Pharmacokinetics of oral ethanamizuril solution in chickens

CHENG Pei-pei, HU Xing-xing, WANG Chun-mei, LIU Ying-chun, WANG Mi, ZHANG Ke-yu, FEI Chenzhong, ZHANG Li-fang, WANG Xiao-yang, ZHENG Wen-li, XUE Fei-qun

Key Laboratory of Veterinary Chemical Drugs and Pharmaceutics, Ministry of Agriculture and Rural Affairs/Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, P.R.China

Abstract Ethanamizuril (EZL) is a novel triazine anticoccidial compound that has high anticoccidial activity in chickens. In order to treat coccidiosis rationally in poultry, a detection method was developed for ethanamizuril in broiler plasma, and then the pharmacokinetics studies were performed in broilers after oral administration of different dose levels. Ethanamizuril was administered as single oral doses at low (0.67 mg kg–1 BW), medium (1.33 mg kg–1 BW) and high (6.67 mg kg–1 BW) levels in which the medium dose was that recommended in clinics. Plasma concentrations of ethanamizuril were determined using ultra-high performance liquid chromatography and the data were analyzed with a non-compartmental model. Peak plasma concentrations of ethanamizuril were (2.16±0.57), (3.91±0.71), and (23.71±5.02) mg L–1 at (5.17±1.80), (4.60±2.12), and(4.60±2.12) h, respectively. The terminal elimination half-lives (t1/2λz) for ethanamizuril were (10.84±2.59), (10.66±2.47), and(13.34±3.10) h after oral administration at low, medium and high doses, respectively. The areas under the concentrationtime curve (AUC0–t) were (37.68±6.87), (73.19±9.18), and (485.76±125.10) mg L–1 h with mean residence times (MRT0–t)of (14.79±3.03), (15.57±3.69), and (20.22±4.01) h at the 3 dosages, respectively. Ethanamizuril was absorbed rapidly and eliminated slowly. A comparison across the dose range indicated that the time to reach peak plasma concentration (Tmax)values were similar while peak plasma concentration (Cmax) and AUC0–t were positively correlated with increasing dosages.This study of the pharmacokinetics of an ethanamizuril solution in chickens provides a theoretical basis for the rational use in the clinic.

Keywords: ethanamizuril solution, coccidiosis, pharmacokinetics, chickens

1. Introduction

Chicken coccidiosis is caused by the apicomplexan protozoaEimeriaspp., a single-celled intracellular parasite. This organism severely impairs chicken development and adversely affects the global poultry industry (Clarket al.2008; Yanget al. 2008). The total cost of coccidiosis is estimated to be nearly US$ 3 billion per annum (Blake and Tomley 2014). The discovery of sulfanilamide in 1939 for the treatment of avian coccidiosis was the flrst step in the development of more than 30 anticoccidial drugs. However,this development has come at the cost of increasingEimeriaresistance (Levine 1939). Therefore, novel anticoccidial drugs are urgently need to develop to control the chicken coccidiosis and ease the drug resistance problem.

Ethanamizuril (EZL) is a novel triazine anticoccidial compound that was independently researched and developed at the Shanghai Veterinary Research Institute,Chinese Academy of Agriculture Sciences (CAAS). Its structure is similar to diclazuril and toltrazuril. Preclinical studies have shown that EZL is safe and possesses high anticoccidial activity with an anticoccidial index(ACI) above 180. The effects of EZL on the endogenous phases ofEimeria tenellaindicated that it primarily targets schizogenesis and gametogenesis (Liuet al. 2016). To facilitate the clinical use of EZL, we developed an oral EZL solution where the drug was combined with a solubilizer,solvent and adjuvant (unpublished data). We studied the pharmacokinetics of this oral EZL solution according to the Guidelines for the Research of Veterinary Chemical Drug and Clinical Pharmacokinetics (CVDE 2012).

2. Materials and methods

2.1. Reagents and chemicals

EZL (purity＞99%, no. 20140401) and oral EZL solution (10 mg mL–1) were prepared by the Shanghai Veterinary Research Institute, CAAS. Toltrazuril sulfoxide standard (purity(99.4±0.2)%) was purchased from Witega Laboratorien(Berlin, Germany). HPLC grade methanol and acetonitrile were purchased form Fisher Scientiflc (Fair Lawn, NJ, USA).

2.2. Experimental animals

Pudong yellow broiler chickens weighing (1.27±0.16) kg were purchased from a local hatchery and acclimated to their new environment for one week prior to the start of the experiments. The animals were caged withad libitumaccess to water and drug-free feed. The protocol conformed to the guidelines of Institutional Animal Care and Use Committee of China and was approved by the Ethics Committee of the Faculty of Veterinary Medicine (Shvriro-2014120884).

2.3. Experimental design and sample collection

The chickens were divided into 3 groups at random in 12 chickens per group. Oral EZL solution was diluted immediately with water at 0.33, 0.67 and 3.33 mg mL–1before oral administration. Feed was withheld from the animals 12 h before drug administration. EZL was administered orallyviastomach tubes at 0.67, 1.33 and 6.67 mg kg–1BW. Blood (0.5 mL) was collected from the wing vein into 2 mL heparinized tubes at 0, 0.5, 1, 2, 4, 6, 8, 12, 24, 36,48, 72, 96, and 120 h after administration. Blood samples were centrifuged at 3 000 r min–1for 10 min to separate the plasma, which was stored at –20°C until analysis.

2.4. Standard solutions

EZL standard solution was prepared in methanol at 2 019. 20 μg mL–1, and then serial diluted in methanol to prepare EZL standard solutions of 403.84, 201.92, 40.38, 10.09,4.00, 1.00 and 0.40 μg mL–1. Samples of these solutions(10 μL) were used to construct calibration curves after spiking blank plasma to flnal concentrations of 0.04, 0.10,0.40, 1.00, 4.00, 20.19 and 40.38 μg mL–1. Toltrazuril sulfoxide (TSO) standard solution was diluted in methanol to a flnal concentration of 203.20 μg mL–1and 10 μL of this was used as an internal standard. A heparin sodium solution was prepared at 1% heparin in saline. All of the working solutions were stored at 4°C and stabled for 1 month.

2.5. Sample preparation

Plasma samples were extracted according to previously described methods (Maet al. 2014; Zhanget al. 2014; Zhaoet al. 2017). Briefly, 1 mL ethylacetate was added in 1.5-mL tubes that contained 100 μL plasma and 10 μL toltrazuril sulfoxide internal standard. The tubes were vortexed for 1 min and then centrifuged at 14 800 r min–1for 10 min.The supernatant was dried under a stream of nitrogen in a 60°C water bath. The residue was dissolved using 100 μL ultra-high performance liquid chromatography (UPLC)initial mobile phase solvent of acetonitrile:water (10:90,v/v), vortexed for 1 min and then flltered through a 0.22-μm membrane fllter (Pall, Port Washington, NY, USA). A total of 10 μL of this solution was used for UPLC-UV analysis.

2.6. Chromatographic conditions

The samples were analyzed by UPLC-UV using an Acquity UPLC System (Waters, Millipore, Medford,MA, USA). The chromatographic column was an Acquity UPLC BEH C18 column (2.1 mm×100 mm,1.7 μm; Waters, Millipore, USA) equipped with a Van Guard BEH C18 pre-column (2.1 mm×5 mm,1.7 μm; Waters, Millipore, USA) maintained at 30°C. The mobile phase was acetonitrile (A) and water (B) and mixed under gradient conditions at a flow rate of 0.2 mL min–1and a detection wavelength of 251 nm. The gradient conditions were as follows: 0 min, 10% A; 0–2 min, 10–50% A; 2–7 min,50–80% A; 7–7.2 min, 80–10% A; 7.2–10 min, 10% A.

2.7. Method validation

The method was validated using the Guidelines for the Research of Veterinary Chemical Drug and Clinical Pharmacokinetics (CVDE 2012) and the US FDA Guidance for Industry Bioanalytical Method Validation (US FDA 2001).The limits of detection (LOD) and limits of quantiflcation(LOQ) of EZL were analyzed by measuring the signal to noise ratios (S/N) in blank matrices (n=6) at 3 and 10 times the baseline values, respectively. The calibration curve was established using spiked blank plasma containing 10 μL EZL standard solution and 10 μL toltrazuril sulfoxide internal standard. This method was linear from 0.04 to 40.38 μg mL–1. Accuracy was expressed as recovery of spiked samples and precision was expressed using withinrun and between-run tests. Three EZL concentration levels(high, 20.19 μg mL–1; medium, 4.04 μg mL–1; low, 0.04 μg mL–1) were prepared on 3 d by different operators. Each concentration included 5 replicates. The stability of the analyte was studied with short-time temperature (room temperature for 8 h) and freeze-thaw (3 cycles) stability tests at 2 concentration levels, 20.19 and 4.04 μg mL–1.

2.8. Pharmacokinetic analysis

The plasma concentration time curve data were analyzed using the pharmacokinetic software DAS 3.0 (WSO2,Mountain View, CA) with a non-compartmental model to obtain pharmacokinetic parameters. The peak plasma concentrations (Cmax) and times to reach peak concentrations (Tmax) were calculated according to individual plasma concentration-time curves. Lambda z (λz) is a flrstorder elimination rate constant associated with the terminal segment of the curve. It was calculated by the linear regression of the terminal data points. The elimination halflife was calculated by the equation t1/2λz=0.693/λz. The area under the concentration-time curve (AUC) was calculated using a linear trapezoidal method. The mean residence time(MRT) was calculated by the equation MRT=AUMC/AUC.Volume of distribution (VZ/F) was the volume of distribution to the fraction of the dose absorbed. The clearance (ClZ/F)was the clearance to the fraction of dose absorbed. The area under the flrst moment curve (AUMC) was deflned as the area under the product of the time and drug concentrationtime curve. It was calculated using the trapezoidal rule and integral methods.

2.9. Statistical analysis

The results were represented as mean±standard deviation(SD). The pharmacokinetic parameters between dosage levels were statistically analyzed using the software of SPSS statistics 22.0 (SPSS Inc., Chicago, IL, USA). Oneway ANOVA followed by Tukey’s multiple comparison tests was applied for group comparison. A value ofP≤0.05 was considered statistically signiflcant.

3. Results

The chromatographic conditions used in this study resulted in EZL and toltrazuril sulfoxide retention times of 3.28 and 4.32 min, respectively. We found no interference in the blank samples (Fig. 1). The LOD and LOQ values for EZL were 0.02 and 0.04 μg mL–1, respectively. We established a calibration curve from 0.04 to 40 μg mL–1that was linear with a regression equation ofY=0.1579X+0.0145 (R2=0.9998) where,Xequals the EZL concentrations andYis the peak area ratio of EZL to the toltrazuril sulfoxide (TSO) (Fig. 2-A). Within-run and between-run precision values were less than 15% and the average EZL recovery rate ranged from 85 to 115% (Table 1).The mean recovery of short-time temperature and freezethaw stability of EZL ranged from 85 to 105% (Table 2).These results indicated that our detection method met the methodology requirements for EZL detection.

We calculated pharmacokinetic parameters at low,medium and high dosages. AUC0–twere (37.68±6.87),(73.19±9.18) and (485.76±125.10) mg h L–1, while Cmaxwere (2.16±0.57), (3.91±0.71) and (23.71±5.02) mg L–1at (5.17±1.80), (4.6±2.12) and (4.6±2.12) h, respectively.The t1/2λzvalues fell from (10.84±2.59) to (13.34±3.10) h with an MRT0–trange of (14.79±3.03) to (20.22±4.01) h(Table 3). The linear relationship of the dose and CmaxwasY=3.6175X–0.564 withR2=0.9993. The relationship between the dose and AUC0–twasY=75.28X–19.434 withR2=0.9992 (Fig. 2-B and C). The plasma concentration-time curves are shown in Fig. 3.

4. Discussion

Triazine drugs that are used to treat chicken coccidiosis include diclazuril, toltrazuril and the toltrazuril metabolite,toltrazuril sulfone, also known as ponazuril. Diclazuril and toltrazuril have been used to treat coccidiosis in chickens,ducks, pigeons, quail, rabbits, piglets, mice, common carp, dogs, cattle and sheep (Van Reeth and Vercruysse 1993; Monlnar and Ostoros 2007; Mundtet al. 2007;Sokolet al. 2014; Stocket al. 2018). They have also been used to cureNeospora caninuminfections in mice and cattle, mouse toxoplasmosis, horse equine protozoal myeloencephalitis andIchthyophthirius multifiliisin rainbow trout (Oncorhynchus mykiss) and chubs (Leuciscus cephalus) (Lindsayet al. 1995; Bentzet al. 1998; Furr and Kennedy 2000; Strohbuschet al. 2009; Ghadaet al. 2014;Oz and Tobin 2014; Qianet al. 2015). The structures of EZL,diclazuril and toltrazuril are similar and they are all triazines drugs with signiflcant anticoccidial effects. Therefore,EZL may be active against other apicomplexan protozoan parasites and this was worth to further study.

Fig. 1 Sample chromatograms. A, blank plasma. B, spiked samples at 1 μg mL–1 ethanamizuril (EZL) and 20.32 μg mL–1 toltrazuril sulfoxide (TSO). C, practical sample collected 1 h after oral administration of the EZL solution at a dose of 1.33 mg kg–1 BW.

Fig. 2 Linear relationships. A, calibration curves. B, dose and peak plasma concentrations (Cmax) relationship. C, dose and area under the concentration-time curve from time zero to the last measured time point (AUC0–t) relationship. EZL, ethanamizuril; TSO,toltrazuril sulfoxide.

We study the pharmacokinetic of oral EZL solution in chickens at 0.67, 1.33 and 6.67 mg kg–1BW. The low dosage (0.67 mg kg–1BW) was the lowest effective dose of EZL in chickens, the medium dosage (1.33 mg kg–1BW) was the recommended dose of EZL in chickens, and the high dosage (6.67 mg kg–1BW) was 5 times of the recommended dose. The pharmacokinetic parameters in Table 3 suggested that the Tmaxand t1/2λzwere similar between dosage levels while Cmaxand AUC0–twere positively correlated with increasing dose. EZL in chickens have linear pharmacokinetic characteristics, which provided valuable reference for clinical rational application. The time to reach peak plasma concentration were (5.17±1.80), (4.60±2.12),and (4.60±2.12) h, and the terminal elimination half-lives(t1/2λz) for ethanamizuril were (10.84±2.59), (10.66±2.47),and (13.34±3.10) h after oral administration at low, medium and high doses, respectively, which suggested that the oral EZL solution was rapidly absorbed and slowly eliminated in chickens. In a preliminary study, EZL was given to chickens with PEG 200 at 0.45, 0.9, 4.5 mg kg–1and Cmaxwas reached at (1.3±0.35), (2.79±0.53), (12.80±2.65) mg L–1at(4.67±2.05), (3.20±1.35) and (4.40±1.24) h, respectively. The t1/2λzvalues were (17.60±6.12), (20.09±9.05), (17.71±3.61) h with AUC0–tvalues of (27.16±8.48), (57.20±16.92) and(309.73±84.94) mg L–1h, respectively (Chenget al. 2018).Compared with the pharmacokinetics of the PEG 200 solution of EZL, the t1/2λzof the oral EZL solution was shorter but other pharmacokinetic characteristics were similar.

Table 1 Accuracy and precision of the UPLC-UV method for ethanamizuril (EZL) detection in chicken plasma

Table 2 Short-term room temperature stability and freeze-thaw stability

Table 3 The pharmacokinetic parameters of ethanamizuril (EZL) solution in chickens analyzed using a non-compartmental model(n=12)

Fig. 3 Plasma concentration-time curves of the ethanamizuril(EZL) solution in chickens after oral administration at 0.67, 1.33 and 6.67 mg kg–1 BW. Error bars are standard deviation (SD).

The pharmacokinetics of diclazuril, toltrazuril as well as toltrazuril sulfone have been previously studied in many animal species, especially in chickens (Stocket al. 2018).Due to the similar structure of EZL, diclazuril and toltrazuril,they might have similar metabolism in chickens. Then, we compared the pharmacokinetic parameters of diclazuril,toltrazuril, and EZL in chickens. Diclazuril administered to chickens and turkeys reached peak plasma concentrations at 6 h after a single oral dose of 1 mg kg–1with elimination half-lives of 50 and 38 h, respectively (Meuldermanset al.1988, 1990). Plasma concentrations of toltrazuril in chickens reached the peak plasma concentrations of (18.04±5.80)and (47.15±9.40) μg mL–1at (4.33±1.51) and (3.67±1.15) h after single oral doses of 10 and 40 mg kg–1, respectively.The t1/2λzvalues were (11.40±4.68) and (11.64±4.08) h and the area under the curve values were (209.46±44.89)and (869.98±226.90) μg h mL–1, respectively (Limet al.2007). A similar study with toltrazuril in chickens after single oral doses of 10 and 20 mg kg–1found toltrazuril Cmaxlevels of (16.4±2.0) and (25.2±1.5) μg mL–1at (5.0±1.2)and (4.7±1.0) h, elimination half-lives of (10.6±1.2) and(10.7±1.6) h, and AUC values of (0.3±0.1) and (0.4±0.1)mg h mL–1, respectively (Kimet al. 2013). These studies demonstrated that Tmaxand t1/2λzwere similar between EZL,toltrazuril and diclazuril in chickens and the pharmacokinetic characteristics of triazine in chickens were similar.

The results of toxicological experiments and environmental toxicology tests have shown that EZL was safe. The EZL residue test in chickens has been studied according to the guideline of US FDA and EMEA (US FDA 2012; EMEA 2012, 2015). We have established the withdrawal time of EZL in chickens at the recommended dosage (unpublished data). EZL is safe to humans and animals based on the recommended doses and the withdrawal periods.

5. Conclusion

This paper presented the pharmacokinetics of oral EZL solution in chickens. In this preparation, EZL was absorbed rapidly and eliminated slowly in chickens. The pharmacokinetic characteristics were similar to EZL when combined with PEG 200. This study provides a reference for applying oral EZL solution in the clinic. EZL has broad application prospects but further pharmacokinetic studies in other animal species are required.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (31472235 and 31272607), the Natural Science Foundation of Shanghai, China (14ZR1449000),the Special Scientiflc Research Fund of Agricultural Public Welfare Profession of China (201303038), the Central Grade Public Welfare Fundamental Science Fund For Scientiflc Research Institute, China (2016JB08 and 2016JB04) and the National Key Technology Research and Development Program of the Ministry of Science and Technology of China(2015BAD11B01-06).