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Pharmacokinetics of Quinocetone and Its Major Metabolites in Swine After Intravenous and Oral Administration
Agricultural Sciences in China
August 2011
2011, 10(8): 1292-1300
Pharmacokinetics of Quinocetone and Its Major Metabolites in Swine After Intravenous and Oral Administration ZHONG Jia-lin, ZHANG Gui-jun, SHEN Xiang-guang, WANG Lin, FANG Bing-hu and DING Huan-zhong Laboratory of Veterinary Pharmacology, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, P.R. China
Abstract The pharmacokinetics of quinocetone and its major metabolites in healthy swine was investigated in this paper. Quinocetone was administered to 8 healthy cross-bread swine intravenously and orally at a dosage of 4 and 40 mg kg-1 body weight respectively in a randomized crossover design test with two-week washout period. A sensitive highperformance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the determination of quinocetone and its metabolite 1-desoxyquinocetone in plasma. Plasma concentration versus time profiles of quinocetone and its metabolite 1-desoxyquinocetone were analyzed by non-compartmental analysis using Winnonlin 5.2 software. Mean maximum concentrations (Cmax) for quinocetone was found to be (0.56±0.13) μg mL-1 at 2.92 h, after oral administration of quinocetone. Mean maximum concentrations (Cmax) for 1-desoxyquinocetone after intravenous or oral administration of quinocetone were (0.0095±0.0012) μg mL-1 at 0.083 h and (0.0067±0.0053) μg mL-1 at 3.08 h. The apparent elimination half-lives (T 1/2) for quinocetone and its metabolite 1-desoxyquinocetone were (2.24±0.24) and (5.23±0.56) h after intravenous administration of quinocetone and (2.91±0.29) and (11.85±2.89) h after oral administration of quinocetone, respectively. Mean areas under the plasma concentration-time curve (AUC0- ) for quinocetone and 1desoxyquinocetone were (2.02±0.15) and (0.2±0.002) μg h mL-1 respectively after intravenous administration of quinocetone, and (3.5±0.79) and (0.053±0.03) μg h mL-1 after oral administration of quinocetone, respectively. Quinocetone was rapidly absorbed and metabolized in swine after oral and intravenous administration. The plasma concentration-time curve (AUC0- ) of 1-desoxyquinocetone were much smaller than those of quinocetone, while the elimination half-lives (T1/2) were much longer than those of quinocetone after intravenously (i.v.) or oral administration. Key words: quinocetone, metabolites, pharmacokinetics, HPLC-MS/MS, swine
INTRODUCTION Quinocetone (QCT), 3-methyl-2-cinnamoylquinoxaline1,4-dioxide, is a new synthetic veterinary drug from the quinoxaline-1,4-dioxide family developed by Lanzhou Institute of Animal Husbandry and Veterinary Drugs, Chinese Academy of Agricultural Sciences (Lanzhou, China). QCT has excellent growth-enhanc-
ing activity when used in pigs, chickens and fishes (Xu et al. 1995; Wang et al. 1995; Li et al. 2010). High safety of QCT in animals has also been reported (Wang et al. 1995; Xu et al. 2005; Zhang et al. 2007; Ban et al. 2010). Considering the fact that carbadox (CBX) and olaquindox (OLA) have been banned or allowed only limited use in food animals because of their toxicities, QCT has good prospects in growth promotion in food animals. In fact, QCT is now
Received 30 January, 2011 Accepted 31 May, 2011 ZHONG Jia-lin, MSc, Tel: +86-20-85284896, E-mail: [email protected]; Correspondence DING Huan-zhong, Associate Professor, Ph D, Tel: +86-20-85284896, Mobile: 13751886811, E-mail: [email protected] © 2011, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(11)60121-1
Pharmacokinetics of Quinocetone and Its Major Metabolites in Swine After Intravenous and Oral Administration
broadly used in China to promote animal growth and prevent infection caused by intestinal pathogen in pigs or chickens. However, only recently the metabolism of QCT was intensively studied in animals and the results showed that QCT was extensively metabolized and 31 metabolites were identified in pig urine. The reduction of the N-O group in quinocetone was the main metabolic pathway observed in swine urine (Shen et al. 2010). Nowadays there have been some records about the pharmacokinetic properties of QCT in animals or fish. However, those studies focused only on the parent drug and metabolites of QCT were not included in the experimental process (Li et al. 2002; Liu et al. 2009; Yang et al. 2010). Since QCT were metabolized much rapidly and extensively (Shen et al. 2010), the profile of main metabolites of QCT should also be considered
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in determining pharmacokinetic parameters in animals. On the basis of results from previous in vivo and in vitro studies by the team of the National Basic Research Program of China (973 Program, 2009CB118800), 1-desoxyquinocetone (Q2), dideoxyquinocetone (Q4), and double-bond reduced metabolite of dideoxyquin ocetone (Q33) were recognized as important metabolites of QCT in pigs (Fig. 1). The objective of the present study was to provide further information on the pharmacokinetics of QCT and these three metabolites (Fig. 1) after oral and intravenous administration. Knowledge of pharmacokinetic properties of a parent drug and its important metabolites is necessary for comprehensive evaluation of corresponding kinetic processes and the determination of the withdrawal periods in foodproducing animals. Such data will be essential for judicious use of QCT as an antibacterial agent in swine.
Fig. 1 Chemical structure of QCT, 1-desoxyquinocetone (Q2), dideoxyquinocetone (Q4), and double-bond reduced metabolite of dideoxyquinocetone (Q33).
MATERIALS AND METHODS
(Millipore, MA, USA). Other reagents were A.R. grade and purchased in China.
Drugs and chemical reagents Animals Quinocetone (90%, for analysis of plasma drug concentration) was purchased from the Sigma Chemical Co. (St. Louis, MO, USA). Quinocetone (98%, for drug administration) was purchased from DEO Pharm. Intermediates Co., Ltd. (Qihe, Shandong Province, China). Dideoxyquinocetone (95%) and 1desoxyquinocetone (95%), were synthesized by the College of Science, China Agricultural University (Beijing, China). Double-bond reduced metabolite of dideoxyquinocetone (95%) was synthesized by the Laboratory of Veterinary Pharmacology, College of Veterinary Medicine, South China Agricultural University (Guangzhou, China). HPLC-grade methanol was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Water was purified using a Milli-Q system
Eight healthy cross-bread (Duroc×Landrace×Large White) castrated swine were used for the studies. The animals were purchased from the Lizhi Agricultural Co. Ltd. (Guangzhou, China) and did not receive any medication for the previous 1 mon. The average body weight of the pigs was (30±3) kg. Pigs were housed indoor and fed daily with drug-free commercial pellet diet. Pigs had free access to drinking water. An acclimation period of one week was observed prior to initiation of the study.
Experimental design A randomized crossover design with a 2-wk washout © 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
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period was used for the study. For i.v. administration, quinocetone was dissolved in N,N-dimethylacetamide and then injected into the swine ear vein at a dosage of 4 mg kg-1 body weight (bw). Blood samples were collected from the superior vena cava by venipuncture into tubes containing heparin before drug application and at 0.083, 0.167, 0.25, 0.333, 0.50, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, and 24 h after i.v. administration. The per oral (p.o.) administrations were carried out by gavage at a dosage of 40 mg kg-1 bw and blood samples were collected into tubes containing heparin prior to and at 0.083, 0.167, 0.25, 0.50, 0.75, 1, 1.5, 2,2.5, 3, 4, 6, 8, 12, and 24 h after quinocetone administration. All blood samples were centrifuged at 930×g for 10 min at room temperature (25°C). The separated plasma samples were kept at -20°C until HPLC-MS/MS analysis.
cursor ion m/z 275.2 and product ions m/z 247.2, 143.1 for dideoxyquinocetone; Precursor ion m/z 279.2 and product ions m/z 261.2, 175.1 for double-bond reduced metabolite of dideoxyquinocetone. For calibration, 0.5 mL blank swine plasma was spiked with 20 μL of a series of diluted quinocetone and its metabolites working standard solutions and analyzed as above. The concentration of quinocetone and it’s metabolites in the prepared standard samples were 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5 μg mL-1. The limit of quantitation, quantitation linearity and recovery of quinocetone and it’s metabolites from plasma were determined. Coefficients of variation (CV, %) within and between HPLC-MS/MS runs were also calculated.
HPLC-MS/MS analysis
Plasma concentration-time data of quinocetone and its metabolites were analyzed with non-compartmental model based on statistical moment theory (Cutler 1978; Yamaoka et al. 1978). A commercially available software program (Winnonlin 5.2, Pharsight Corporation, CA, USA) was used to estimate the pharmacokinetic parameters. The maximum plasma concentration (Cmax) and the time to reach this concentration (Tmax) were taken directly from the plasma concentration-time profiles. The area under the concentration-time curve (AUC0- ) and the area under the first moment curve (AUMC0- ) were calculated using the linear/logarithmic trapezoidal rule up to the last determined concentration, and were extrapolated to infinity (Gibaldi and Perrier 1982). The first order rate constant associated with the terminal (log-linear) portion of the curve (λz) was estimated using linear regression of the terminal log-linear portion of the plasma concentration-time profile and the terminal half-life (tl/2) was calculated as ln(2)/λz. Mean residence time (MRT) was determined as the ratio of AUMC0- to AUC0- and apparent total body clearance (CL/F) was determined by dividing the administered qnocetone dose by the AUC0- . The apparent volume of distribution during the terminal elimination phase (Vz/F) was calculated by the formula: Vz/F=Dose/(λz×AUC0- ). The steady-state volume of distribution (Vss) was estimated from the formula: Vss=Di.v. ×MRT/AUC0- (Benet and Galeazzi 1979), where Di.v.
Quinocetone and its metabolites concentrations in plasma were simultaneously determined using a HPLCMS/MS method with an API 4000 HPLC-MS/MS system. Briefly, an aliquot of 0.5 mL plasma was deproteinized with 0.5 mL acetonitrile, vigorously vortexed for 2 min, and then followed by centrifugation for 10 min at 20 620×g. The supernatant was filtered through a 0.22 μm microbore cellulose membrane and then 20 mL of sample was injected onto the API 4000 HPLC-MS/MS system from ABI Corporation (Sunnyvale, USA). Chromatography was carried out using a phenomenex C18 column (150 mm×2 mm, 5 μm); the mobile phase consisted of A (water/formic acid, 100:0.2, v/v), and B (acetonitrile),while the flow rate was 0.25 mL min-1. The gradient elution program of the mobile phases were as follows: 0 to 1 min, mobile phase A 90 to 40%, mobile phase B 10 to 60%; 1 to 7 min, mobile phase A 40 to 20%, mobile phase B 60 to 80%; 7 to 7.1 min, phase A 20 to 90%, mobile phase B 80 to 10%; 7.1 to 13 min, mobile phase A 90%, mobile phase B 10%. Mass spectrometric parameters were electrospray ionization source, multiple reaction monitoring; Precursor ion m/z 307.1 and product ions m/z 273.1, 131.1 for quinocetone; Precursor ion m/z 291.1 and product ions m/z 245.2, 159.0 for 1-desoxyquinocetone; Pre-
Pharmacokinetic calculations
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
Pharmacokinetics of Quinocetone and Its Major Metabolites in Swine After Intravenous and Oral Administration
is the intravenously administrated quinocetone dose. The bioavailability (F) for oral administration was calculated by the formula F=(Dosei.v.×AUCp.o.)/(Dosep.o. ×AUCi.v.)×100%
RESULTS The method developed in this study was selective for the substance analyzed and no endogenous interference was observed on chromatograms. The elution times were approximately 7, 8.1, 8.2, and 9.6 min for QCT, Q33, Q2, and Q4, respectively (Fig. 2). The LOQ were 0.002 μg mL-1 for QCT and the metabolites (S/N>10) respectively. QCT and its metabolites quantitations were linear within a range of 0.001-0.5 μg mL-1 (r2 0.99) respectively. The intra-assay and inter-assay coefficients of variation of QCT and the metabolites were <15%. The recoveries of QCT and metabolites from plasma samples were between 80-95%. The plasma concentration vs. time curves of QCT and 1-desoxyquinocetone in pig plasma after i.v. and oral administration are shown in Figs. 3 and 4, respectively. QCT concentration descended rapidly in pig plasma after intravenous administration. As a result, QCT could not be detected in plasma 24 h after administration and the average concentration of QCT were only 6.73 ng mL-1 12 h after i.v. administration. For p.o. administration, low Cmax of QCT was attained and the average concentrations of QCT were 52.7 and 4.5 ng mL -1 12 and 24 h after oral administration, respectively. The concentrations of 1-desoxyquin ocetone in plasma were much lower than QCT for both i.v. and p.o. administration of QCT. The main pharmacokinetic parameters of QCT and 1-desoxyquinocetone calculated from the plasma data are listed in Table. Dideoxyquinocetone were detected only in several plasma samples from a pig with a much low concentration and pharmacokinetic parameters could not be calculated due to sparse concentrationtime data. In all pig plasma samples, double-bond reduced metabolite of dideoxyquinocetone could not be detected. Cmax of QCT were observed to be (3.62±0.31) and (0.56±0.13) μg mL-1 at 0.083 and (2.92±0.65) h for i.v. and p.o. administration, respectively. C max of 1desoxyquinocetone in plasma were observed to be
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(0.0095±0.0012) and (0.0067±0.0053) μg mL -1 at 0.083 and (3.08±0.75) h after administration for i.v. and oral dosing, respectively. Much smaller AUCs and longer elimination half-lives of 1-desoxyquinocetone than those of QCT were also observed in this study. The results of present studies showed that QCT was rapidly transformed to 1-desoxyquinocetone in pigs after i.v. or oral administration.
DISCUSSION Although many metabolites of QCT were found in urine of pig after single oral administration of QCT at a single dose of 500 mg kg-1 bw, there is no information about the pharmacokinetics for metabolites of QCT in animals. So some of important metabolites of QCT were synthesized for investigation of pharmacokinetics of QCT in animals by the team of the National Basic Research Program of China (973 Program, 2009CB118800). Additionally, we developed a high selective and sensitive HPLC-MS/MS method to simultaneously determine QCT and its 3 metabolites of QCT (1-desoxyq uinocetone, dideoxyquinocetone, double-bond reduced metabolite of dideoxyquinocetone) in pig plasma, and thus will provide a comprehensive pharmacokinetic profile of QCT in animals. In this study, areas under the plasma concentrationtime curve (AUC0- ) of QCT were 2.02 μg h mL-1 for i.v. administration (4 mg kg-1 bw) and 3.05 μg h mL-1 for oral (40 mg kg-1 bw) administration respectively. The bioavailability was calculated to be 17.33% after oral administration, suggesting that only minor part of QCT was absorbed in gastrointestinal tract after oral administration. The terminal elimination half-lives (t 1/2λz) of QCT were 2.24 h for i.v. administration and 2.91 h for oral administration respectively, showing that QCT was rapidly eliminated in pig plasma. Low Cmax of QCT (0.56 μg mL-1) was observed in pig plasma for oral administration in this study. Consistently, low Cmax of QCT in animals or fish were also reported by others, for example, 0.28 μg mL-1 in swine (p.o., 30 mg kg-1 bw), 0.452 μg mL-1 in carp (p.o., 50 mg kg-1 bw) and 0.029 μg mL-1 in channel catfish (p.o., 50 mg kg-1 bw). Elimination half-life of QCT were reported to be 5.05 h in pigs (p.o., 30 mg kg-1 bw), 4.66 h in chickens (p.o., 31.15 mg kg-1 bw), 35.87 h in carp (p.o., 50 mg kg-1
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
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(Continued on next page) Fig. 2 A, chromatogram of blank plasma sample. B, chromatogram of standard solution of QCT, Q33, Q2 and Q4; retention time 7, 8.1, 8.2, and 9.6 min respectively (5 ng mL-1). C, chromatogram of plasma spicked with QCT, Q33, Q2, and Q4 (5 ng mL-1). D, chromatogram of plasma 1 h after intravenous administration of QCT. E, mass spectrogram of Q33. F, mass spectrogram of Q2.
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
Pharmacokinetics of Quinocetone and Its Major Metabolites in Swine After Intravenous and Oral Administration
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Fig. 2 (Continued from preceding page)
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Fig. 3 The mean plasma concentration-time curves of QCT after a single intravenous and oral administration of QCT at the dosage of 4 and 40 mg kg-1 bw respectively in swine (n=8).
Fig. 4 The mean plasma concentration-time curves of 1-desoxyquinocetone after a single intravenous and oral administration of QCT at the dosage of 4 and 40 mg kg-1 bw respectively in swine (n=8).
Table Plasma pharmacokinetic parameters of QCT and 1-desoxyquinocetone in swine following i.v. and p.o. administration of QCT at the dosage of 4 and 40 mg kg-1 bw respectively Parameter (unit) λz (h-1) t1/2λz (h) Tmax (h) Cmax (μg mL-1) AUC0-¡Þ (h μg mL-1) CL/F (L h kg-1) MRT (h) F (%)
p.o.
i.v.
QCT
1-Desoxyquinocetone
QCT
0.25±0.033 2.91±0.29 2.92±0.65 0.56±0.13 3.50±0.79 13.70±2.36 5.12±0.70 17.33%
0.074±0.013 11.85±2.89 3.08±0.75 0.0067±0.0053 0.053±0.030
0.33±0.035 2.24±0.24 0.083 3.62±0.31 2.02±0.15 1.9±0.31 1.03±0.13
8.63±0.76
1-Desoxyquinocetone 0.14±0.015 5.23±0.56 0.083 0.0095±0.0012 0.020±0.0020 3.28±0.18
Data are mean±SD. n=8.
© 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
Pharmacokinetics of Quinocetone and Its Major Metabolites in Swine After Intravenous and Oral Administration
bw) and 31.46 h in channel catfish (p.o., 50 mg kg-1 bw) (Li et al. 2002; Li et al. 2005; Yang et al. 2010). There are some differences between the values of elimination half-life of QCT obtained in this study (2.91 h for p.o. administration and 2.24 h for oral administration) and values reported by others in above-mentioned studies. One of the reasons may be the poor selectivity of analytic method adopted in those studies to distinguish QCT from its metabolites in plasma of animals or fishes. In pig plasma, low Cmax of 1-desoxyquinocetone were observed to be 0.0095 μg mL -1 at 0.083 h after i.v. administration and 0.0067 μg mL-1 at 3.08 h after p.o. administration of QCT. So the results showed that QCT was much rapidly but only partly transformed to 1-desoxyquinocetone in pigs. Interestingly, elimination half-life of 1-desoxyquinoceton (5.23, 11.85 h for i.v. and p.o. administration, respectively) were longer than those of QCT. So close attention should be paid to this metabolite (1-desoxyquinocetone) when we identify the residue marker of QCT in animal tissues. Dideoxyquinocetone could be detected only in several plasma samples from a pig with much lower values. This result is in accordance with the low concentrations of dideoxyquinocetone in carp after oral administration of QCT reported by Yang et al. (2010). In China, QCT is widely used as drug additive to promote animal growth and has good effect in treating clinical infections such as Swine Dysentery, Colibacillosis and Salmonellosis. The results from this study showed that QCT was transformed to metabolites and concentrations of QCT in the plasma were low after oral administration. Therefore, further investigation will be carried out in next step, such as pharmacological effect and toxicity of metabolites of QCT, pharmacokinetics of QCT and its major metabolites in animal tissue.
CONCLUSION QCT and its main metabolites in swine plasma were determined by HPLC-MS/MS method which could be used for future pharmacokinetic studies. Pharmacokinetic results showed that QCT was metabolized rapidly in swine after p.o. and i.v. administration and was eliminated quickly. QCT had a low bioavailability of
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17.33% after oral administration. The metabolite 1desoxyquinoceton had a lower plasma concentration than QCT but a longer half-life (T1/2), especially after oral administration. Further studies should be conducted to investigate the pharmacokinetics of QCT and its major metabolites in animal tissue.
Acknowledgements This study was supported by the National Basic Research Program of China (973 Program, 2009CB118805) and the Key Technologies R&D Program of China during the 11th Five-Year Plan preiod (2009BADB7B05-03).
References Ban M M, Zhang K Y, Jiang S X, Xue F Q. 2010. Cytotoxicity of quinocetone and olaquindox on human liver cells. Chinese Journal of Veterinary Science, 30, 1517-1521. (in Chinese) Benet L Z, Galeazzi R L. 1979. Noncompartmental determination of the steady-state volume of distribution. Journal of Pharmaceutical Sciences, 68, 1071-1074. Cutler D J. 1978. Theory of the absorption time, an adjunct to conventional bioavailability studies. Journal of Pharmacy and Pharmacology, 30, 476-478. Gibaldi M, Perrier D. 1982. Pharmacokinetics. 2nd ed. Marrcel Dekker, New York. pp. 409-417. Li J Y, Zhou X Z, Li J S, Zhao R C, Miao X L, Zhang J Y. 2005. The pharmacokinetics of quinocetone in pigs. Chinese Journal of Veterinary Drug, 39, 1-3. (in Chinese) Li J W, Huang F, Zhou Y P, Liu J. 2010. Effect of quinocetone on growth of allogynogentic crucian and its metabolism in vivo. Cereal & Feed Industry, 5, 45-48. (in Chinese) Li J Y, Li J S, Xu Z Z, Du X L, Miao X L, Zhao R C, Liu J X, Xue F Q. 2002. Studies on the pharmacokinetics of quinocetone in pigs and chickens. Acta Veterinaria et Zootechnica Sinica, 34, 94-97. (in Chinese) Liu Y T, Guo D F, Yang L, Yang H, Yuan K P, Ai X H. 2009. Study on residues of quinocetone in Jian carp (Cyprinus carpio var. jian) and channel catfish (Ictalurus punctatus). Journal of Hydroecology, 2, 95-97. (in Chinese) Shen J Z, Yang C Y, Wu C M, Feng P S, Wang Z H, Li Y, Li Y S, Zhang S X. 2010. Identification of the major metabolites of quinocetone in swine urine using ultra-performance liquid chromatography/electrospray ionization quadrupole time-offlight tandem mass spectrometry. Rapid Communications in Mass Spectrometry, 24, 375-383. Wang Y C, Zhao R C, Yan X L, Li J S, Du X L, Zhang S Z, Xue F Q, Xu Z Z, Liang J P, Zhang J Y. 1995. Carcinogenicity study of quinocetone on mice. Chinese Journal of Veterinary
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Science and Technology, 25, 24-25. (in Chinese) Wang Y C, Zhao R C, Yan X L, Li J S, Xu Z Z, Xue F Q, Liang J
Technology, 25, 37-39. (in Chinese) Yamaoka K, Nakagawa T, Uno T. 1978. Statistical moments in
P, Yao S L, Wang F L. 1995. Effect of quinocetone on growth of chickens. Chinese Journal of Veterinary Science and
pharmacokinetics. I. Pharmacokinet. Biopharm, 6, 547-558. Yang L,Wang K Y, Ai X H,Yuan K P, Liu Y T. 2010.
Technology, 25, 36-38. (in Chinese) Xu J N, Wang Q K, Cui T, Huang Q Y, Wang J G. 2005. Studies
Pharmacokinetics and tissue distribution of quinocetone in carp and channel catfish. Chinese Veterinary Science, 40,
on the subchronic oral toxicity of quinocetone. Chinese Journal of Veterinary Drug, 39, 10-15. (in Chinese)
537-542. (in Chinese) Zhang W, Peng D P, Huang L L, Wang Y L, Yuan Z H. 2007.
Xu Z Z, Li J S, Zhao R C, Wang Y C, Xue F Q, Liang J P, Yan X L, Du X L, Zang J Y, He Z L. 1995. Effect of quinocetone on
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growth of piglets. Chinese Journal of Veterinary Science and (Managing editor ZHANG Juan)
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August 2011
2011, 10(8): 1292-1300
Pharmacokinetics of Quinocetone and Its Major Metabolites in Swine After Intravenous and Oral Administration ZHONG Jia-lin, ZHANG Gui-jun, SHEN Xiang-guang, WANG Lin, FANG Bing-hu and DING Huan-zhong Laboratory of Veterinary Pharmacology, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, P.R. China
Abstract The pharmacokinetics of quinocetone and its major metabolites in healthy swine was investigated in this paper. Quinocetone was administered to 8 healthy cross-bread swine intravenously and orally at a dosage of 4 and 40 mg kg-1 body weight respectively in a randomized crossover design test with two-week washout period. A sensitive highperformance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed for the determination of quinocetone and its metabolite 1-desoxyquinocetone in plasma. Plasma concentration versus time profiles of quinocetone and its metabolite 1-desoxyquinocetone were analyzed by non-compartmental analysis using Winnonlin 5.2 software. Mean maximum concentrations (Cmax) for quinocetone was found to be (0.56±0.13) μg mL-1 at 2.92 h, after oral administration of quinocetone. Mean maximum concentrations (Cmax) for 1-desoxyquinocetone after intravenous or oral administration of quinocetone were (0.0095±0.0012) μg mL-1 at 0.083 h and (0.0067±0.0053) μg mL-1 at 3.08 h. The apparent elimination half-lives (T 1/2) for quinocetone and its metabolite 1-desoxyquinocetone were (2.24±0.24) and (5.23±0.56) h after intravenous administration of quinocetone and (2.91±0.29) and (11.85±2.89) h after oral administration of quinocetone, respectively. Mean areas under the plasma concentration-time curve (AUC0- ) for quinocetone and 1desoxyquinocetone were (2.02±0.15) and (0.2±0.002) μg h mL-1 respectively after intravenous administration of quinocetone, and (3.5±0.79) and (0.053±0.03) μg h mL-1 after oral administration of quinocetone, respectively. Quinocetone was rapidly absorbed and metabolized in swine after oral and intravenous administration. The plasma concentration-time curve (AUC0- ) of 1-desoxyquinocetone were much smaller than those of quinocetone, while the elimination half-lives (T1/2) were much longer than those of quinocetone after intravenously (i.v.) or oral administration. Key words: quinocetone, metabolites, pharmacokinetics, HPLC-MS/MS, swine
INTRODUCTION Quinocetone (QCT), 3-methyl-2-cinnamoylquinoxaline1,4-dioxide, is a new synthetic veterinary drug from the quinoxaline-1,4-dioxide family developed by Lanzhou Institute of Animal Husbandry and Veterinary Drugs, Chinese Academy of Agricultural Sciences (Lanzhou, China). QCT has excellent growth-enhanc-
ing activity when used in pigs, chickens and fishes (Xu et al. 1995; Wang et al. 1995; Li et al. 2010). High safety of QCT in animals has also been reported (Wang et al. 1995; Xu et al. 2005; Zhang et al. 2007; Ban et al. 2010). Considering the fact that carbadox (CBX) and olaquindox (OLA) have been banned or allowed only limited use in food animals because of their toxicities, QCT has good prospects in growth promotion in food animals. In fact, QCT is now
Received 30 January, 2011 Accepted 31 May, 2011 ZHONG Jia-lin, MSc, Tel: +86-20-85284896, E-mail: [email protected]; Correspondence DING Huan-zhong, Associate Professor, Ph D, Tel: +86-20-85284896, Mobile: 13751886811, E-mail: [email protected] © 2011, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(11)60121-1
Pharmacokinetics of Quinocetone and Its Major Metabolites in Swine After Intravenous and Oral Administration
broadly used in China to promote animal growth and prevent infection caused by intestinal pathogen in pigs or chickens. However, only recently the metabolism of QCT was intensively studied in animals and the results showed that QCT was extensively metabolized and 31 metabolites were identified in pig urine. The reduction of the N-O group in quinocetone was the main metabolic pathway observed in swine urine (Shen et al. 2010). Nowadays there have been some records about the pharmacokinetic properties of QCT in animals or fish. However, those studies focused only on the parent drug and metabolites of QCT were not included in the experimental process (Li et al. 2002; Liu et al. 2009; Yang et al. 2010). Since QCT were metabolized much rapidly and extensively (Shen et al. 2010), the profile of main metabolites of QCT should also be considered
1293
in determining pharmacokinetic parameters in animals. On the basis of results from previous in vivo and in vitro studies by the team of the National Basic Research Program of China (973 Program, 2009CB118800), 1-desoxyquinocetone (Q2), dideoxyquinocetone (Q4), and double-bond reduced metabolite of dideoxyquin ocetone (Q33) were recognized as important metabolites of QCT in pigs (Fig. 1). The objective of the present study was to provide further information on the pharmacokinetics of QCT and these three metabolites (Fig. 1) after oral and intravenous administration. Knowledge of pharmacokinetic properties of a parent drug and its important metabolites is necessary for comprehensive evaluation of corresponding kinetic processes and the determination of the withdrawal periods in foodproducing animals. Such data will be essential for judicious use of QCT as an antibacterial agent in swine.
Fig. 1 Chemical structure of QCT, 1-desoxyquinocetone (Q2), dideoxyquinocetone (Q4), and double-bond reduced metabolite of dideoxyquinocetone (Q33).
MATERIALS AND METHODS
(Millipore, MA, USA). Other reagents were A.R. grade and purchased in China.
Drugs and chemical reagents Animals Quinocetone (90%, for analysis of plasma drug concentration) was purchased from the Sigma Chemical Co. (St. Louis, MO, USA). Quinocetone (98%, for drug administration) was purchased from DEO Pharm. Intermediates Co., Ltd. (Qihe, Shandong Province, China). Dideoxyquinocetone (95%) and 1desoxyquinocetone (95%), were synthesized by the College of Science, China Agricultural University (Beijing, China). Double-bond reduced metabolite of dideoxyquinocetone (95%) was synthesized by the Laboratory of Veterinary Pharmacology, College of Veterinary Medicine, South China Agricultural University (Guangzhou, China). HPLC-grade methanol was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Water was purified using a Milli-Q system
Eight healthy cross-bread (Duroc×Landrace×Large White) castrated swine were used for the studies. The animals were purchased from the Lizhi Agricultural Co. Ltd. (Guangzhou, China) and did not receive any medication for the previous 1 mon. The average body weight of the pigs was (30±3) kg. Pigs were housed indoor and fed daily with drug-free commercial pellet diet. Pigs had free access to drinking water. An acclimation period of one week was observed prior to initiation of the study.
Experimental design A randomized crossover design with a 2-wk washout © 2011, CAAS. All rights reserved. Published by Elsevier Ltd.
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period was used for the study. For i.v. administration, quinocetone was dissolved in N,N-dimethylacetamide and then injected into the swine ear vein at a dosage of 4 mg kg-1 body weight (bw). Blood samples were collected from the superior vena cava by venipuncture into tubes containing heparin before drug application and at 0.083, 0.167, 0.25, 0.333, 0.50, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, and 24 h after i.v. administration. The per oral (p.o.) administrations were carried out by gavage at a dosage of 40 mg kg-1 bw and blood samples were collected into tubes containing heparin prior to and at 0.083, 0.167, 0.25, 0.50, 0.75, 1, 1.5, 2,2.5, 3, 4, 6, 8, 12, and 24 h after quinocetone administration. All blood samples were centrifuged at 930×g for 10 min at room temperature (25°C). The separated plasma samples were kept at -20°C until HPLC-MS/MS analysis.
cursor ion m/z 275.2 and product ions m/z 247.2, 143.1 for dideoxyquinocetone; Precursor ion m/z 279.2 and product ions m/z 261.2, 175.1 for double-bond reduced metabolite of dideoxyquinocetone. For calibration, 0.5 mL blank swine plasma was spiked with 20 μL of a series of diluted quinocetone and its metabolites working standard solutions and analyzed as above. The concentration of quinocetone and it’s metabolites in the prepared standard samples were 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5 μg mL-1. The limit of quantitation, quantitation linearity and recovery of quinocetone and it’s metabolites from plasma were determined. Coefficients of variation (CV, %) within and between HPLC-MS/MS runs were also calculated.
HPLC-MS/MS analysis
Plasma concentration-time data of quinocetone and its metabolites were analyzed with non-compartmental model based on statistical moment theory (Cutler 1978; Yamaoka et al. 1978). A commercially available software program (Winnonlin 5.2, Pharsight Corporation, CA, USA) was used to estimate the pharmacokinetic parameters. The maximum plasma concentration (Cmax) and the time to reach this concentration (Tmax) were taken directly from the plasma concentration-time profiles. The area under the concentration-time curve (AUC0- ) and the area under the first moment curve (AUMC0- ) were calculated using the linear/logarithmic trapezoidal rule up to the last determined concentration, and were extrapolated to infinity (Gibaldi and Perrier 1982). The first order rate constant associated with the terminal (log-linear) portion of the curve (λz) was estimated using linear regression of the terminal log-linear portion of the plasma concentration-time profile and the terminal half-life (tl/2) was calculated as ln(2)/λz. Mean residence time (MRT) was determined as the ratio of AUMC0- to AUC0- and apparent total body clearance (CL/F) was determined by dividing the administered qnocetone dose by the AUC0- . The apparent volume of distribution during the terminal elimination phase (Vz/F) was calculated by the formula: Vz/F=Dose/(λz×AUC0- ). The steady-state volume of distribution (Vss) was estimated from the formula: Vss=Di.v. ×MRT/AUC0- (Benet and Galeazzi 1979), where Di.v.
Quinocetone and its metabolites concentrations in plasma were simultaneously determined using a HPLCMS/MS method with an API 4000 HPLC-MS/MS system. Briefly, an aliquot of 0.5 mL plasma was deproteinized with 0.5 mL acetonitrile, vigorously vortexed for 2 min, and then followed by centrifugation for 10 min at 20 620×g. The supernatant was filtered through a 0.22 μm microbore cellulose membrane and then 20 mL of sample was injected onto the API 4000 HPLC-MS/MS system from ABI Corporation (Sunnyvale, USA). Chromatography was carried out using a phenomenex C18 column (150 mm×2 mm, 5 μm); the mobile phase consisted of A (water/formic acid, 100:0.2, v/v), and B (acetonitrile),while the flow rate was 0.25 mL min-1. The gradient elution program of the mobile phases were as follows: 0 to 1 min, mobile phase A 90 to 40%, mobile phase B 10 to 60%; 1 to 7 min, mobile phase A 40 to 20%, mobile phase B 60 to 80%; 7 to 7.1 min, phase A 20 to 90%, mobile phase B 80 to 10%; 7.1 to 13 min, mobile phase A 90%, mobile phase B 10%. Mass spectrometric parameters were electrospray ionization source, multiple reaction monitoring; Precursor ion m/z 307.1 and product ions m/z 273.1, 131.1 for quinocetone; Precursor ion m/z 291.1 and product ions m/z 245.2, 159.0 for 1-desoxyquinocetone; Pre-
Pharmacokinetic calculations
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Pharmacokinetics of Quinocetone and Its Major Metabolites in Swine After Intravenous and Oral Administration
is the intravenously administrated quinocetone dose. The bioavailability (F) for oral administration was calculated by the formula F=(Dosei.v.×AUCp.o.)/(Dosep.o. ×AUCi.v.)×100%
RESULTS The method developed in this study was selective for the substance analyzed and no endogenous interference was observed on chromatograms. The elution times were approximately 7, 8.1, 8.2, and 9.6 min for QCT, Q33, Q2, and Q4, respectively (Fig. 2). The LOQ were 0.002 μg mL-1 for QCT and the metabolites (S/N>10) respectively. QCT and its metabolites quantitations were linear within a range of 0.001-0.5 μg mL-1 (r2 0.99) respectively. The intra-assay and inter-assay coefficients of variation of QCT and the metabolites were <15%. The recoveries of QCT and metabolites from plasma samples were between 80-95%. The plasma concentration vs. time curves of QCT and 1-desoxyquinocetone in pig plasma after i.v. and oral administration are shown in Figs. 3 and 4, respectively. QCT concentration descended rapidly in pig plasma after intravenous administration. As a result, QCT could not be detected in plasma 24 h after administration and the average concentration of QCT were only 6.73 ng mL-1 12 h after i.v. administration. For p.o. administration, low Cmax of QCT was attained and the average concentrations of QCT were 52.7 and 4.5 ng mL -1 12 and 24 h after oral administration, respectively. The concentrations of 1-desoxyquin ocetone in plasma were much lower than QCT for both i.v. and p.o. administration of QCT. The main pharmacokinetic parameters of QCT and 1-desoxyquinocetone calculated from the plasma data are listed in Table. Dideoxyquinocetone were detected only in several plasma samples from a pig with a much low concentration and pharmacokinetic parameters could not be calculated due to sparse concentrationtime data. In all pig plasma samples, double-bond reduced metabolite of dideoxyquinocetone could not be detected. Cmax of QCT were observed to be (3.62±0.31) and (0.56±0.13) μg mL-1 at 0.083 and (2.92±0.65) h for i.v. and p.o. administration, respectively. C max of 1desoxyquinocetone in plasma were observed to be
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(0.0095±0.0012) and (0.0067±0.0053) μg mL -1 at 0.083 and (3.08±0.75) h after administration for i.v. and oral dosing, respectively. Much smaller AUCs and longer elimination half-lives of 1-desoxyquinocetone than those of QCT were also observed in this study. The results of present studies showed that QCT was rapidly transformed to 1-desoxyquinocetone in pigs after i.v. or oral administration.
DISCUSSION Although many metabolites of QCT were found in urine of pig after single oral administration of QCT at a single dose of 500 mg kg-1 bw, there is no information about the pharmacokinetics for metabolites of QCT in animals. So some of important metabolites of QCT were synthesized for investigation of pharmacokinetics of QCT in animals by the team of the National Basic Research Program of China (973 Program, 2009CB118800). Additionally, we developed a high selective and sensitive HPLC-MS/MS method to simultaneously determine QCT and its 3 metabolites of QCT (1-desoxyq uinocetone, dideoxyquinocetone, double-bond reduced metabolite of dideoxyquinocetone) in pig plasma, and thus will provide a comprehensive pharmacokinetic profile of QCT in animals. In this study, areas under the plasma concentrationtime curve (AUC0- ) of QCT were 2.02 μg h mL-1 for i.v. administration (4 mg kg-1 bw) and 3.05 μg h mL-1 for oral (40 mg kg-1 bw) administration respectively. The bioavailability was calculated to be 17.33% after oral administration, suggesting that only minor part of QCT was absorbed in gastrointestinal tract after oral administration. The terminal elimination half-lives (t 1/2λz) of QCT were 2.24 h for i.v. administration and 2.91 h for oral administration respectively, showing that QCT was rapidly eliminated in pig plasma. Low Cmax of QCT (0.56 μg mL-1) was observed in pig plasma for oral administration in this study. Consistently, low Cmax of QCT in animals or fish were also reported by others, for example, 0.28 μg mL-1 in swine (p.o., 30 mg kg-1 bw), 0.452 μg mL-1 in carp (p.o., 50 mg kg-1 bw) and 0.029 μg mL-1 in channel catfish (p.o., 50 mg kg-1 bw). Elimination half-life of QCT were reported to be 5.05 h in pigs (p.o., 30 mg kg-1 bw), 4.66 h in chickens (p.o., 31.15 mg kg-1 bw), 35.87 h in carp (p.o., 50 mg kg-1
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(Continued on next page) Fig. 2 A, chromatogram of blank plasma sample. B, chromatogram of standard solution of QCT, Q33, Q2 and Q4; retention time 7, 8.1, 8.2, and 9.6 min respectively (5 ng mL-1). C, chromatogram of plasma spicked with QCT, Q33, Q2, and Q4 (5 ng mL-1). D, chromatogram of plasma 1 h after intravenous administration of QCT. E, mass spectrogram of Q33. F, mass spectrogram of Q2.
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Pharmacokinetics of Quinocetone and Its Major Metabolites in Swine After Intravenous and Oral Administration
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Fig. 2 (Continued from preceding page)
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Fig. 3 The mean plasma concentration-time curves of QCT after a single intravenous and oral administration of QCT at the dosage of 4 and 40 mg kg-1 bw respectively in swine (n=8).
Fig. 4 The mean plasma concentration-time curves of 1-desoxyquinocetone after a single intravenous and oral administration of QCT at the dosage of 4 and 40 mg kg-1 bw respectively in swine (n=8).
Table Plasma pharmacokinetic parameters of QCT and 1-desoxyquinocetone in swine following i.v. and p.o. administration of QCT at the dosage of 4 and 40 mg kg-1 bw respectively Parameter (unit) λz (h-1) t1/2λz (h) Tmax (h) Cmax (μg mL-1) AUC0-¡Þ (h μg mL-1) CL/F (L h kg-1) MRT (h) F (%)
p.o.
i.v.
QCT
1-Desoxyquinocetone
QCT
0.25±0.033 2.91±0.29 2.92±0.65 0.56±0.13 3.50±0.79 13.70±2.36 5.12±0.70 17.33%
0.074±0.013 11.85±2.89 3.08±0.75 0.0067±0.0053 0.053±0.030
0.33±0.035 2.24±0.24 0.083 3.62±0.31 2.02±0.15 1.9±0.31 1.03±0.13
8.63±0.76
1-Desoxyquinocetone 0.14±0.015 5.23±0.56 0.083 0.0095±0.0012 0.020±0.0020 3.28±0.18
Data are mean±SD. n=8.
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Pharmacokinetics of Quinocetone and Its Major Metabolites in Swine After Intravenous and Oral Administration
bw) and 31.46 h in channel catfish (p.o., 50 mg kg-1 bw) (Li et al. 2002; Li et al. 2005; Yang et al. 2010). There are some differences between the values of elimination half-life of QCT obtained in this study (2.91 h for p.o. administration and 2.24 h for oral administration) and values reported by others in above-mentioned studies. One of the reasons may be the poor selectivity of analytic method adopted in those studies to distinguish QCT from its metabolites in plasma of animals or fishes. In pig plasma, low Cmax of 1-desoxyquinocetone were observed to be 0.0095 μg mL -1 at 0.083 h after i.v. administration and 0.0067 μg mL-1 at 3.08 h after p.o. administration of QCT. So the results showed that QCT was much rapidly but only partly transformed to 1-desoxyquinocetone in pigs. Interestingly, elimination half-life of 1-desoxyquinoceton (5.23, 11.85 h for i.v. and p.o. administration, respectively) were longer than those of QCT. So close attention should be paid to this metabolite (1-desoxyquinocetone) when we identify the residue marker of QCT in animal tissues. Dideoxyquinocetone could be detected only in several plasma samples from a pig with much lower values. This result is in accordance with the low concentrations of dideoxyquinocetone in carp after oral administration of QCT reported by Yang et al. (2010). In China, QCT is widely used as drug additive to promote animal growth and has good effect in treating clinical infections such as Swine Dysentery, Colibacillosis and Salmonellosis. The results from this study showed that QCT was transformed to metabolites and concentrations of QCT in the plasma were low after oral administration. Therefore, further investigation will be carried out in next step, such as pharmacological effect and toxicity of metabolites of QCT, pharmacokinetics of QCT and its major metabolites in animal tissue.
CONCLUSION QCT and its main metabolites in swine plasma were determined by HPLC-MS/MS method which could be used for future pharmacokinetic studies. Pharmacokinetic results showed that QCT was metabolized rapidly in swine after p.o. and i.v. administration and was eliminated quickly. QCT had a low bioavailability of
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17.33% after oral administration. The metabolite 1desoxyquinoceton had a lower plasma concentration than QCT but a longer half-life (T1/2), especially after oral administration. Further studies should be conducted to investigate the pharmacokinetics of QCT and its major metabolites in animal tissue.
Acknowledgements This study was supported by the National Basic Research Program of China (973 Program, 2009CB118805) and the Key Technologies R&D Program of China during the 11th Five-Year Plan preiod (2009BADB7B05-03).
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