Potential pharmacokinetic effect of rifampicin on enrofloxacin in broilers: Roles of P-glycoprotein and BCRP induction by rifampicin

Potential pharmacokinetic effect of rifampicin on enrofloxacin in broilers: Roles of P-glycoprotein and BCRP induction by rifampicin Mengjie Guo,∗,† Xiaohua Dai,∗,‡ Dongmin Hu,∗ Yu Zhang,∗ Yong Sun,∗ Weilong Ren,∗ and Liping Wang∗,1 ∗

Laboratory of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu Province, 210095, PR China; † Center for Safety Evaluation of Drugs, Science and Technology Division, Nanjing University of Traditional Chinese Medicine, Nanjing 210029, China; and ‡ College of Food Science and Pharmacy, Xinjiang Agricultural University, Urumqi 830052, China

Key words: P-gp, BCRP, broilers, rifampicin, induction 2016 Poultry Science 0:1–7 http://dx.doi.org/10.3382/ps/pew148

INTRODUCTION

now there is scanty information available for veterinary clinicians to consider drug-drug interactions associated with ABC transporters in order to assist them in their decision paradigms. P-gp can modulate the pharmacokinetics of drugs resulting in drug-drug interactions of different compounds (Cox et al., 2002; Lin and Yamazaki, 2003; Endres et al., 2006). Strong evidence shows that expression and activity of P-gp can be inhibited or induced by dietary components, hormones, and several xenobiotics (Barnes, 2001; Kim and Benet, 2004; Petropoulos et al., 2010). BCRP also play an important role in drug interactions, as it is involved in drug absorption and disposition (Kruijtzer et al., 2002; Endres et al., 2006). Rifampicin has been shown not only to induce intestinal cytochrome P450 3A4 enzyme but also to elicit a significant increase of intestinal P-gp in humans and rodents (Westphal et al., 2000; Kota et al., 2010). A distinct pregnane X receptor (PXR) binding site, DR4 nuclear receptor response element, has been identified and shown essential for Abcb1 induction by rifampicin

P-glycoprotein (P-gp, encoding gene Abcb1) and Breast Cancer Resistance Protein (BCRP, encoding gene Abcg2), belonging to ATP binding cassette (ABC) transporters, play a key role in modulating the bioavailability of oral drugs in humans and rodents (Zhang and Benet, 2001; Daood et al., 2008; Hua et al., 2012). Expression of P-gp in various organs of farm animals is reported worldwide (Barnes, 2001; Tyden et al., 2009; Ballent et al., 2013; Guo et al., 2013; Hasibu et al., 2014). Some drugs used in veterinary medicine, including ivermectin, erythromycin, and fluoroquinolones, are substrates for P-gp (Sikri et al., 2004; Eriksson et al., 2006; el-Ashmawy et al., 2011). Therefore, the relevance for understanding the roles of P-gp and BCRP transporters in veterinary medicine is evident; however, up to  C 2016 Poultry Science Association Inc. Received December 8, 2015. Accepted March 7, 2016. 1 Corresponding author: [email protected]

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rifampicin. Further analysis revealed that the variation tendencies of Abcb1, Abcg2, and CYP3A37 expression levels were significantly correlated with CXR mRNA expression levels in liver, kidney, jejunum, and ileum. Coadministration of rifampicin significantly changed the pharmacokinetic behavior of enrofloxacin orally administered by showing clearly lower AUC0-∞ , AUC0-t , and Cmax as well as longer Tmax. The bioavailability of orally administered enrofloxacin was decreased from 72.5% to 24.8% by rifampicin. However, rifampicin did not significantly change the pharmacokinetics of enrofloxacin following intravenous administration. Our study shows that rifampicin up-regulated the small intestinal level of P-gp and BCRP and suggests that P-gp and BCRP are key factors that affected pharmacokinetic behavior of orally administered enrofloxacin by limiting its absorption from the intestine in broilers.

ABSTRACT P-glycoprotein (P-gp, encoding gene Abcb1) and Breast Cancer Resistance Protein (BCRP, encoding gene Abcg2) are transport proteins that play a major role in modulating the bioavailability of oral drugs in humans and rodents. It has been shown that rifampicin is the typical inducer of P-gp in rodents by activating the nuclear receptor. However, its effect on Abcb1, Abcg2, CYP3A, and chicken xenobiotic-sensing orphan nuclear receptor (CXR) mRNA expression in broilers is poorly understood. This study explored the effect of rifampicin on mRNA expression of Abcb1, Abcg2, CYP3A37, CXR as well as its effect on the pharmacokinetics of enrofloxacin in broilers. The mRNA levels of Abcb1, Abcg2, CYP3A37, and CXR were significantly increased in the liver (except Abcg2), kidney, jejunum, and ileum (P < 0.05) but not significantly changed in the duodenum (P > 0.05) after treated with

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MATERIALS AND METHODS Animals and Reagents Ross 308 broilers (1-day-old, male and female randomly) were purchased from a local commercial poultry farm (Nanjing, China). All birds were kept at 25 ◦ C, had free access to a standard commercial feed (without additives) and water. All birds were treated following the protocol approved by Nanjing Agricultural University Animal Care and Use Committee (SYXKSu 2011-0036). Rifampicin was purchased from Sigma (St. Louis, MO), the purity of which was more than 97%. The analytical standard of enrofloxacin used for method validation was bought from China Institute of Veterinary Drug Control and enrofloxacin hydrochloride (ENRO, bulk drug) used for administration was purchased from Shangyu Jingxin Pharmaceutical Co., Ltd. All the other highest quality reagents were bought commercially.

male). One group remained untreated and served as a control, another group was pretreated with rifampicin (single oral administration 500 mg for each bird). Chickens were slaughtered by carbon dioxide asphyxiation machine after rifampicin administered 15 hours. Tissue sampled from all birds included the liver, kidney, duodenum, jejunum, and ileum. All samples after dissection were snap-frozen in liquid nitrogen and stored at −70◦ C until further analysis. Total RNA was isolated from each sample according to the previous method (Guo et al., 2013). Briefly, frozen tissue samples (60 ∼ 80 mg) were homogenized in 1 mL of extract Trizol reagent (Takara, Tokyo, Japan). Samples were then centrifuged for 5 min at 12,000 ×g at a temperature setting of 4 ◦ C. The supernatant was added to 300 μL of chloroform and mixed vigorously using a vortex mixer for 15 s and centrifuged for 20 min. A 400-μL aliquot of the upper aqueous phase was taken out and mixed with an equal volume of isopropanol. Samples were placed at −20 ◦ C for at least 30 min and were then centrifuged at 4 ◦ C, and 12,000 × g for 20 min. The pellets were washed twice with 75% alcohol, dissolved in DEPC (diethyl pyrocarbonate) water and RNA concentrations were determined by optical density values set at wave of 260 and 280 nm using Nanodrop 2000 (Thermo, Waltham). All RNA samples were stored at −80 ◦ C. Single-stranded cDNAs were synthesized from 2 μg total RNA using the M-MLV (Promega, Madison) and stored at −20◦ C until analysis by real-time reverse transcription polymerase chain reaction (RT-PCR). β -actin was chosen as a housekeeping gene for liver and kidney, while villin served as a housekeeping gene for small intestine. Primer pairs specific for P-gp, CXR, CYP3A37, and housekeeping gene β -actin were designed as previously described (Guo et al., 2014). Primer pairs specific for BCRP and villin were designed according to the following references (Su et al., 2014). Real-time RT- PCR was performed using CFX (BioRad, California, America). Each assay included a negative control without a cDNA template. All samples were run in duplicate. Cycling parameters utilized were as follows: denaturing at 94◦ C for 5 min, degenerating at 94◦ C for 30 s, annealing at 58◦ C for 30 s, and elongating at 72◦ C for 30 s for a total of 30 cycles. The 2−ΔΔCt method (Livak and Schmittgen, 2001) was used to analyze the real-time RT-PCR data. Values of mRNA abundance were expressed as the fold change relative to the average value of one group. The statistical method for calculating Spearman coefficients was used to analyze the correlation between genetic changes for each treatment.

Sample Collection and Reverse Transcription Polymerase Chain Reaction Analysis

Pharmacokinetic Analysis of Enrofloxacin in Broilers

Twelve 42-day-old chickens were divided into 2 groups (6 chickens per group, half male and half fe-

Twenty 42-day-old birds were chosen to be randomly allocated among 4 treatment groups (3 male and

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in the human colon carcinoma cell line LS174T (Kota et al., 2010). The effect is similar to Abcg2 in intestine and liver (Albermann et al., 2005).The chicken genome does not include PXR or constitutive androstane receptor (CAR) genes like mammals; however, it does have the chicken X receptor (CXR) gene, which appears homologous and has functional similarity to the PXR and CAR. The CXR acts as the main xenobiotic-sensing nuclear receptor in the chicken (Handschin et al., 2000; Handschin et al., 2001). Considering the species difference, whether rifampicin has an effect on P-gp and BCRP gene expression via inducing CXR expression in chicken remains unknown. Additionally, evidence suggests that the pharmacokinetics of enrofloxacin, commonly used in veterinary clinics, is associated with the expression level of P-gp in broilers and BCRP in dairy animals (Pulido et al., 2006; Guo et al., 2014). Recent results of our experiments indicated that rifampicin affects the transport of enrofloxacin in Caco-2 cells (in press). The extralabel use of rifampicin is common in the poultry industry in China for the treatment of intestinal infection caused by E. coli (Chen, 2006). Nonetheless, little is known about whether rifampicin influences disposal of enrofloxacin by modulating the expression of P-gp, BCRP, and CYP3A in broilers. Therefore, we aimed to evaluate the effect of rifampicin on the mRNA expression of P-gp, BCRP, CYP3A and CXR and assessed the expected interaction between rifampicin and enrofloxacin.

P-GP- AND BCRP-INDUCED INTERACTION BETWEEN ENROFLOXACIN AND RIFAMPICIN

HPLC Method for Detection of Enrofloxacin in Plasma An Agilent 1200 high-performance liquid chromatography (HPLC) system was utilized to detect plasma concentrations of enrofloxacin as previously reported (Guo et al., 2013). The mobile phase comprised 950 mL phosphate-triethylamine buffer (pH = 3.5) and 167 mL acetonitrile. A C18 reversed-phase column (4.6 × 250 mm) was used for separation. The UV detector was set at 278 nm and the flow rate of the mobile phase was 1.2 mL/min. Twenty microliters of the sample was injected into the HPLC. Drugs were quantified by measuring the peak area. The method validation was performed using the following parameters: limit of detection (LOD, estimated as 3 times noise value), limit of quantification (LOQ, estimated as 10 times noise value), precision, recovery, correlation coefficients of the calibration curves. The precision was evaluated by intra-day and interday precision for 3 d at concentrations of 0.05, 1, and 10 μg/mL. The accuracy was evaluated by the rate of recovery by adding 3 standard levels (0.05, 1, and 10 μg/mL). The stability of enrofloxacin was measured by precision under 4 conditions as follows: short-term placement at room temperature (25◦ C,4 h), repeated freezing and thawing 3 times, long-term placement (−20◦ C, 2 weeks) and placement at room temperature before injection (25◦ C, 13 h). Each concentration

of parallel processed 5 samples. The standard curve of enrofloxacin was prepared at concentrations of 0.02, 0.1, 0.2, 0.5, 1, 2 and 5 μg/mL with each parallel processing 5 samples. Pharmacokinetic parameters were calculated using non-compartmental analysis with a computer program (WinNonlin 6.1, Phoenix Software, Los Angeles, California). The systemic bioavailability (F, %) of enrofloxacin after oral administration was determined as follows: F = AUC0-∞ p.o. ·Dosep.o. /AUC0-∞ i.v. ·Dosei.v. × 100%, where AUCp.o. and AUCi.v. are the areas under the plasma concentration curves.

Data Analysis All data were presented as mean ± S.E.M. Student’s t test was conducted for the comparison between groups with a significance level of P < 0.05.

RESULTS Effect of Rifampicin on mRNA Expression Levels of Abcb1, Abcg2 and CYP3A37 in Liver, Kidney, and Small Intestines After treatment with rifampicin, the expression of Abcb1, Abcg2 and CYP3A37 were detected in different tissues. Compared to the controls, Abcb1 mRNA was significantly up-regulated in the liver (P = 0.001), kidney (P = 0.008), jejunum (P = 0.012), and ileum (P = 0.003) after treatment with rifampicin. The duodenum, however, did not show significant differences (P > 0.05) (Figure 1A). Similarly, the CYP3A37 level was also significantly increased in the liver (P = 0.000), kidney (P = 0.021), jejunum (P = 0.029), and ileum (P = 0.039) in rifampicin pretreated group (Figure 1B). The Abcg2 level was significantly increased in the kidney (P = 0.018), jejunum (P = 0.017), and ileum (P = 0.007), with no effects shown by the liver and duodenum in the rifampicin-pretreated group (Figure 1C). The mRNA expression of Abcg2 after treatment with rifampicin also significantly increased in kidney and small intestines, however, it was not significantly increased in the liver (P > 0.05). The levels of housekeeping gene mRNAs did not differ between the 2 experimental groups.

Effect of Rifampicin on mRNA Expression Levels of CXR in Liver, Kidney and Small Intestines As shown in Figure 1D, the CXR mRNA was significantly increased in the liver (P = 0.004) and kidney (P = 0.004), jejunum (P = 0.038), and ileum (P = 0.031). Abcb1 expression level was highly correlated with CXR expression level in the liver (r = 0.827, P = 0001), kidney (r = 0.895, P = 0.000), jejunum (r = 0.666, P = 0.035), and ileum (r = 0.798, P = 0.01). CYP3A37

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2 female per group). The first group received a single dose of 10 mg/kg body weight (BW) of enrofloxacin orally via the crop by tube gavages. The second group was first orally dosed with rifampicin (500 mg for each bird) and 15 hours later, with enrofloxacin (10 mg/kg BW). The third group received a single dose of 10 mg/kg BW of enrofloxacin intravenously (i.v.) utilizing the left brachialis vein. The fourth group was first orally dosed with rifampicin (500 mg for each bird) and fifteen hours later, enrofloxacin was administered intravenously (10 mg/kg BW). Blood samples were collected prior to enrofloxacin administration and at each time point of 0.083, 0.25, 0.33, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 12 h, 24 h and 48 h following the final administration of enrofloxacin. Blood samples were immediately centrifuged at 3,000× g for 15 min and the plasma were stored at −20◦ C until analysis. The extract method for enrofloxacin from the plasma was according to our previous work (Guo et al., 2013). The method was briefly as follows: the frozen sample were removed from the refrigerator for natural thawing and centrifuged at 2,000× g for 5 min. 0.5 mL of the supernatant and 2 mL of acetonitrile were vortexed for 2 min followed by centrifugation to separate the organic and water phases. A nitrogen evaporator was used to make the supernatant dry. After that the residue was resuspended with mobile phase solution. The mixture (200 μL) was collected and analyzed by HPLC.

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expression level also showed highly significant correlation with CXR expression levels in liver (r = 0.744, P = 0.006), kidney (r = 0.719, P = 0.008), jejunum (r = 0.814, P = 0.008), and ileum (r = 0.821, P = 0.024). A similar result was occurred between Abcg2 and CXR. Abcg2 expression levels showed significantly high correlation with CXR expression levels in the kidney (r = 0.850, P = 0.002), jejunum (r = 0.941, P = 0.000), and ileum (r = 0.635, P = 0.048). It suggests there is a strong correlation between Abcb1 and CYP3A37 mRNA expression with the CXR level.

Effect of Rifampicin on Pharmacokinetics of Orally and I.V. Administered Enrofloxacin The mean plasma concentration-time profiles of enrofloxacin (10 mg/kg BW) orally administered alone or coadministered with rifampicin (500 mg for each bird) are shown in Figure 2 and the relevant pharmacokinetic parameters are listed in Table 1. The combination enrofloxacin/rifampicin caused significant changes in the pharmacokinetic behavior of enrofloxacin in broilers via

Method Validation for Detection of Enrofloxacin The LOQ and LOD of enrofloxacin was 0.05 and 0.02 μg/mL, respectively. After detection for the 3 concentrations of enrofloxacin for 3 d under the experimental conditions, the intra-day and inter-day coefficients of variation were less than 11%. The recovery rates of 3 concentrations of enrofloxacin were all more than 81%. In the stability test, the precision of enrofloxacin were all less than 15% under 4 different conditions, which comply with the testing requirements for biological samples. The correlation coefficients for the calibration curves were 0.9997.

Figure 2. Plasma concentration-time profiles of enrofloxacin after single oral administration at 10 mg/kg BW alone and coadministered with rifampicin (500 mg for each bird, single oral administration). Data represent mean ± S.E.M (n = 5).

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Figure 1. Expression of Abcb1 (A), CYP3A37 (B), Abcg2 (C), and CXR (D) mRNA in broilers with and without rifampicin, as detected by real-time RT PCR. β -actin and villin were used as reference genes for normalization (n = 6). ∗∗ (P < 0.01) and ∗ (P < 0.05) difference between control and rifampicin-treated broilers.

P-GP- AND BCRP-INDUCED INTERACTION BETWEEN ENROFLOXACIN AND RIFAMPICIN

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Table 1. Pharmacokinetic parameters of enrofloxacin orally administered in broilers (mean ± S.E.M., n = 5). Parameters Tmax (h) Cmax (μ g/mL) AUC0-t (h·μ g/mL) AUC0-∞ (h·μ g/mL) t1/2λ (h) Vz/F (L/kg) Cl /F (L/h/kg) MRT0-∞ (h) F (%)

ENRO

ENRO+RFP

1.80 ± 0.20 2.07 ± 0.21 27.29 ± 2.51 30.57 ± 1.97 16.5 ± 2.22 8.15 ± 1.49 0.33 ± 0.02 21.2 ± 2.14 72.5%

5.75 ± 1.03∗∗ 0.75 ± 0.12∗∗ 8.81 ± 2.43∗∗ 9.64 ± 2.59∗∗ 10.3 ± 2.67 15.68 ± 3.77 1.43 ± 0.52 15.6 ± 3.80 24.8%

P-value 0.004 0.002 0.001 0.000 0.114 0.083 0.126 0.221

Table 2. Pharmacokinetic parameters of enrofloxacin intravenously administered in broilers (mean ± S.E.M., n = 5). Parameters AUC0-t (h·μ g/mL) AUC0-∞ (h·μ g/mL) t1/2λ (h) Vz/F(L/kg) Cl/F (L/h/kg) MRT0-∞ (h)

ENRO 39.08 42.18 15.84 5.42 0.24 15.44

± ± ± ± ± ±

1.48 1.84 1.47 0.45 0.01 1.20

ENRO+RFP

P-value

± ± ± ± ± ±

0.575 0.425 0.071 0.229 0.361 0.471

36.95 38.83 11.90 4.52 0.26 14.26

3.28 3.45 1.03 0.50 0.02 0.94

oral administration. The lower enrofloxacin plasma concentrations over the whole drug-detection period were observed, and a significant decrease by 2.76-fold in the Cmax (P < 0.05) was displayed in rifampicin-treated group compared with the enrofloxacin alone group. In parallel, the AUC0-∞, and AUC0-t of enrofloxacin was significantly decreased following concomitant administration of rifampicin (3.17-fold and 3.10-fold, respectively, P < 0.05). The differences in Tmax between the 2 groups also achieved statistical significance (P < 0.05). Although the differences in t1/2λ , MRT0-∞, CL/F and Vz/F between the 2 groups did not achieve statistical significance, they tended to be shorter (t1/2λ , MRT0-∞ ) or higher (CL/F and Vz/F) in the presence of rifampicin. In contrast, the parameters and plasma concentration-time profiles of enrofloxacin after i.v. administration were not apparently changed by rifampicin (Table 2, Figure 3). The estimated bioavailabilities of enrofloxacin after oral administrations were 72.5% and 24.8% for alone and coadministered with rifampicin, respectively. It further showed that the presence of rifampicin did not significantly affect the elimination of enrofloxacin from body tissues, even though the expression of the Abcb1 gene was highly expressed in liver and kidney.

Figure 3. Plasma concentration-time profiles of enrofloxacin after single i.v. administration at 10 mg/kg BW alone and administered with rifampicin (500 mg for each bird, single oral administration). Data represent mean ± S.E.M (n = 5).

DISCUSSION A well-documented problem associated with rifampicin usage in humans is its influence on the disposition of other drugs by inducing P-gp and CYP through PXR activation (Greiner et al., 1999; Westphal et al., 2000). Rifampicin is not approved for using in broilers; however, its extra-label use is a common practice in poultry in China (Chen, 2006). The possible pharmacoepigenetic interaction of rifampicin with enrofloxacin may occur, which is of high practical relevance for therapeutics. Therefore, drug-drug interactions between rifampicin and other drugs should be carefully evaluated in veterinary clinics. In our study, it was revealed initially that rifampicin up-regulated P-gp and CYP3A37 gene mRNA in liver, kidney, and some parts of small intestines, and the mRNA changes of both genes were correlated with CXR mRNA in broilers. Similar findings in both in vivo and in vitro studies have shown that rifampicin increases the expression of P-gp and CYP via PXR in human and rodents (Matheny et al., 2004; Kota et al., 2010; Liu et al., 2012). Further experiments with RNA interference to the CXR will be performed to confirm whether or not the relationship remains. Considering that enrofloxacin is the substrate of P-gp (Schrickx and Fink-Gremmels, 2007; Guo et al., 2014), we further investigated the influence of rifampicin on the pharmacokinetics of enrofloxacin in broilers to examine potential drug interactions between them. In this study, plasma concentrations of enrofloxacin (1.79 μg/mL) were measured after single oral administration to broilers at 10 mg/kg bw which confirmed results of past studies (2.44 and 1.88 μg/mL) by Anadon et al. (Anadon et al., 1995) and Knoll et al(Knoll et al., 1999), respectively. We found that Cmax , AUC0-∞, AUC0-t and bioavailability of orally administered enrofloxacin were significantly decreased by coadministration of rifampicin. With respect to intravenously administered enrofloxacin, concurrent administration of rifampicin did not significantly change the parameters of pharmacokinetics in particular t1/2λ , AUC0-∞ and the CL/F.

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∗ P < 0.05, ∗∗ P < 0.01 significant difference between parameters of enrofloxacin in the presence and absence of rifampicin in healthy broilers. Tmax , time to reach peak concentration; Cmax , the peak concentration; AUC0-t , area under the curve up to the last measurable concentration; AUC0-∞ , area under the plasma concentration-time curve from zero to infinity; t1/2λ , the elimination half-life; Vz/F, apparent volume of distribution per fraction of the dose absorbed; Cl/F, plasma clearance per fraction of the dose absorbed; MRT0-∞ , mean residence time; F: bioavailability

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ACKNOWLEDGMENTS The authors would like to thank Professor Kuenzel from University of Arkansas for critically reviewing the manuscript. The authors would like to thank all other laboratory members for their kind assistance during the collection of blood samples. The study was supported in

part by Natural Science Foundation of Jiangsu Province of China (No. BK2012771), Qinlan Project of Jiangsu Province (2014) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

REFERENCES Albermann, N., F. H. Schmitz-Winnenthal, K. Z’Graggen, C. Volk, M. M. Hoffmann, W. E. Haefeli, and J. Weiss. 2005. Expression of the drug transporters MDR1/ABCB1, MRP1/ABCC1, MRP2/ABCC2, BCRP/ABCG2, and PXR in peripheral blood mononuclear cells and their relationship with the expression in intestine and liver. Biochem. Pharmacol. 70:949–958. Anadon, A., M. R. Martinez-Larranaga, M. J. Diaz, P. Bringas, M. A. Martinez, M. L. Fernandez-Cruz, M. C. Fernandez, and R. Fernandez. 1995. Pharmacokinetics and residues of enrofloxacin in chickens. Am. J. Vet. Res. 56:501–506. Ballent, M., M. R. Wilkens, L. Mate, A. S. Muscher, G. Virkel, J. Sallovitz, B. Schroder, C. Lanusse, and A. Lifschitz. 2013. Pglycoprotein in sheep liver and small intestine: gene expression and transport efflux activity. J. Vet. Pharmacol. Ther. 36:576– 582. Barnes, D. M. 2001. Expression of P-glycoprotein in the chicken. Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 130:301–310. Chen, S. S. 2006. The clinic effects of rifampicin on infections caused by E. coli in chicken. Anim. Health. 5:30–31. Collett, A., J. Tanianis-Hughes, and G. Warhurst. 2004. Rapid induction of P-glycoprotein expression by high permeability compounds in colonic cells in vitro: a possible source of transporter mediated drug interactions? Biochem. Pharmacol. 68:783–790. Cox, D. S., K. R. Scott, H. Gao, and N. D. Eddington. 2002. Effect of P-glycoprotein on the pharmacokinetics and tissue distribution of enaminone anticonvulsants: analysis by population and physiological approaches. J. Pharmacol. Exp. Ther. 302:1096–1104. Daood, M., C. Tsai, M. Ahdab-Barmada, and J. F. Watchko. 2008. ABC transporter (P-gp/ABCB1, MRP1/ABCC1, BCRP/ABCG2) expression in the developing human CNS. Neuropediatrics. 39:211–218. el-Ashmawy, I. M., A. F. el-Nahas, and A. E. Bayad. 2011. Teratogenic and cytogenetic effects of ivermectin and its interaction with P-glycoprotein inhibitor. Res. Vet. Sci. 90:116–123. Endres, C. J., P. Hsiao, F. S. Chung, and J. D. Unadkat. 2006. The role of transporters in drug interactions. Eur. J. Pharm Sci. 27:501–517. Eriksson, U. G., H. Dorani, J. Karlsson, H. Fritsch, K. J. Hoffmann, L. Olsson, T. C. Sarich, U. Wall, and K. M. Schutzer. 2006. Influence of erythromycin on the pharmacokinetics of ximelagatran may involve inhibition of P-glycoprotein-mediated excretion. Drug Metab. Dispos. 34:775–782. Greiner, B., M. Eichelbaum, P. Fritz, H. P. Kreichgauer, O. von Richter, J. Zundler, and H. K. Kroemer. 1999. The role of intestinal P-glycoprotein in the interaction of digoxin and rifampin. J. Clin. Invest. 104:147–153. Guo, M., S. Bughio, Y. Sun, Y. Zhang, L. Dong, X. Dai, and L. Wang. 2013. Age-related P-glycoprotein expression in the intestine and affecting the pharmacokinetics of orally administered enrofloxacin in broilers. PLoS One. 8:e74150. Guo, M., Y. Sun, Y. Zhang, S. Bughio, X. Dai, W. Ren, and L. Wang. 2014. E. coli infection modulates the pharmacokinetics of oral enrofloxacin by targeting P-glycoprotein in small intestine and CYP450 3A in liver and kidney of broilers. PLoS One. 9:e87781. Handschin, C., M. Podvinec, and U. A. Meyer. 2000. CXR, a chicken xenobiotic-sensing orphan nuclear receptor, is related to both mammalian pregnane X receptor (PXR) and constitutive androstane receptor (CAR). Proc. Natl. Acad. Sci. U.S.A. 97:10769– 10774. Handschin, C., M. Podvinec, J. Stockli, K. Hoffmann, and U. A. Meyer. 2001. Conservation of signaling pathways of xenobioticsensing orphan nuclear receptors, chicken xenobiotic receptor, constitutive androstane receptor, and pregnane X receptor, from birds to humans. Mol. Endocrinol. 15:1571–1585.

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Accordingly, the decreased oral bioavailability by rifampicin could be mainly due to the limitation of intestinal absorption via P-gp and BCRP induction by rifampicin rather than reduced renal and/or hepatic elimination of enrofloxacin, despite the significantly increased expression of Abcb1 and Abcg2 in liver and kidney by rifampicin. It has been reported that enrofloxacin is metabolized to ciprofloxacin via de-ethylation of the ethyl group on the piperazine ring (Tyczkowska et al., 1989). It is mainly excreted as a parent drug and its metabolite by glomerular filtration and tubular secretion in the kidney, however, the rate of de-ethylation is various in different species (Vancutsem et al., 1990; McKellar and Benchaoui, 1996). Here, we also tried to detect its metabolite ciprofloxacin in plasma, however, following both i.v. and oral administration of enrofloxacin, low concentrations were detected in most plasma samples, in particular after oral administration. It indicates the deethylation rate of enrofloxacin is low in broilers, which is in agreement with the previous results (Knoll et al., 1999). The increased CYP mRNA through CXR modulation by rifampicin has little effect on decreased oral bioavailability of enrofloxacin in this study. More importantly, the findings of the study are the opposite of what has been reported in some literatures, specifically with respect to the short time period after rifampicin treatment on the modulation of P-gp. Noppers et al. (2011) and Kusuhara et al. (2013) independently reported that rifampicin is an inhibitor of Pglycoprotein function after a short time exposure. However, it has been reported that an 8-h pre-treatment with single dose of rifampicin is a sufficient period to induce the metabolism of nifedipine (Ndanusa et al., 1997), which is in line with our findings. An in vitro study also got similar results to this study (Collett et al., 2004). In conclusion, our results are evidence that the concomitant application of rifampicin at the given dose and duration with enrofloxacin caused major interactions in broilers. Specifically, the ability to transport enrofloxacin from the gut into the circulation is greatly decreased, establishing a major role for P-gp and BCRP of the intestine by rifampicin in that process. It is unfortunate that our investigation was limited to the effect of rifampicin on the mRNA expression of P-gp and BCRP in broilers due to a lack of a suitable antibody for detecting chicken P-gp and BCRP through the western blotting method. Further research will be needed to prepare such an antibody to continue this study of chicken P-gp and BCRP protein.

P-GP- AND BCRP-INDUCED INTERACTION BETWEEN ENROFLOXACIN AND RIFAMPICIN

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