Pharmacokinetics of marbofloxacin following intramuscular administration at different doses in sheep

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Small Ruminant Research

journal homepage:www.elsevier.com/locate/smallrumres

Pharmacokinetics of marbo

ﬂoxacin following intramuscular administration

at di

ﬀerent doses in sheep

Feray Altan

a,⁎

, Orhan Corum

b

, Duygu Durna Corum

b

, Semih Altan

c

, Kamil Uney

d a_{Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Dicle, Diyarbakir 21280, Turkey}

b_{Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Kastamonu, Kastamonu 37200, Turkey} c_{Department of Surgery, Faculty of Veterinary Medicine, University of Dicle, Diyarbakir 21280, Turkey}

d_{Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Selcuk, Konya 42031, Turkey}

A R T I C L E I N F O Keywords: Marboﬂoxacin Pharmacokinetics Diﬀerent dose Sheep A B S T R A C T

The pharmacokinetics of marbofloxacin (MBX) was determined following the intramuscular administration at the doses of 2, 4, 6, and 10 mg/kg in twenty-four healthy sheep. In parallel design, sheep were randomized to 2, 4, 6, and 10 mg/kg dose groups of six animals per group. High performance liquid chromatography method for determination of MBX in sheep plasma was used. Pharmacokinetic parameters were calculated by a non-com-partmental method. The dose-normalized the area under the concentration-versus-time curve (AUC0-∞) and dose-normalized maximum plasma concentration (Cmax) in 10 mg/kg dose group were significantly higher than other dose groups. The elimination half-life (t1/2λz) of marbofloxacin in 10 mg/kg dose group was significantly longer than other dose groups. MBX exhibited dose-proportional pharmacokinetics and was well tolerated after 2, 4, 6 and 10 mg/kg doses in sheep. The 2, 4, 6, and 10 mg/kg doses of MBX could be administered in the treatment of infections caused by susceptible pathogens in sheep. However, additional studies are needed to identify whether MBX is efficient in sheep of naturally infected with susceptible bacteria.

1. Introduction

Fluoroqinolones are more frequently used in the treatment of an-imal disease because of its broad spectrum of activity (Brown, 1996). Marbofloxacin (MBX) is third-generation fluoroquinolone used and developed only for veterinary medicine (Bryskier and Chantot, 1995). It has bactericidal efficacy against gram-positive and gram-negative mi-crobial pathogens of sheep (Sidhu et al., 2010;Skoufos et al., 2007). The antibacterial spectrum of MBX includes Pasteurella multocida (P. multocida), Mannheimia haemolytica (M. haemolytica), Mycoplasma bovis, Escherichia coli, Salmonella spp., Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae (Meunier et al., 2004). The re-commended doses for the treatment of diseases caused susceptible pa-thogens ranges from 2 to 3 mg/kg MBX in lambs (Skoufos et al., 2007). The MBX has been shown to have a large volume of distribution, a long elimination half-life, and a high bioavailability, which makes to the distribution of organism and the applicability of daily (Brown, 1996;Fitton, 1992). The clinical efficacy of the fluoroquinolone is de-pendent on the concentration of drug in relation to the minimum in-hibitory concentration (MIC) of the pathogen. The optimize drug

dosage ofﬂuoroquinolones can be achieved by integrating the phar-macokinetic and pharmacodynamic parameters, such as the area under the concentration-versus-time curve (AUC)/MIC and the maximum plasma concentration (Cmax)/MIC (McKellar et al., 2004).

Several studies have been published regarding the pharmacokinetics of MBX on sheep (Altan et al., 2018;Karademir et al., 2015;Sidhu et al., 2010); however, to the best of our knowledge, no information is re-garding the pharmacokinetics and linearity at diﬀerent dose of MBX in sheep. Therefore, the purpose of this study was to determine the pharmacokinetics and tolerability of MBX, administered in-tramuscularly at 2, 4, 6, and 10 mg/kg doses in sheep.

2. Materials and methods 2.1. Animals

The Ethics Committee of the University of Dicle, Diyarbakir, Turkey approved the use of the animals for this study and all study protocols. Prior to the start of this study, the sheep were assigned to healthy based on a physical examination, complete blood count, and serum

https://doi.org/10.1016/j.smallrumres.2019.03.016

Received 13 January 2019; Received in revised form 22 March 2019; Accepted 24 March 2019 ⁎_{Corresponding author.}

E-mail addresses:feray.altan@dicle.edu.tr(F. Altan),orhancorum46@hotmail.com(O. Corum),ddurna@kastamonu.edu.tr(D. Durna Corum),

semih.altan@dicle.edu.tr(S. Altan),kuney@selcuk.edu.tr(K. Uney).

Small Ruminant Research 174 (2019) 88–91

Available online 25 March 2019

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biochemistry panel. Twenty-four healthy sheep (Akkaraman, weighing 55 ± 2 kg and 2 ± 0.5 years) were selected for this study. Animals were housed and fed daily with a rye grass and clover hay mix and a supplementary concentrate. Water was given ad libitum. At the end of the study sheep were continued their normal life.

2.2. Study design

The study was performed according to a randomized parallel pharmacokinetic design. Twenty-four sheep were randomly allocated into four dose groups of six animals per group. The MBX (Marbox®, CEVA, Turkey) was administered into the semitendinosus muscle at single 2, 4, 6 and 10 mg/kg doses to each sheep. Blood samples (2 mL) were collected in tubes containing lithium heparin as an anticoagulant via IV catheter (22 G, 0.9 x 25 mm, Bicakcilar Medical Devices Industry and Trade Co., Istanbul, Turkey) placed into the left Vena jugularis be-fore drug administration (0 min) and at 5, 10, 15, 20, 25, 30, and 45 min and 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 10, 12, 18, 24, 36 and 48 h fol-lowing drug administration. The plasma concentration of MBX is ana-lysed up to 36 h only at doses of 2, 4, and 6 mg/kg, whereas this is determined up to 48 h at 10 mg/kg dose. Blood samples were cen-trifuged at 4000 × g for 10 min. After the centrifugation, plasma was transferred into polypropylene tubes and stored at −70 °C until the assay.

2.3. Analytical method

The plasma MBX concentration was determined using the high-performance liquid chromatography–ultraviolet detection (HPLC–UV) method modified previously (Potter et al., 2013; Real et al., 2011). Briefly, frozen serum samples were allowed to reach at room tem-perature. The 400μL of acetonitrile was added to a 200 μL of each plasma sample, and then was vortexed for 30 s. After centrifuged at 10.000 g for 10 min, the supernatant (100μL) was harvested and added to 100μL pure water of each supernatants, and transferred into auto-sampler vials, and a volume of 10μL was injected into an HPLC system (Shimadzu, Tokyo, Japan). The HPLC analysis was controlled using a Shimadzu-LC system equipped with a CBM-20A controller, LC-20AT pump, DGU-20A degasser, SIL-20A auto-sampler, SPD-20A UV–VIS detector, and CTO-10A column oven. Chromatographic separation was conducted with a Gemini C18 column (250 × 4.6 mm; internal dia-meter, 5μm; Phenomenex, Torrance, CA, USA) at 280 nm wavelength. The mobile phase was consisted of acetonitrile and triethylamine (0.04%) + ortho-phosphoric acid (0.04%) (v/v 17/83%). Theflow rate was set at 1 mL/min. The total analysis time for samples was 12 min. Data was analysed with a PC-controlled LC solution software program (Shimadzu, Japan).

The MBX was prepared in pure water to give a stock solution of 1 mg/mL. The calibration standards of blank plasma spiked with MBX were linear between 0.04 and 20μg/mL ( r2> 0.9996). The limits of

detection and the limit of quantification were 0.02 and 0.04 μg/mL, respectively. The interday percent coefficients of variation (CV) for different concentrations of MBX were as follows: < 3.97% (0.1 μg/ mL), < 2.88% (1μg/mL), and < 1.65% (10 μg/mL). The intraday per-cent CV for different conper-centrations of MBX were as follows: < 1.26.% (0.1μg/mL), < 2.80% (1 μg/mL), and < 1.26% (10 μg/mL). The re-covery was 98–104%.

2.4. Pharmacokinetic analysis

Plasma concentration versus time curves were described using the Phoenix WinNonlin V 6.1.0.173 software (Pharsight, Certara, St. Louis, MO, USA). The pharmacokinetic parameters were determined using a non-compartmental model analysis using WinNonlin. Elimination rate constant (λz) was estimated by least squares regression of the logarithm of plasma concentration versus time curve over the terminal elimina-tion phase. The maximum plasma concentraelimina-tion (Cmax) and time to reach Cmax(Tmax) were obtained directly from the plasma concentra-tion-time curve. The elimination half-life (t1/2λz) was calculated as ln (2)/λz. AUC was estimated by the log-trapezoidal rule.

2.5. Statistical analysis

Statistical analyses were carried out with the SPSS 22.0 (IBM Corp, Armonk, NY). Tmaxwas presented as a median. For time parameters (MRT and t1/2λz), the harmonic mean and the standard deviation (SD) were computed. The Mann Whitney U test was used to MRT and t1/2λz parameters for significant differences between dose groups. Other pharmacokinetic parameters (mean ± SD) were evaluated with one-way variance analysis (ANOVA), and statistical differences between groups were detected with a Tukey’s test after normalization of AUC0–∞ and Cmaxvalues to the 2 mg/kg dose. A p value of < 0.05 was accepted as statistically significant.

3. Results

The pharmacokinetic data for MBX after intramuscular adminis-tration of 2, 4, 6 and 10 mg/kg doses in sheep are presented inTable 1. Mean concentration-time curves of MBX after the intramuscular ad-ministration at 2, 4, 6 and 10 mg/kg doses are shown inFig. 1. No general or local side effects were observed in sheep treated with MBX. In this study, we didn't use AUC0-∞and Cmaxfor statistical di ffer-ences, but the dose-normalized AUC0-∞and Cmaxto the 2 mg/kg dose were used instead of it. Following intramuscular administration of 2, 4, 6 and 10 mg/kg doses, the AUC0-∞ and Cmaxof MBX in plasma in-creased proportionally to the doses from 2 to 10 mg/kg. The dose-normalized AUC0-∞and Cmaxof MBX in 10 mg/kg dose groups were found significantly higher than other dose groups (p < 0.05). The Tmax was 0.75 h in the all dose groups. Following intramuscular adminis-tration of MBX at 2, 4, 6 and 10 mg/kg doses, the t1/2λz was 14.54, Table 1

Pharmacokinetic parameters (Mean ± SD) of marboﬂoxacin following intramuscular administration at 2, 4, 6, and 10 mg / kg doses in sheep (n = 6).

Parameters 2 mg/kg 4 mg/kg 6 mg/kg 10 mg/kg t1/2ʎz(h) (HM) 14.54 ± 1.75b 15.43 ± 1.85b 15.71 ± 1.95b 16.82 ± 1.09a MRT0-∞(h) (HM) 14.74 ± 1.46 14.60 ± 1.66 13.73 ± 1.27 14.35 ± 0.60 ClT(L/h/kg) 0.14 ± 0.06b 0.15 ± 0.04b 0.14 ± 0.07b 0.12 ± 0.04a Tmax(h) (M) 0.75 ± 0.00 0.75 ± 0.00 0.75 ± 0.00 0.75 ± 0.00 Cmax(μg/mL) 2.01 ± 0.08 3.91 ± 0.16 6.74 ± 0.25 13.21 ± 0.58 AUC0-24(h*μg/mL) 11.19 ± 0.48 20.81 ± 0.80 34.11 ± 2.34 65.23 ± 2.24 AUC0-∞(h*μg/mL) 14.00 ± 0.63 26.23 ± 0.79 41.96 ± 2.25 81.06 ± 3.06 Cmax(μg/mL) (N)x 2.01 ± 0.08c 1.96 ± 0.08c 2.25 ± 0.08b 2.64 ± 0.12a AUC0-∞(h*μg/mL) (N)x 14.00 ± 0.63b 13.12 ± 0.40b 13.99 ± 0.75b 16.21 ± 0.61a a, b, c, d_{Varied character in the same row are statistically signi}_{ﬁcantly diﬀerent (P < 0.05).}x_{AUC and C}

maxvalues were dose-normalized by 2 mg/kg.

t1/2ʎz; elimination half-life, MRT; mean residence time, CLT; total clearance, Tmax; time to reach the maximum concentration, Cmax; peak plasma concentration, AUC; area under the concentration-versus time curve, HM; harmonic means, M; median,∞; inﬁnitive, N; normalized.

F. Altan, et al. Small Ruminant Research 174 (2019) 88–91

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15.43, 15.71, and 16.82 h, respectively. The total clearance (ClT) was 1.23 (L/h/kg) after intramuscular administration of 10 mg/kg dose of MBX. The t1/2λz and ClT of MBX in the 10 mg/kg dose group were signiﬁcantly diﬀerent from 2, 4, and 6 mg/kg dose groups (p < 0.05). 4. Discussion

Lower respiratory tract infections, such as pneumonia, which caused economic loss because of excessive weight loss and infertility, are im-portant respiratory diseases in sheep (Bell, 2008;Martin, 1996). The occurrence rate of bacterial pneumonia results from a complex inter-action of infectious agents (M. haemolytica and P. multocida) in sheep, and antimicrobial drugs are used to treat this infections (Alley, 2002). The effective treatment of MBX based on the observation of only clin-ical results have been reported for respiratory infections in lambs (Skoufos et al., 2007). However, studies based solely on monitoring clinical outcomes are defective as they may cause an effect such as underestimation of the efficacy of very good drugs and overestimation of the effectiveness of weak drugs (McKellar et al., 2004). Pharmaco-kinetic parameters, together with pharmacodynamic parameters can provide more valid information about the assessment of the efficacy of antibacterial drugs against infectious agents (Theuretzbacher, 2012). These approaches have ensured new strategies for forecast drug dosage that the make optimum efficacy and minimise emergence of resistant pathogens (Aliabadi and Lees, 2002;Altan et al., 2018). This situation can be solved by changes to the dosage regimen such as increasing and decreasing the recommended dose. The use of higher doses of flour-oqinolones generally facilitates access to targeted Cmax/MIC and AUC/ MIC indices against susceptible isolates (Boothe et al., 2006). Therefore, ascending dose of the MBX was used in this study. For these reasons, MBX was administered on the doses of 2, 4, 6, and 10 mg/kg in sheep. There have been some studies to describe the pharmacokinetics of MBX in sheep (Sidhu et al., 2010). However, the pharmacokinetics of ascending dose of MBX has not been previously determined in the sheep to the author’s knowledge. The findings of the current study propose that an effect of different doses may be present on some pharmacoki-netic parameters of MBX in sheep. Side effects, such as local or systemic adverse reactions, were not determined in sheep following in-tramuscular administrations of MBX at 2, 4, 6, and 10 mg/kg doses.

In this study, the Cmaxwas higher and Tmaxwas longer in the 2 mg/ kg dose group than those previously reported following the in-tramuscular administration in sheep (Sidhu et al., 2010). The Cmax value reﬂects the rate and degree of absorption from the administration site of the drug according to the elimination rate (Trepanier, 2013). In this study, the t1/2λzof MBX after intramuscular injection at 2 mg/kg

dose was 14.54 h, which was consistent with the previously reported values (9.50–12.64 h) after intramuscular administration in lambs (Altan et al., 2018) and in sheep (Karademir et al., 2015), but were much longer than that previously obtained after intramuscular (3.65 h) administrations in sheep ((Sidhu et al., 2010). The ClT(0.14 L/h/kg) values of MBX after 2 mg/kg IM administration obtained in this study was lower than those reported for sheep (ClT0.44 L/h/kg)(Sidhu et al., 2010). The long t1/2λzand slow ClTfollowing intramuscular adminis-tration of MBX may be outcome of the slowly absorption of drug from the intramuscular injection site during the elimination process (Toutain and Bousquet-Melou, 2004).

The pharmacokinetics of MBX over the dose range 2–10 mg/kg showed dose-proportional pharmacokinetics with Cmax and AUC0-∞ values increasing in direct proportion to increase in dosage. The Tmax (0.75 h) was similar in dose groups. However, the t1/2λzand ClTof MBX in the 10 mg/kg dose group were significantly different from 2, 4 and 6 mg/kg dose groups (p < 0.05). The estimation of terminal half-life is highly sensitive to especially LOQ of analytical method (Toutain and Bousquet-Melou, 2004). The long t1/2λz and slow ClT of MBX in the 10 mg/kg dose group is related to that the plasma concentration of MBX is analyzed up to 36 h only at doses of 2, 4, and 6 mg/kg, whereas this is determined up to 48 h at 10 mg/kg dose. Therefore, t1/2λzof MBX at 10 mg/kg dose was better estimated than that at 2, 4, and 6 mg/kg doses. In addition, long t1/2λzand slow ClTof MBX in the 10 mg/kg dose group might be related to the differences between animals in this par-allel-group study.

Forﬂuoroquinolones, the AUC0-24 /MIC and Cmax/MIC literature values for bactericidal activity and to reduce the risk of the emergence of resistance are 125 and 10, respectively (Aliabadi and Lees, 2002; Sidhu et al., 2010). In this study, the 2, 4, 6, and 10 mg/kg doses of MBX provided AUC0-24/MIC value of≥125 for bacteria with MIC value of≤0.09, 0.17, 0.27 and 0.52 μg/mL, respectively, and Cmax/MIC value of≥10 for bacteria with MIC value of ≤0.20, 0.35, 0.67 and 1.32 μg/ mL, respectively, in sheep.

5. Conclusions

In conclusion, this is theﬁrst study to compare the diﬀerent dose pharmacokinetics of MBX in sheep. The MBX exhibited dose-propor-tional pharmacokinetics and was well tolerated after 2, 4, 6, and 10 mg/kg doses in sheep. The 2, 4, 6, and 10 mg/kg doses of MBX could be administered in the treatment of infections caused by susceptible pathogens in sheep. Therefore, we are intending to use, before sug-gesting these results for use in clinical studies, the more number of bacteria strains isolated from sheep for our future

pharmacokinetic-Fig. 1. Semi-logarithmic plasma concentration–time curves following single intramuscular administrations of marboﬂoxacin at dose of 2 mg/kg, 4 mg/kg, 6 mg/kg, and 10 mg/kg in sheep (n = 6).

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pharmacodynamic studies. Conﬂict of interest

All authors declare that they have no conﬂicts of interest. Acknowledgments

Authors are grateful to Scientiﬁc Research Council of Dicle University (DUBAP Project No: Veteriner.17.020) for providing the fund and facilities for this experiment. Presented in abstract form at the 34th World Veterinary Association Congress taking place at the Centre de Convencions Internacional de Barcelona (CCIB) from May 5 to 8, 2018, in Barcelona, Spain. Thanks are due to Ceva Animal Health Inc. Turkey for supplying MB pure substance.

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