < 25% (Adams et al., 1987; Afifi & Abo El-Sooud, 1997; Abd El-Aty et al., 2004). This low extent is consistent with the moderate Vd. Since no IV bolus was administered, Cl was not corrected for the absolute bioavailability (Fabs). Therefore, the mean Cl found in our study (0.74 L/kg/h) could be even lower. There is a wide range in Cl values reported among avian species, from 0.3 to 0.6 L/kg/h in larger birds (turkey and Muscovy ducks) to 1.6 L/kg/h in broiler chickens, 3.9 L/kg/h in pigeons, and 5.3 L/kg/h in quails (Afifi & Abo El-Sooud, 1997; El-Banna, 1998; Switala et al., 2007; Ismail & El-Kattan, 2009). A mean plasma t1/2el of 6.27 h in turkeys was comparable to that reported for Muscovy ducks (El-Banna, 1998), whereas it was twice as long as reported in chickens (Afifi & Abo El-Sooud, 1997; Shen et al., 2003; Ismail & El-Kattan, 2009). Also Switala et al. (2007) found a lower t1/2el value, i.e., 3.76 h in turkeys. In the present study, almost all FF was eliminated from the body at 24 h p.a. Regarding the plasma concentration-time profile of the continuous drinking water medication, the plasma concentrations balanced around 1 μg/mL during the whole drug administration period (5 d). After stopping the medicated drinking water administration, FF was rapidly eliminated from plasma. Lung and PELF Pharmacokinetics Despite that concentrations of FF in tissues have been reported in avian (Anadón et al., 2008; Chang et al., 2010) as well as in mammalian species (Lane et al., 2008), only one study described the PK characteristics of FF in lung tissue. After intramuscular administration of 20 mg FF/kg BW to pigs (Liu et al., 2002), the mean Cmax and tmax of FF in lung tissue were comparable to turkeys, 2.64 μg/g and 1 h compared to 2.46 μg/g and 2 h for turkeys and pigs, respectively. This confirms the rapid distribution of FF from plasma to lung tissue. However, a great discrepancy can be observed between turkeys and pigs concerning the elimination of FF from lung tissue. In pigs, the mean t1/2el of FF from lung tissue was 38.5 h, whereas in turkeys a mean t1/2el of 5.96 h was observed. This could be attributed to the pharmaceutical formulation of the used intramuscular injection of FF in the study of Liu et al. (2002). They used a long-acting formulation, which resulted in a delayed absorption period and a prolonged elimination time. In order to compare the concentrations in either lung tissue or PELF to the plasma concentrations, lung/plasma and PELF/plasma ratios were calculated. For the continuous drinking water administration experiment, the lung/plasma concentration ratio was above 1 during the treatment, confirming the high affinity of FF for the respiratory tract. Throughout the administration period, the FF lung concentrations increased. After stopping the treatment, only low FF lung concentrations and no plasma concentrations could be detected. In all PELF samples, the FF concentrations were below the LOQ. It is likely that these low concentrations in PELF are related to the low concentrations in plasma and lung tissue. After the oral bolus administration, the lung/plasma concentration ratio was above 1 only at 6 h and 24 h p.a. This would imply a more rapid elimination from plasma compared to lung tissue, although the t1/2el of FF in plasma and lung are comparable. After 8 h, the plasma concentrations increased with 0.7 μg/mL, and this is also reflected in a higher lung FF concentration at 12 h (increase of 0.9 μg/g). This phenomenon could be explained by enterohepatic circulation of FF (Pasmans et al., 2008). The high concentration of FF in bile from chickens confirms this suggestion (Afifi & Abo El-Sooud, 1997). The same authors found after multiple oral doses of 30 mg/kg BW/d to chickens for 5 successive d detectable lung concentrations (20 μg/g) until 48 h after the last dose. The Cmax reported in that study was similar to the mean Cmax in the present study, i.e., 2.80 μg/g compared to 2.64 μg/g, respectively. In contrast to continuous drinking water medication, FF was detectable in PELF after a single bolus administration. However, the FF PELF/plasma concentration ratios were very low (range between 0.55 and 0.08). The low FF concentrations in PELF could be due to the collection method of PELF and/or to the anatomy and physiology of the respiratory tract of birds (Watteyn et al., 2015). Birds have a typical arrangement of the respiratory system, with flow-through lungs and bronchi that are connected with the air sacs via ostia. In contrast with a bronchoalveolar lavage in mammals, the used technique in this study was based on flushing the ex vivo lungs. The flush solution, heparin-saline, was distributed in and immediately flushed out of the lung through the ostia. These differences with the mammalian respiratory tract have an influence on the collection method for PELF. CONCLUSION FF was very rapidly absorbed and distributed to the lung tissue after a single oral bolus administration in turkeys. However, FF as such is not applicable for drinking water medication, as the water solubility is very low (only 1 mg/mL), which implies an elaborate preparation, including stirring and sonication, of the medicated drinking water. Therefore, efforts should be made to develop and register a soluble pharmaceutical FF formulation for poultry. Acknowledgements The authors wish to thank Jelle Lambrecht* and Matteo Stecca‡ for their excellent laboratory assistance. 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Poultry ScienceOxford University Press

Published: Apr 1, 2018

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