Disposition kinetics and bioavailability of danofloxacin in Mehsana buffalo calves after parenteral administration

Introduction

Buffaloes (Bubalus bubalis) constitute a vital component of the livestock sector in India, contributing substantially to milk and meat production. Bacterial infections, particularly those involving the respiratory system, continue to cause significant economic losses in buffalo production systems. Effective antimicrobial therapy, based on sound pharmacokinetic principles, is essential for optimizing therapeutic outcomes and minimizing antimicrobial resistance. Danofloxacin is a third-generation fluoroquinolone developed exclusively for veterinary use and exhibits potent bactericidal activity against major Gramnegative respiratory pathogens (Sartini et al., 2021; Stipkovits & Szathmary, 2012) [10,11] . Its favourable pharmacokinetic properties, including high lipid solubility, extensive tissue penetration, and prolonged post-antibiotic effect, make it suitable for use in food-producing animals. However, pharmacokinetic behaviour may vary among species and breeds, necessitating breed-specific evaluation. The present study was therefore designed to characterize the pharmacokinetics of danofloxacin following intravenous and intramuscular administration in Mehsana buffalo calves to support rational dosage optimization in this breed.

The experimental work was approved by the Institutional Animal Ethics Committee (Reg.

No. 734/GO/Re/SL/03/CCSEA) under protocol number VETCOLL/IAEC/2024/23/PROTOCOL-02 dated 21/12/2024. The trial was carried out at the Large Animal Experimental Facility of the College of Veterinary Science and Animal Husbandry, located in Banaskantha district of North Gujarat (24.323272°N, 72.297212°E; 154.52 m AMSL), which experiences a tropical semi-arid climate with temperatures ranging between 25-35 °C during the study period. Animals were managed as per standard husbandry practices, provided with adequate green fodder, dry roughage, concentrate feed, and clean drinking water ad libitum. They were acclimatized and closely monitored for 10 days before the experiment, confirmed healthy through clinical examination, and individually identified using ear tags for the entire study duration.

Drug, Chemicals and Reagents

Danofloxacin 98% (CAS# 119478-55-6) was procured from JK Chemicals, Vapi, Gujarat (India). Water, methanol, acetonitrile, perchloric acid (70%), and formic acid were HPLC grade and purchased from S. D. Fine Chemicals Ltd., Mumbai.

Experimental Protocol

For the pharmacokinetic evaluation of danofloxacin in Mehsana buffalo calves, a solution was prepared by dissolving 500 mg of danofloxacin mesylate powder in 10 mL of sterile water for injection and administered at a dose of 5.0 mg/kg body weight for both intravenous (IV) and intramuscular (IM) routes. All injections were given using disposable 18G × 38 mm needles, with IV dosing through the jugular vein and IM administration into the trapezius muscle of the neck region. Blood samples (up to 3.0 mL) were collected into heparinized tubes from the contralateral jugular vein at predetermined intervals: after IV treatment at 0 min, 2 min (0.033 h), 5 min (0.083 h), 15 min (0.25 h), 30 min (0.5 h), and 1, 2, 4, 8, 12, 24, 36, and 48 hours; and following IM dosing at 0 min, 5 min (0.083 h), 15 min (0.25 h), 30 min (0.5 h), and 1, 2, 4, 8, 12, 24, 36, 48, and 72 hours. Plasma was separated by centrifugation at 2856 g for 5 minutes at 4 °C, transferred carefully into labelled 2 mL polypropylene microcentrifuge tubes, and stored at -4 °C to avoid degradation and freeze-thaw effects until chromatographic analysis.

Analytical Method

In this experiment, an Ultra-High Performance Liquid Chromatography (UHPLC) system (Dionex Ultimate 3000 ® , Thermo Fisher Scientific, Germany) was employed. The setup included a UV detector, a gradient solvent delivery pump, and a manual sample injector. Separation of analytes was achieved using a reverse-phase C-18 column (ODS-3V, GL Science Inc., Japan; 250 cm × 4.6 mm ID), operated at ambient laboratory temperature (20-30 °C). A 20 µL injection loop was utilized for sample introduction, and the chromatographic results were analyzed using Chromeleon™ software (version 6.8).

Chromatographic Conditions

For chromatographic analysis, a mobile phase composed of 0.01 M formic acid buffer and acetonitrile (79:21, v/v) was utilized, with the flow rate set at 1.0 mL/min.

The formic acid buffer (0.01 M) was prepared by adding 460 µL of formic acid to one litre of HPLC-grade water, followed by pH adjustment to 3.0 using 230 µL of triethylamine. The buffer was then filtered through a 0.47 µm membrane filter using vacuum filtration and subsequently degassed in an ultrasonic bath for 10 minutes (Frontline Ultrasonic Cleaner, Ahmedabad, India). The separation was performed under isocratic conditions with UV detection at 282 nm. Samples (20 µL) were injected manually using a Hamilton ® syringe #710 (Sigma-Aldrich, USA). Each chromatographic run lasted for 15 minutes, and danofloxacin typically eluted at approximately 5.5 ± 0.3 minutes.

Sample Extraction Procedure

The extraction of danofloxacin from plasma was performed using a modified protocol based on Abo-EL-Sooud et al. (2017) [1] . Plasma samples were first equilibrated to room temperature, after which 100 µL of each sample was pipetted into a 2 mL Eppendorf ® microcentrifuge tube. To precipitate plasma proteins, 100 µL of 20% perchloric acid-prepared by combining 2.86 mL of 70% perchloric acid with 7.14 mL of chilled acetonitrile-was added. The mixture was vortexed for 2 minutes at 658 g and then centrifuged at 11,424 g for 10 minutes at 4 °C using a refrigerated centrifuge. The resulting clear supernatant was carefully transferred into a fresh microcentrifuge tube, and a 20 µL portion of this extract was injected into the UHPLC system for quantification.

Pharmacokinetic Analysis

The plasma concentration-time profiles obtained from each buffalo calf were evaluated using a non-compartmental analysis approach to determine the pharmacokinetic (PK) parameters of danofloxacin. Parameters were calculated separately for both intravenous and intramuscular routes of administration, and the results were presented as mean ± standard error (SE). Non-compartmental analysis was selected as it provides reliable PK estimates without the need to assume any specific compartmental model. The calculations were based on the area under the plasma concentration-time curve (AUC) and the area under the firstmoment curve (AUMC), determined using the trapezoidal rule. Parameters included β, t½β, AUC, AUMC, MRT, Vd(area), Vdss, ClB, Cmax, Tmax, and bioavailability (F).

Validation of Analytical Method

The UHPLC method developed for quantifying danofloxacin in buffalo plasma was validated by evaluating linearity, extraction recovery, accuracy, precision, and sensitivity in accordance with USFDA-CDER (1994) and ICH (2005) guidelines. The calibration curve for danofloxacin over the range of 0.1-8.0 µg/mL showed excellent linearity with an average R² value of 0.9988. The mean recovery at low, medium, and high concentrations was 84.94%, 91.41%, and 94.03%, respectively, with an overall recovery of 90.12%, confirming efficient analyte extraction. Accuracy values ranged from 91.66% to 99.23%, indicating reliable measurement of true analyte concentrations. Intraday precision (% CV) ranged between 4.21-6.66%, while inter-day precision ranged between 3.30-3.72%, demonstrating good repeatability and reproducibility. The lower limit of quantification (LOQ) was established at 0.1 µg/mL within the validated linear range, ensuring reliable quantification of danofloxacin in all plasma samples analyzed.

Results and Discussion

Intravenous Administration

After a single intravenous (IV) dose of danofloxacin at 5.0 mg/kg body weight in Mehsana buffalo calves, the mean plasma concentration at the first sampling time point (0.033 h; 2 minutes) was 9.55 µg/ml, indicating rapid systemic availability immediately following IV administration. Thereafter, plasma concentrations declined sharply during the early post-administration period, with mean values of 5.27 µg/ml at 0.083 h and 3.43 µg/ml at 0.25 h, reflecting a rapid distribution of danofloxacin from the central compartment into peripheral tissues. This initial steep decline was followed by a more gradual reduction in plasma concentrations at later time points, with mean values of 2.95, 1.83, and 1.26 µg/ml at 0.50, 1, and 2 hours, respectively, suggesting a transition from the distribution phase to the elimination phase. Subsequently, plasma concentrations continued to decrease progressively, reaching 0.71, 0.43, and 0.26 µg/ml at 4, 8, and 12 hours, respectively, indicative of sustained elimination of the drug from systemic circulation. The lowest detectable plasma concentration (0.22 µg/ml) was observed at 12 hours in calf no. 3, while the average minimum concentration across all six calves at this time point was 0.26 µg/ml. Plasma drug levels were not detectable in any samples collected beyond 12 hours, including those obtained at 36 and 48 hours (Table1). In the present study, a comprehensive pharmacokinetic evaluation of danofloxacin was conducted following a single intravenous administration at a dose of 5 mg/kg body weight in clinically healthy Mehsana buffalo calves. The elimination rate constant (β) was determined to be 0.13 ± 0.01 h⁻¹, demonstrating a moderate rate of drug removal from systemic circulation. This value is closely aligned with β estimates reported in Nili-Ravi (0.21 ± 0.02 h⁻¹) and Kundhi buffaloes (0.20 ± 0.04 h⁻¹) by Manzoor et al. (2017) [8] , and comparable to that observed in cattle (0.13 ± 0.01 h⁻¹) by Corum et al. (2019) [3] , confirming that danofloxacin displays similar elimination kinetics across large ruminant species. [8] may be attributed to differences in physiological status, metabolic capacity, and hepatic microcirculation between geographically distinct buffalo breeds. Comparatively, large variability in half-life among ruminants such as 4.67 ± 0.45 h in goats (Aliabadi et al., 2003) [2] and 3.27 h in sheep (Escudero et al., 2007) [7] reinforces species-dependent influences on disposition kinetics, while the markedly prolonged half-life reported in cattle (17.47 ± 0.60 h) by Corum et al. (2019) [3] suggests lower total clearance and more extensive distribution in bovines. In this study, the mean residence time (MRT) was calculated to be 5.77 h, signifying that danofloxacin remained within the biological system for a considerable period before complete elimination. MRT results were in agreement with the obtained half-life and indicate favorable pharmacokinetic behavior for achieving therapeutic coverage in systemic infections. The area under the plasma concentration-time curve (AUC₀-∞), an essential indicator of overall drug exposure and bioavailability, was 12.69 ± 0.23 µg•h/mL. This value indicates sustained systemic availability following IV administration and is slightly higher than those documented in Nili-Ravi buffaloes (10.96 ± 2.30 µg•h/mL) and Kundhi buffaloes (8.31 ± 1.39 µg•h/mL) by Manzoor et al. (2017) [8] . [7,2] may be attributed to breed differences, age variation, dose regimens, plasma protein binding affinity, and metabolic activity. Conversely, the higher AUC reported in cattle (15.50 ± 0.20 µg•h/mL) by

Corum et al. (2019) [3] [8] but lower when compared to the values recorded in cattle (Corum et al., 2019) [3] , suggesting that distribution dynamics may vary depending on species-specific anatomical and physiological factors, including extracellular fluid [2,7] , reflecting faster metabolic turnover and renal excretion in small ruminants. In contrast, cattle exhibit slower clearance (0.18 ± 0.01 L/kg/h; Corum et al., 2019) [3] , which contributes to their prolonged elimination half-life. Differences in hepatic enzyme activity, renal blood flow, physiological maturity, disease status, and genetic variability are key contributors to the wide variability in clearance values observed across species, as supported by Elazab et al. (2020) and De la Puente et al. (2024) [6,4] .

Intramuscular Administration

The plasma concentrations of danofloxacin following a single intramuscular dose of 5.0 mg/kg body weight in six buffalo calves, along with the corresponding mean values and standard error (SE), are presented in Table 3. The initial plasma samples collected 5 minutes after intramuscular administration exhibited a relatively high mean concentration of danofloxacin (0.97 µg/ml). The mean plasma drug concentration subsequently increased, reaching a peak value of 2.01 µg/ml at 1 hour, and then declined steadily, suggesting rapid distribution after achieving sufficient plasma levels. At later time points, plasma samples demonstrated a continuous decrease in drug concentration, with a low mean value of 0.22 µg/ml at 24 hours. No drug concentration was detected in plasma samples collected at 36 and 48 hours. Following intramuscular administration of danofloxacin at 5.0 mg/kg body weight in Mehsana buffalo calves, the pharmacokinetic assessment revealed a mean maximum plasma concentration (Cmax) of 2.09 µg/mL, attained at a mean Tmax of 0.83 h, indicating rapid absorption from the injection site. Individual variations were observed, with four calves achieving Cmax at 1.0 h and two at 0.5 h. The Cmax obtained in the present study is consistent with the dosedependent pharmacokinetic behavior of danofloxacin and is higher than that reported at lower dose levels in buffaloes by Sappal et al. (2009) [9] and El-Gendy & Tohamy (2007) [5] . Lower Cmax values have also been documented in goats and sheep (Aliabadi et al., 2003; Escudero et al., 2007) [2,7] , highlighting interspecies differences in systemic exposure, which may be attributed to variation in muscle perfusion, absorption rates, and species-specific physiology.

The elimination rate constant (β) following IM administration was 0.10 h⁻¹, with a corresponding elimination half-life (t½β) of 6.92 h, which was longer compared to the half-life observed after intravenous administration in this study (5.24 h). Such prolongation supports a slower elimination phase and suggests possible flip-flop kinetics, where the absorption rate becomes the rate-limiting determinant for overall drug disposition. The t½β value observed in this study is within the range reported for buffaloes by Sappal et al. (2009) [9] and reflects moderate persistence of danofloxacin in systemic circulation. Comparatively shorter half-lives reported in sheep and goats (Escudero et al., 2007; Aliabadi et al., 2003) [7,2] indicate more rapid elimination in smaller ruminants, whereas the substantially prolonged half-life in cattle (Corum et al., 2019) [3] may reflect slower metabolic and renal clearance in that species. The mean residence time (MRT) of 10.08 h further supports the prolonged systemic exposure achieved via the intramuscular route, contributing to extended antimicrobial action. The extent of systemic exposure quantified through AUC₀-∞ was 17.04 µg•h/mL and the corresponding AUMC was 175.29 µg•h²/mL. These values were notably higher compared to those reported in Nili-Ravi and Kundhi buffaloes by Manzoor et al. (2017) [8] , as well as those documented in goats and sheep ( [3,9,8,2,7] reveals considerable interspecies variability, primarily influenced by lipid solubility, tissue perfusion, extracellular fluid volume, and protein binding dynamics. The mean apparent total body clearance scaled by bioavailability (ClB/F) was estimated at 0.30 ± 0.02 L/kg/h, indicating a moderate rate of drug elimination from the body. This value showed close similarity to clearance reported for buffaloes by El-Gendy & Tohamy (2007) [5] and Manzoor et al. (2017) [8] , suggesting that danofloxacin is efficiently eliminated without excessive accumulation risks. However, differences in clearance between species, particularly the lower clearance in cattle reported by Corum et al. (2019) [3] , can be attributed to species-specific variations in hepatic metabolism, renal blood flow, and overall metabolic activity. These findings emphasize the relevance of pharmacokinetic profiling for dose optimization in food-producing animals to prevent drug residues.

The intramuscular bioavailability (F) of danofloxacin in this study was calculated as 127%, indicating excellent systemic availability and suggesting prolonged absorption kinetics likely due to depot formation at the injection site. A similar high bioavailability trend has been observed in cattle (Corum et al., 2019) [3] , supporting enhanced absorption and sustained systemic persistence in large ruminants. Interstudy variations in bioavailability may also depend on formulation properties, injection site factors, and breed-specific physiological characteristics.

Conclusion

The present study successfully characterized the pharmacokinetics of danofloxacin following single-dose intravenous and intramuscular administration in Mehsana buffalo calves. Danofloxacin displayed rapid systemic distribution and extensive tissue penetration after IV dosing, accompanied by moderate clearance and sustained therapeutic plasma levels. Intramuscular administration resulted in rapid absorption, high systemic exposure, prolonged elimination, and notably high bioavailability, indicating efficient uptake from the injection site. Overall, both administration routes demonstrated favourable pharmacokinetic behaviour, with IM administration offering practical advantages for field use due to excellent absorption and prolonged persistence. These findings support the suitability of danofloxacin for treating systemic and respiratory bacterial infections in buffalo calves and provide valuable pharmacokinetic data for rational dosage optimization in this important indigenous breed.

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