Pharmacokinetic and tissue analyses of levofloxacin in sheep (Ovis aries Linnaeus) after multiple-dose administration

Research in Veterinary Science 128 (2020) 124–128

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Pharmacokinetic and tissue analyses of levofloxacin in sheep (Ovis aries Linnaeus) after multiple-dose administration

T

Irene Sartinia, Beata Łebkowska-Wieruszewskab, Tae Won Kimc, Andrzej Lisowskid, ⁎ Amnart Poapolathepe, Mario Giorgif, a

Department of Veterinary Medicine, University of Sassari, Sassari, Italy Department of Pharmacology, Toxicology and Environmental Protection, University of Life Sciences, Lublin, Poland c College of Veterinary Medicine, Chungnam National University, Daejeon, South Korea d Institute of Animal Breeding and Biodiversity Conservation, University of Life Sciences, Lublin, Poland e Department of Pharmacology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand f Department of Veterinary Sciences, University of Pisa, Via Livornese (lato monte), San Piero a Grado, 56122 Pisa, Italy b

A R T I C LE I N FO

A B S T R A C T

Keywords: Levofloxacin Sheep Minimum inhibitory concentration (MIC) Absolute oral bioavailability

The aim of this study was to assess the pharmacokinetic profile of LFX in sheep after intravenous (IV) and oral (PO) administration of 2 mg/kg LFX once a day for 5 days and to evaluate its tissue depletion in the muscles, heart, liver, lungs, and kidneys. Twenty healthy female sheep were randomly divided into two equal groups. Each group was further randomly subdivided into two equal subgroups (n = 5). Group 1 was used for blood collection and underwent a crossover design (2 × 2 Latin square). Group 2 was randomly subdivided into two equal subgroups (n = 5) for IV and PO route respectively, and used for tissue collection. A single sheep was sacrificed at each time point and the organs were harvested. Samples were analyzed using a validated HPLC method with fluorescence detection. LFX administered orally was rapidly absorbed with a peak plasma concentration of 2866 ± 239 ng/mL and an absolute oral bioavailability of 114 ± 27.7%. The pharmacokinetic estimates were comparable between PO and IV administration. According to the pharmacokinetic/pharmacodynamic surrogate index (area under the curve / minimum inhibitory concentration) of 100–125, LFX has the potential to be an effective treatment for infections caused by bacteria with a MIC of 0.049–0.061 μg/mL. LFX was detected for up to 48 h in all the tissues samples. The kidney had the highest LFX concentration after IV and PO administration. The AUCtissue/plasma ratio was lower than 1 in all tissues indicating absence of LFX tissue accumulation.

Introduction Fluoroquinolones are extensively used in human and veterinary medicine for the treatment of a variety of bacterial infections (Davis and Bryson 1994; Martinez et al. 2006). Levofloxacin (LFX), a third generation fluoroquinolone, is active against a wide range of aerobic gram-positive and gram-negative organisms and demonstrates moderate activity against anaerobes (Blondeau 1999; Klesel et al. 1995; Martinez et al. 2006). Like most fluoroquinolones, LFX can inhibit bacterial DNA replication and transcription through reversible binding with the enzyme DNA gyrase (Wolfson and Hooper 1989). LFX is widely used in humans, but is not registered for veterinary use. In humans, the drug is distributed in body fluids and tissues, including saliva and skin (Langtry and Lamb 1998; Sheikh et al. 2010). LFX has been reported to enhance the activity of the microsomal ⁎

enzymes in the liver (Dwivedi et al. 2011). Fluoroquinolones are used in veterinary medicine after strong evidences for their efficacy and when there are no alternative treatment options. The interest in the use of fluoroquinolones in veterinary medicine has led to the initiation of several studies on the pharmacokinetics of LFX in various animal species, such as goats (Goudahand and Abo-El-Sooud 2009), chickens (Lee et al. 2017), cats (Albarellos et al. 2005), horses (Goudah et al. 2008), rats (Cheng et al. 2002), rabbits (Destache et al. 2001), camels (Goudah 2009.), and calves (Kumar et al. 2009; Dumka and Srivastava 2006, 2007). Numerous studies have been performed on the pharmacokinetics of LFX in healthy (Goudah and Hasabelnaby., 2010; Patel et al. 2012a) and febrile sheep (Patel et al. 2012b), following single dose administration. LFX displayed a large volume of distribution and extensive penetration in the tissues of sheep (Patel et al. 2012a) due to its high lipophilicity and low protein binding (Goudah and Hasabelnaby 2010).

Corresponding author. E-mail address: [email protected] (M. Giorgi).

https://doi.org/10.1016/j.rvsc.2019.11.008 Received 30 September 2019; Received in revised form 15 November 2019; Accepted 19 November 2019 0034-5288/ © 2019 Elsevier Ltd. All rights reserved.

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Twenty-gram samples of the muscle, heart, liver, lung, and kidney were obtained, washed in saline, placed in criobags, and immediately stored at −20 °C until further analysis. HPLC instrumentation. The HPLC system was an LC system (Jasco, Japan) consisting of a high-pressure mixer pump (model PU 980 Plus), a spectrofluorometric detector (model 2020 Plus), an autosampler (model AS 950), and a Peltier system (model CO-4062) with a variable loop set at 50 μL. Data was processed using the CromNav 2.0 software (Jasco, Inc.). The chromatographic separation assay, modified from Lee et al. (2017), was performed using a Gemini analytical column (250 × 4.6 mm inner diameter, 5 μm particle size, Phenomenex, Torrance, California, USA) at 25 °C. The mobile phase consisted of ACN: aqueous solution (20: 80 v/v %) at a flow rate of 1 mL/min. The aqueous solution consisted of potassium dihydrogenphosphate (0.02 M), phosphoric acid (0.006 M), and tetraethyl amine (0.012 M) in water (pH = 4.0). Excitation and emission wavelengths were set at 295 and 490 nm, respectively. Sample extraction. The procedure for plasma sample extraction was based on the methods described by Giorgi et al. (2013) and Lee et al. (2017). Briefly, aliquots (0.2 mL) of plasma were combined with 0.1 mL of IS (10 μg/ mL) and 0.8 mL of 0.1 M phosphate buffer at pH 7.1. After adding 4 mL of a mixture of trichloromethane and isopropanol (5: 1 v/v), the samples were shaken at 60 oscillations/min for 10 min and centrifuged at 4000 ×g for 5 min. Four milliliters of the organic layer was transferred into a clean tube and dried at 40 °C under a nitrogen stream. The residue was dissolved in 0.2 mL mobile phase, vortexed, and an aliquot (50 μL) was injected into the chromatographic system. The liver, kidneys, lungs, heart, and muscles were thawed and immediately dissected into small pieces. A total of 1 g per tissue sample was placed into a 5 mL plastic tube and 3 mL of 0.1 M phosphate buffer at pH 7.1 was added. The suspensions were homogenized using a tissue homogenizer (Tearor, Biospec, Bartlesville, USA) for approximately 40 s. Aliquots of 200 μL were processed as described for plasma samples. Quantification. The analytical method was validated in all the complex matrices evaluated. Standard curves were plotted using standard LFX concentrations and the ratio of LFX / IS peak areas. Linearity of the regression curves in the range of 2.5–5000 ng/mL for both, plasma and organs, were assessed based on the residual plot, fit test, and back calculation (within 20% of the known amount). Limit of detection (LOD) and limit of quantitation (LOQ) were determined as analyte concentrations producing signal-to-noise ratios of 3 and 10, respectively. The quantitative HPLC methods were validated by examining the consistency of the results (within-run and between-runs), correlation (coefficient of determination of the standard curve), and extraction efficiency of the assay. Pharmacokinetic analysis. The pharmacokinetic calculations were carried out using ThothPro™ 4.2 software, (ThothPro™, Gdansk, Poland). LFX plasma concentration was modeled for each subject using a non-compartmental approach. Maximum plasma concentration (Cmax) of LFX and the time required to reach Cmax (Tmax) were obtained from the data. The elimination halflife (t1/2λz) was calculated using linear least squares regression analysis of the concentration-time curve. The area under the concentration-time curve (AUC0-inf) was calculated using the logarithmic trapezoidal method and the linear-up log-down rule for IV and PO administration, respectively. Using the abovementioned values, the apparent volume of distribution at steady state (Vss = dose x AUMC / AUC2), mean residence time (MRT = AUMC / AUC), and systemic clearance (Cl = dose / AUC) were determined. Mean absorption time (MAT) was calculated as MRTpo - MRTiv. Pharmacokinetic estimates were calculated only if the individual value of AUCrest% was lower than 20% of AUC0-∞ and R2 (square of coefficient of determination) of the terminal phase regression

Antibacterial drugs usually follow a multiple dose therapy, with potential drug accumulation. Pharmacokinetic data on multiple dose administration of LFX and tissue residue depletion in sheep have not previously been available. Thus, this study aims to assess the pharmacokinetic profile of LFX and its residue depletion in the muscle, heart, liver, lung, and kidney tissues in sheep after IV and PO administration of 2 mg/kg LFX once a day for 5 days. Material and methods Chemicals and reagents. Pure LFX and enrofloxacin as internal standard (IS) with a standard purity of 99.0% were purchased from Sigma-Aldrich (Milan, Italy). High Performance Liquid Chromatography (HPLC)-grade acetonitrile (ACN), methanol (MeOH), trichloromethane (CHCl3), and isopropanol (C3H8O) were procured from Merck. Tetraethylamine was obtained from Sigma-Aldrich (St Louis, MI, US). Orthophosphoric acid, sodium dihydrogen phosphate, and potassium dihydrogen phosphate were purchased from Carlo Erba Reagents (Milan, Italy). Deionized water was produced using a Milli-Q Millipore Water System (Millipore, Darmstadt, Germany). All the other reagents and materials were of analytical grade and were acquired from commercial sources. The aqueous and organic components of the mobile phase were degassed under pressure and mixed in the HPLC system. The mobile phases were filtered through 0.2 μm cellulose acetate membrane filters (Sartorius Stedim Biotech, Goettingen, Germany) with a solvent filtration apparatus. Animal experiments. Twenty healthy adult female sheep (Ovis aries Linnaeus) Swiniarka breed, with body weights ranging from 26.9 to 40.0 kg (age 5–8 years) were used in the study. The sheep were determined to be clinically healthy based on a physical examination and full chemical and hematological analyses. They were assessed by a qualified veterinarian (L-W B), who certificated the health, absence of recent drug treatment, and parasites in sheep. Animal experiments were conducted at the University of Life Sciences, Lublin, Poland. Before the start of the study, sheep were acclimatized for 7 days in an animal shed. Feed (alfalfa hay) and water were given ad libitum. Animals included in the study did not get any history of previous drug administration. The animal experiment was approved by the animal welfare ethics committee of the University of Lublin and carried out in accordance with the European law (Directive 2010/63/EU) (authorization number 90/2018). The animals were randomly divided into two groups (n = 10). Group one was randomly subdivided into two equal subgroups (n = 5) and underwent a crossover design (2 × 2 Latin square). Subgroup 1 (sheep A-E) were administered LFX IV 2 mg/kg every 24 h for 5 days (levofloxacin TEVA 5 mg/mL; Teva pharmaceutical, Hungary) while subgroup 2 (sheep 1–5) received the same dosage of LFX PO (levofloxacin ACCORD 250 mg/tablet; Accord Healthcare Limited UK). The oral doses were prepared by weighing and partitioning the marketed drug. Blood samples were collected in vacutainer lithium heparin tubes (BD, Vaud, Switzerland) from the left jugular vein 12, 24, 36, 48, 60, 72, 84, and 96 h after the first administration and 5, 15, 30, 45 min and 1, 2, 4, 6, 8, 10, 12, 24, and 48 h after the last drug administration in both subgroups. After a 7-day wash out period, the subgroups were rotated, and the experiment was repeated. Blood samples were immediately placed on ice for 30 min, centrifuged at 1500 ×g and the harvested plasma was stored at −20 °C until further analysis. Group two (n = 10/group) was randomly subdivided into two equal subgroups (n = 5): subgroups 3 (sheep F-J) and 4 (sheep 6–10). They received the same 5-day treatment as subgroups 1 and 2, respectively. In both these subgroups, a single animal was sacrificed (shot with a captive bolt pistol) 6, 12, 24, 36, and 48 h after drug administration. 125

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line was > 0.85 (). The penetration of LFX into the selected tissue was determined by comparing the AUC (AUC0–48 tissue/plasma) ratios between tissues and plasma after PO and IV administration. PO absolute oral bioavailability (F %) was calculated using the following formula: F % = (AUCPO) / (AUCIV) x 100. Statistical analysis Pharmacokinetic variables were evaluated using Student's t-test to determine statistically significant differences between groups (Kolmogorov-Smirnov test for Tmax). Pharmacokinetic parameters are presented as mean ± SD (normality was tested by Shapiro-Wilk test). Differences were considered significant if p < .05. All analyses were conducted using GraphPad Prism (GraphPad Software, La Jolla, California, USA). Fig. 1. Semi logarithm mean ± SD LFX plasma concentration vs time curve upon IV (−o-) and PO (−●-) administration of LFX (2 mg/kg once a day for 5 days) in sheep (n = 10). Please note that units on the x- and y-axes are reported using two different scales. Blood during the first 4 days after the first administration has been withdraw every 12 h, while after the last administration more frequently.

Results Validation of the analytical method. The analytical method validation parameters for plasma and organs were within the stipulated limits requested from the guidelines for the analytical method validation (Anonymous 2009) (Table 1). Animals. A licensed veterinarian (B L-W) evaluated the health of the animals. The sheep did not exhibit visible immediate or delayed (up to 7 days), local or systemic adverse effects. Pharmacokinetics. Fig. 1 depicts the progressive mean plasma concentration of LFX. The graphs for IV and PO administration show similar pharmacokinetic profiles. Table 2 shows the main PK estimates analyzed using a noncompartmental model. No significant pharmacokinetic differences were observed between the IV and PO estimates. Orally administered LFX was rapidly absorbed with a peak plasma concentration of 2866 ng/mL and a complete absolute oral bioavailability (F %). Tissue residues. Fig. 2 shows the progressive changes in tissue concentrations of LFX on IV (a) and PO (b) administration in sheep. LFX was detected in all the tissues. Its concentration, highest at the first collection time point (6 h), decreased consistently for 48 h after IV and PO administration. The kidney showed highest LFX residue concentration, followed by the liver, muscle, lung, and heart. Tissue concentration of LFX in each organ was consistently higher after IV administration than after PO administration at corresponding time points. Fig. 3 shows the AUC (0–48) tissue/plasma ratio determined upon IV and PO administration of LFX in each organ.

Table 2 Pharmacokinetic parameters after IV and PO administration of LFX (2 mg/kg once a day for 5 days) in sheep (n = 10). IV

PO

Parameter

Unit

Mean

SD

Mean

SD

AUC(0-t) AUC(0-inf) kel MRT t1/2λz Cmax Tmax§ Cl Vss MAT F

μg*h/L μg*h/L 1/h h h ng/mL h L/kg*h L/kg h %

10,531 10,788 0.244 3.24 4.06 – – 0.192 0.56 – –

1246 1051 0.162 0.98 2.41 – – 0.023 0.18 – –

10,753 12,159 0.241 4.25 3.76 2866 0.75 – – 1.46 114.7

1731 1723 0.126 1.65 1.73 239 (0.75–2) – – 0.86 27.7

AUC0-t = area under the curve from T = 0 to last time point, AUC0-inf = area under the curve from T = 0 to infinity, kel = terminal phase rate constant, MRT = mean residence time, t1/2kel = terminal half-life, Cmax = peak plasma concentration, Tmax = time of peak concentration, Cl = plasma clearance, Vss = volume of distribution, MAT = mean absorption time, F = bioavailability, §Median value and min-max range.

it should be taken into account that in clinical practice, many infections require a longer duration of therapy. This may lead to potential toxicity from drug accumulation (Leekha et al. 2011). From this perspective, no pharmacokinetic studies have been performed for IV and PO administration of multiple doses of LFX in sheep.

Discussion Most studies on antibacterial drugs, in human and veterinary medicine, are performed using a single administration design. However,

Table 1 Analytical method validation parameters for the detection of LFX in the plasma and tissues of sheep. Parameter

Unit

Plasma

Liver

Kidney

Lung

Muscle

Heart

Range Calibration equation

ng/mL

2.5–5000 y = 0.0027× + 0.0162

R2

0.997

2.5–5000 y = 0.8992× 0.0273 0.996

2.5–5000 y = 0.9849× 0.0299 0.998

2.5–5000 y = 0.0028× 0.0036 0.996

2.5–5000 y = 0.0037× + 0.0059

Correlation coefficient Intre-day precision Interday-precision LOQ LOD Recovery

2.5–5000 y = 0.00× 0.0392 0.998

% % ng/mL ng/mL %

< 9.6 < 7.9 2.5 1 98 ± 7

< 7.3 < 8.7 2.5 1 94 ± 6

< 8.9 < 6.7 2.5 1 85 ± 9

< 11.2 < 10.9 2.5 1 87 ± 8

< 9.0 < 10.4 2.5 1 95 ± 9

< 8.8 < 9.6 2.5 1 93 ± 8

126

0.996

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Albarellos 2019, 5 mg/kg), chickens (780 ng/mL) (Lee et al. 2017, 5 mg/kg), and cats (940 ng/mL) (Albarellos et al. 2005, 10 mg/kg), also reported lower Cmax values (normalized for the dose). After PO administration, Tmax was attained more quickly compared to a single IM (1.64 h) or SC (1 h) dose administration (Goudah and Hasabelnaby 2010; Patel et al. 2012a). Tmax was also achieved more quickly in this study than those reported after oral administration in dogs (1.82 h) (Landoni and Albarellos 2019) and cats (1.6 h) (Albarellos et al. 2005), but was comparable to that of chickens (0.88 h) (Lee et al. 2017). The absolute PO F % in this study was > 100%, which may be caused by multiple factors, such as errors during the in-life phase of the experiment; during the sampling, preparation, and conservation of samples; or due to the analytical technique used (Toutain and BousquetMélou, 2004a). The variability in animal response might also played a role as well as the involvement of some transporter-mediated secretion (Ward et al. 2004) However, several other studies on fluoroquinolones have reported a similar phenomenon (Lynch et al. 1994; Lee et al. 2017; Giorgi et al. 2013). The t1/2λz observed in the present study (around 4 h) was considered short. The amount of drug in the body was approximately 1.5% of the initial dose, 24 h after administration. After this, once-a-day LFX administration did not induce any further accumulation of the drug in the plasma. This was further confirmed by the steady LFX plasma concentration observed at each collection point after drug administration (Fig. 1). However, the t1/2λz value was higher than those reported in healthy sheep that were administered LFX PO by Goudah and Hasabelnaby (2010) (3.58 ± 0.30 h) and Patel et al. (2012a) (1.73 ± 0.04 h). Since t1/2λz is a hybrid parameter, its gross comparison with those reported in other studies is not appropriate (Toutain and Bousquet-Mélou, 2004b). For a more suitable comparison, t1/2λz needs to be split into two primary parameters: volume of distribution and clearance. Although widespread distribution, high values of CL resulted in short half-life of LFX which do not lead to accumulation of the drug in line with findings reported below. CL (0.19 ± 0.02 L / kg * h) was rapid and Vss (0.56 ± 0.18 L / kg) relatively high, indicating widespread distribution of LFX in body tissues. The CL value was similar to that found in a previous study performed in sheep by Goudah and Hasabelnaby (2010) (0.20 ± 0.05 L / kg * h), but slower than that found by Patel et al. (2012a) (0.55 ± 0.02 L / kg * h). The Vss value was lower than those found previously (0.86 ± 0.23 L / kg, Goudah and Hasabelnaby 2010; 0.92 ± 0.08 L / kg, Patel et al. 2012a). Effective dosing regimens for concentration-dependent antibacterials can be evaluated according to the PK/PD surrogate, AUC / MIC value. For fluoroquinolones, it should be at least 100 to 125 in gram-negative bacilli and 25 to 30 for S. pneumoniae (Levison and Levison 2009). The MIC of LFX has not yet been determined for bacteria isolated from sheep. If an MIC of 0.12 μg/mL is considered in order to cover most of the susceptible organisms (Dumka and Srivastava 2006; Lee et al. 2017), the range of MIC in this study giving an AUC / MIC ratio > 100 was 0.049–0.061 μg/mL. The calculation of this range included the AUC0–24 value corrected for the plasma protein binding reported in sheep (23.74%; Goudah and Hasabelnaby 2010). Clinically speaking the dosage regimen used in the present study is not suitable for clinically relevant isolates. LFX penetration into body tissues and fluids has been reported to be rapid and widespread after oral administration (Davis and Bryson 1994). In this study, the concentration of drug in the tissues was detectable up to 48 h after administration in all the organs and at all time points tested. Tissue analysis showed that the highest LFX concentration was in the kidney followed by the liver. The lung, muscle, and heart had lower concentrations of LFX. This profile is consistent with that observed in poultry (Bisht et al. 2018; Kumar et al. 2017; Lee et al. 2017). The tissue residue concentrations observed here are comparable with those reported by Bisht et al. (2018) in the muscle, liver, and

Muscle

1000

Heart

Concentration LVX (ng/g)

Liver Kidney Lung

100

10

1 102

114

126 Time (h)

138

(a) 1000

Muscle Heart

Concentration LVX (ng/g)

Liver Kidney Lung

100

10

1 102

114

126 Time (h)

138

(b)

AUC (0-48) tissue/plasma ratio

Fig. 2. Tissue concentrations of LFX (n = 1/ each time point) upon IV (a) and PO (b) administration (2 mg/kg once a day for 5 days) in the muscle, heart, liver, kidney, and lungs of sheep after the last (5th) administration.

1

PO IV

0.5

0 Kidney

Liver

Muscle

Heart

Lung

Fig. 3. AUC(0–48) tissue/plasma ratio after IV and PO administration of LFX (2 mg/ kg once a day for 5 days) in the kidney, liver, muscle, heart and lungs of sheep.

No statistical differences were found in the pharmacokinetic estimates after IV and PO administrations as early reported earlier (Albarellos et al. 2005). The Cmax value (2866 ng/mL) upon PO administration of LFX in sheep reported in this study (normalized for the dose) was higher than those previously reported for intramuscular (IM) (1550 ng/mL) (Goudah and Hasabelnaby 2010) and subcutaneous (SC) (1040 ng/mL) (Patel et al. 2012a) administrations. Other investigations performed on domestic animal species, such as dogs (1280 ng/mL) (Landoni and

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kidney, but lower than those reported by Kumar et al. (2017) in the muscle and Lee et al. (2017) in the muscle, lung, kidney, and liver. These might be due to species-specific differences. Drug penetration in tissues can be evaluated using the AUC(0–48) tissue/plasma ratio. A ratio over 1 indicates relatively higher concentrations of a drug in the tissues than in blood, with the potential for accumulation in the tissues (Bellmann et al. 2004). In the present study tissue levels were compared with plasma levels of other individuals. The inter-individual variability and the different shapes of the AUCs (different number of sampling points) may have caused some inaccuracy in the AUCtissue/AUCplasma ratio assessment. This ratio was lower than 1 in all tissues tested in this study. LFX showed limited tissue penetration in the muscle, heart, lung, and liver of sheep, with low AUC(0–48) tissue/plasma ratios of 0.1–0.3 after both, PO and IV administration. The AUC(0–48) tissue/plasma ratio was higher in the kidney (0.7, PO; 0.8, IV), suggesting that LFX accumulation in the organs would not potentially be a limitation. However, as the residue study was carried out using a small sample size, these data should be confirmed by further analyses.

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Conclusions IV and PO administration of a single dose of 2 mg/kg of LFX every 24 h for 5 days showed no significant differences in the pharmacokinetic parameters in sheep. The treatment was considered effective for bacteria with an MIC of 0.049–0.061 μg/mL that is lower than the MIC reported for ruminant pathogens. Tissue residues were highest in the kidney and liver, but the AUC0–48 tissue/plasma ratios indicated that LFX is not likely to accumulate in the plasma or organs. Acknowledgments None of the authors has any financial or personal relationships that could inappropriately influence or bias the content of this paper. This work was supported by University of Pisa (ex 60%). Authors acknowledge ThothPro (Gdansk, Poland) for supplying the software used for the pharmacokinetic analysis. References Albarellos, G.A., Ambros, L.A., Landoni, M.F., 2005. Pharmacokinetics of levofloxacin after single intravenous and repeat oral administration to cats. J. Vet. Pharmacol. Ther. 28 (4), 363–369. Anonymous, 2009. Guideline on validation of bioanalytical methods. In: EMEA/CHMP/ EWP/192217/2009. Bellmann, R., Kuchling, G., Dehghanyar, P., Zeitlinger, M., Minar, E., Mayer, B.X., Müller, M., Joukhadar, C., 2004. Tissue pharmacokinetics of levofloxacin in human soft tissue infections. Br. J. Clin. Pharmacol. 57 (5), 563–568. Bisht, P., Ahmad, A.H., Mishra, A., Sharma, S., 2018. Pharmacokinetics and tissue residue study of levofloxacin following multiple dose intramuscular administration in poultry. Int. J. Curr. Microbiol. App. Sci. 7 (7), 2614–2618. Blondeau, J.M., 1999. Expanded activity and utility of the new fluoroquinolones: a review. Clin. Ther. 21, 3–40. Cheng, F.C., Tsai, T.R., Chen, Y.F., Hung, L.C., Tsai, T.H., 2002. Pharmacokinetic study of levofloxacin in rat blood and bile by microdialysis and high-performance liquid chromatography. J. Chromatogr. A 961 (1), 131–136. Davis, R., Bryson, H.M., 1994. Levofloxacin: a review of its antibacterial activity, pharmacokinetics and therapeutic efficacy. Drugs 47 (4), 677–700. Destache, C.J., Pakiz, C.B., Larsen, C., Owens, H., Dash, A.K., 2001. Cerebrospinal fluid penetration and pharmacokinetics of levofloxacin in an experimental rabbit

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