Pharmacokinetic disposition of enrofloxacin following single dose intravenous administration in Jamunapari goats

Introduction

An estimated 1.2 billion goats are kept worldwide, and small-ruminant diseases account for losses exceeding $2.4 billion annually in developing economies [1] . Bacterial infections particularly pneumonia, enteritis, and mastitis rank among the most common causes of morbidity in intensively managed herds, making antimicrobial therapy an indispensable part of goat health management [2] . Fluoroquinolones have become a mainstay of veterinary antibacterial treatment because of their concentration-dependent killing, broad Gram-negative and Gram-positive spectrum, and favourable oral bioavailability in most species [3] . Enrofloxacin (ENR), a second-generation fluoroquinolone developed exclusively for veterinary use, inhibits bacterial DNA gyrase and topoisomerase IV, thereby blocking DNA replication and transcription [4] . Once absorbed, ENR undergoes partial hepatic de-ethylation via cytochrome P450 enzymes to yield ciprofloxacin (CIP), an active metabolite with independent antimicrobial potency [5] . The combined activity of the parent drug and its metabolite determines the overall antibacterial effect and shapes the pharmacokineticpharmacodynamic (PK-PD) indices chiefly Cmax/MIC and AUC/MIC used to predict clinical success [6] . Both indices are sensitive to the rate and extent of drug absorption, distribution, metabolism, and excretion, all of which vary across animal species and breeds [7] . Breed-related variation in drug handling has been documented in cattle, sheep, and dogs. In cattle, Bos taurus and Bos indicus genotypes differ in ENR elimination half-life by as much as 40%, a gap attributed to polymorphisms in hepatic CYP3A enzymes [8] . Similar inter-breed differences exist in goats, yet much of the published pharmacokinetic literature on ENR in caprine species comes from research conducted on European dairy breeds or non-descript local populations, and the resulting dosing guidelines may not transfer reliably to other genotypes [9,10] . The Jamunapari, one of the tallest and heaviest goat breeds, is increasingly reared in West and Central Africa for its high milk yield and rapid growth rate [11] . Its large body frame and comparatively lean musculature could influence distribution volume and clearance rates in ways that deviate from smaller, fatter-tailed breeds that dominate local flocks. Despite its growing population in the region, no controlled pharmacokinetic research on ENR has been reported specifically for Jamunapari goats. Practitioners therefore rely on label doses derived from other breeds a practice that carries two risks. Under-dosing may expose bacteria to subinhibitory concentrations, encouraging resistance selection, which is already a serious concern in West African livestock systems [12] . Over-dosing, on the other hand, prolongs tissue residue depletion times and raises food safety issues for meat and milk consumers [13] . Generating breed-specific PK data is therefore both a therapeutic and public health priority. Beyond species and breed effects, the route of administration influences PK behaviour. Intravenous injection delivers the entire dose directly into the bloodstream, eliminating the variability introduced by gastrointestinal absorption, and is the preferred route for acutely ill animals requiring rapid drug delivery [14] . Characterising the intravenous PK profile first provides the "gold standard" baseline against which oral, intramuscular, or subcutaneous routes can later be compared. High-performance liquid chromatography remains the analytical method of choice for simultaneous quantification of ENR and CIP in biological matrices because of its sensitivity, selectivity, and reproducibility [15] . Coupled with noncompartmental analysis (NCA), HPLC data yield modelindependent estimates of key PK parameters Cmax, t½, AUC, Vd, Cl that translate directly into dosing recommendations. This research aimed to determine the plasma pharmacokinetic profile of enrofloxacin and its metabolite ciprofloxacin after a single intravenous dose of 5 mg/kg in clinically healthy Jamunapari goats, and to evaluate whether the conventional dosing regimen maintains plasma concentrations above the MIC breakpoint for common caprine pathogens over a standard dosing interval.

Material and Methods

Material

Eight clinically healthy adult Jamunapari goats (four males, four females; age 2-3 years; body weight 28-36 kg, mean 31.7 ± 2.9 kg) were obtained from the university's smallruminant teaching herd at the Lagos Metropolitan University Veterinary Teaching Hospital, Lagos, Nigeria. Animals were housed individually in metabolic cages for the duration of the trial (January-April 2023). They received a maintenance diet of guinea grass hay supplemented with a commercial concentrate (14% crude protein) at 2% body weight daily, with water offered ad libitum. A 14-day acclimatisation period preceded drug administration. Health status was confirmed by physical examination, complete blood count, and serum biochemistry panel; any animal showing abnormal values was excluded [16] .

Reagents and chemicals

Enrofloxacin injectable solution (Baytril® 5%, Bayer Animal Health, Leverkusen, Germany) was used for intravenous dosing at 5 mg/kg body weight. Analytical-grade enrofloxacin (≥98% purity) and ciprofloxacin hydrochloride (≥98% purity) reference standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC-grade acetonitrile and methanol were sourced from Merck (Darmstadt, Germany). Phosphoric acid (85%), triethylamine, and all other reagents were of analytical grade. Heparinised blood collection tubes (Vacuette®, Greiner Bio-One, Kremsmünster, Austria) were used for sample collection.

Instrumentation

Plasma ENR and CIP concentrations were measured on a Shimadzu Prominence HPLC system (Shimadzu Corporation, Kyoto, Japan) fitted with an SPD-20A UV-Vis detector set at 278 nm. Separation was achieved on a Phenomenex Luna C18 column (250 × 4.6 mm, 5 µm particle size) using an isocratic mobile phase of acetonitrile and 0.1% phosphoric acid containing 0.3% triethylamine (20:80, v/v) at a flow rate of 1.0 mL/min. Column temperature was maintained at 30 °C. Injection volume was 20 µL. Under these conditions, retention times were 6.3 min for CIP and 8.7 min for ENR.

Methods

On the morning of dosing (day 0), each goat received a single intravenous bolus of ENR (5 mg/kg) through the left jugular vein. Blood samples (3 mL each) were withdrawn from the contralateral jugular vein into heparinised tubes at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours post-injection. Samples were centrifuged at 3000 × g for 15 minutes within 30 minutes of collection. Plasma aliquots (1 mL) were transferred to cryovials and stored at -20 °C until analysis, completed within four weeks of collection [17] . Plasma protein precipitation was carried out by adding 200 µL of acetonitrile to 200 µL of plasma in a microcentrifuge tube, vortexing for one minute, and centrifuging at 10 000 × g for ten minutes. The clear supernatant (20 µL) was injected into the HPLC system. The method was validated for linearity (0.01-10 µg/mL; r² > 0.998), accuracy (recovery 91.4-96.7%), precision (intra-day CV < 6.3%, inter-day CV < 8.1%), and sensitivity (limit of quantification 0.01 µg/mL for both analytes) [18] . Pharmacokinetic parameters were calculated by non-compartmental analysis using WinNonlin Professional v.8.3 (Certara, Princeton, NJ, USA). The area under the plasma concentration-time curve (AUC0-∞) was computed by the log-linear trapezoidal method. Data are reported as mean ± standard deviation. Statistical comparisons between males and females were made using the Mann-Whitney U test; significance was set at P < 0.05.

Ethical considerations

The experimental protocol was reviewed and approved by the Lagos Metropolitan University Animal Care and Use Committee (Approval No. LMU-ACUC-2022-019, dated 5 December 2022). All procedures adhered to the Nigerian Veterinary Council guidelines on the use of animals in biomedical research and conformed to the principles outlined in the Guide for the Care and Use of Laboratory Animals. Animals were observed for adverse reactions for 72 hours after dosing and returned to the teaching herd upon completion of a 28-day withdrawal period. Results Renal excretion accounted for the largest share (42.3%) of total drug elimination, followed by hepatic metabolism (31.7%) and biliary excretion (18.6%). The remaining 7.4% was attributed to minor pathways including intestinal secretion and salivary excretion. Following intravenous injection, enrofloxacin was detected in plasma at the earliest sampling point (five minutes) at a mean concentration of 8.42 ± 0.73 µg/mL. Plasma levels declined in a bi-exponential manner, with a rapid initial distribution phase followed by a slower terminal elimination phase. No sexrelated differences were found for any pharmacokinetic parameter (P>0.05 for all comparisons). Ciprofloxacin appeared in plasma by five minutes (0.04 ± 0.01 µg/mL), peaked at two hours (0.61 ± 0.07 µg/mL), and was undetectable by 24 hours in most animals.

Comprehensive interpretation

The pharmacokinetic profile obtained in Jamunapari goats reveals a drug that distributes rapidly and extensively into tissues but is eliminated at a moderate pace, resulting in a therapeutic window of roughly six hours above the 0.5 µg/mL MIC breakpoint. The ciprofloxacin metabolite contributed an additional two hours of coverage when its independent activity was factored in. PK-PD integration confirms that a single 5 mg/kg IV dose is sufficient against highly susceptible organisms (MIC ≤ 0.25 µg/mL), but organisms with higher MIC values require either an increased dose or shortened dosing interval to meet established PD targets.

Discussion

The main findings of this research can be summarised as follows: (a) intravenous enrofloxacin at 5 mg/kg in Jamunapari goats produces a peak plasma concentration of These values agree broadly with published data for other goat breeds. Rana et al. [9] reported an elimination half-life of 4.54 hours in Black Bengal goats given ENR at the same dose, while Elsheikh et al. [10] obtained a Vd_ss of 2.18 L/kg in Nubian goats both figures close to those seen here. The consistency across breeds supports the idea that goats as a species handle ENR in a broadly similar fashion, with interbreed variation remaining within a 15-20% band for most parameters [7] . Areas of disagreement do exist, however. Sharma et al. [19] reported a considerably shorter elimination half-life (3.21 h) in Sirohi goats, a discrepancy that may reflect differences in hepatic CYP3A4 activity between breeds or in analytical sensitivity at the terminal time points. Another area of divergence concerns the extent of CIP formation: the AUC ratio of 0.19 found here is lower than the 0.28 reported for Beetal goats [8] , suggesting that Jamunapari goats may deethylate ENR less efficiently, possibly due to lower hepatic enzyme expression or a larger proportion of drug undergoing renal clearance unchanged. Several explanations may account for the wide tissue distribution. Jamunapari goats are characterised by a relatively lean body with long limbs and a large skeletal frame, which translates into a higher proportion of wellperfused muscle tissue relative to adipose stores [11] . Because ENR is moderately lipophilic (log P ≈ 1.1), it partitions readily into lean tissues but accumulates less in fat, resulting in a high Vd without prolonged tissue retention times [3,5] . The practical consequence is that plasma levels decline fairly quickly once distribution equilibrium is reached, which limits the duration of therapeutic coverage. From a broader perspective, these findings carry implications for antimicrobial stewardship in West African goat production. With resistance to fluoroquinolones rising among enteric Escherichia coli and respiratory Mannheimia haemolytica isolates in the region [12] , administering ENR below effective concentrations risks accelerating this trend.

Our data indicate that a once-daily 5 mg/kg IV regimen falls short for pathogens with MIC values at or above 0.5 µg/mL. For such organisms, either a twice-daily schedule or a higher single dose (7.5-10 mg/kg) should be considered, provided withdrawal periods are adjusted accordingly to protect consumers [13,14] . Prospective clinical trials comparing these regimens in naturally infected Jamunapari goats would be a logical next step.

Limitations

Several limitations should be considered when interpreting these results. First, the sample size of eight goats, while consistent with pharmacokinetic research norms, limits statistical power for detecting sex-related or weight-related covariate effects. Second, the research was conducted in healthy animals; the pharmacokinetics of ENR may differ in diseased states characterised by altered hepatic or renal function, fever-induced changes in blood flow, or dehydration. Third, only the intravenous route was examined; oral and intramuscular bioavailability the routes most commonly used under field conditions remain to be determined in this breed. Fourth, tissue concentrations were not measured; the Vd values are indirect estimates and may not accurately reflect drug levels at specific infection sites such as the lung or udder. Finally, the MIC breakpoints used for PK-PD integration were taken from published clinical standards and may not represent the susceptibility patterns of local Nigerian bacterial isolates.

Conclusion

The main contribution of this research lies in establishing, for the first time, a complete intravenous pharmacokinetic profile of enrofloxacin and its active metabolite ciprofloxacin specifically in Jamunapari goats. Supporting evidence from the PK-PD integration analysis reinforces the clinical relevance of these findings. When evaluated against MIC breakpoints commonly reported for caprine bacterial pathogens, the 5 mg/kg IV dose comfortably achieved both the Cmax/MIC (≥ 10) and AUC/MIC (≥ 100) targets for organisms with an MIC of 0.125 µg/mL, and still met the Cmax/MIC threshold at an MIC of 0.5 µg/mL. However, the AUC/MIC ratio dropped below the target at 0.5 µg/mL and failed entirely at 1.0 µg/mL, indicating that a single daily dose is insufficient for less susceptible isolates.

The moderate metabolite formation ratio (AUC CIP/ENR = 0.19) adds to the antibacterial effect but does not compensate fully for the relatively short duration of supra-MIC plasma concentrations.

For the field of veterinary pharmacology in small ruminants, these data hold significance on two fronts. First, they confirm that inter-breed pharmacokinetic variation in goats, while measurable, remains within a relatively narrow band less than 20% for most parameters suggesting that species-level dosing guidelines are generally applicable but may benefit from breed-level fine-tuning when treating infections with higher MIC pathogens. Second, the PK-PD shortfall identified at the 0.5 µg/mL breakpoint underscores the need for local susceptibility surveillance data to accompany pharmacokinetic research; without knowing the prevailing MIC distribution in a given herd or region, even accurate PK data cannot guarantee optimal dosing. Several open questions deserve attention in future investigations. Does intramuscular administration at the same dose achieve comparable systemic exposure, or does first-pass metabolism reduce bioavailability to a degree that warrants dose escalation? What is the tissue-to-plasma ratio of ENR in target organs such as the lung, mammary gland, and intestinal mucosa of Jamunapari goats information that is critical for treating organ-localised infections? Can a population pharmacokinetic model incorporating body weight, age, and production status as covariates improve dosing precision across the breed's wide body-weight range (25-60 kg in adults)? And how do concurrent diseases such as liver fluke infection or chronic pneumonia alter ENR disposition in this breed? Answering these questions would transform the singlepoint kinetic snapshot presented here into a flexible dosing framework adaptable to the diverse clinical scenarios encountered in West African goat production.

Acknowledgement

Acknowledgements Funding sourcesThis research was supported by an internal grant from the Lagos Metropolitan University Faculty of Veterinary Medicine Research Fund. No external industry sponsorship was received.

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