Pharmacokinetics of Orbifloxacin in Mehsana Goats after Intravenous and Intramuscular Administration

Single-dose pharmacokinetics of orbifloxacin (2.5 mg/kg bodyweight) were determined in clinically normal female mehsana goats (n=6) following intravenous and intramuscular administration. Orbifloxacin concentrations were determined by high performance liquid chromatography with ultraviolet detection. The concentration–time data were analyzed by non-compartmental kinetic method. Following a single intravenous injection, an elimination half-life (t1/2β) of 8.63 ± 0.130 h. Steady-state volume of distribution (Vdss) and total body clearance (ClB) were 2.99 ± 0.038 L/kg and 0.187 ± 0.002 L/kg/h, respectively. Following intramuscular administration, the elimination rate constant (β), the area under the curve from zero to infinity (AUC0-∞) and the mean absorption time (MAT) were 0.019 ± 0.001 h-1, 19.66 ± 0.216 μg·h/mL and 7.618 ± 0.549 h, respectively. The peak plasma concentration (Cmax) of 1.76 ± 0.010 μg/mL was achieved at 1.00 ± 0.00 h. The mean residence time (MRT) was 21.07 ± 0.478 h and the absolute bioavailability were 155.5 ± 2.487%, respectively. Orbifloxacin could be useful for the treatment of bacterial infections in goats that are sensitive to this drug. Pharmacokinetics of Orbifloxacin in Mehsana Goats after Intravenous and Intramuscular Administration


Introduction
Fluoroquinolones developed over the past few years have greater potency, a broader spectrum of activity, greater in vivo efficacy against resistant organisms and possess a better safety profile than other antimicrobial agents. Orbifloxacin is a third generation synthetic fluoroquinolone, an antimicrobial drug developed extensively for use in veterinary medicine [1,2] and it exhibits high bactericidal activity against Gram-positive, Gram-negative and Mycoplasma spp. [3,4] ( Table 1). It is commonly used for infections of the skin.

Experimental animals
Twelve healthy adult mehsana goats weighing between 25-35 kg and aged 2-4 years were used in the present study. The animals were procured from Livestock Research Station, Sardarkrushinagar Dantiwada Agricultural University, Sardarkrushinagar, Gujarat, India. Sheep were kept at optimal nutritional conditions and had access to ad libitum water. The animals were kept under observations throughout the study period. The experimental protocol was approved by Institutional Animal Ethics Committee of Sardarkrushinagar Dantiwada Agricultural University, Sardarkrushinagar, Gujarat, India.

Drugs and chemicals
Orbifloxacin was obtained from Intas pharmaceuticals Ltd., Ashram road, Ahmedabad. Water, methanol, potassium dihydrogen orthophosphate, acetonitrile, orthophosphoric acid, disodium hydrogen phosphate and triethylamine of HPLC grade were purchased from S. D. Fine Chem. Ltd, Mumbai.

Experimental design and dosing
Twelve animals were divided into two groups -Group I and Group II as 6 animals in each group. Single dose intravenous pharmacokinetics study (Group I) and single dose intramuscular pharmacokinetic study (Group II) of orbifloxacin were performed in mehsana goats. Group I animals were administered with orbifloxacin at 2.5 mg/kg body weight by intravenous route and Group II animals were administered with orbifloxacin at 2.5 mg/kg body weight by intramuscular route.

Analytical method
Plasma samples were analyzed by using HPLC system equipped with G1312A pump, UV detector, SIL6B autoinjector and CTO6A Plasma proteins were precipitated by adding 1000 µL plasma and 1000 µL acetonitrile. The mixture was vortexed for 10 s and centrifuged at 1600 rpm for 10 min. Then, 250 µL of supernatant was diluted with 750 µL 0.067 M disodium hydrogen phosphate buffer (pH 7.5). Chromatographic separation was performed by using Chromatopak Peerless Basic C 18 column (250 × 4.6 mm, 5 µ) at 40°C with an injection volume of 80 µL. The mobile phase (43:57) consisted of 430 volumes of buffer and 570 volumes of methanol with a flow rate of 1.5 mL/min. Buffer was prepared by dissolving 6.8 g of potassium dihydrogen orthophosphate in 1000 mL of water and by adding 3 mL of triethylamine. Then, pH adjusted to 2.5 with orthophosphoric acid. UV detection was performed at a wavelength of 320 nm.

Validation of analytical methods
Blank plasma samples were analyzed to check the absence of interference in the elution position of orbifloxacin. Stock solution of 1 mg/mL orbifloxacin was prepared by dissolving 100 mg powder form of pure drug in 100 mL of HPLC water. Further dilutions of the stock solution were prepared and spiked into blank plasma to produce calibration curves at different concentrations. Linearity was determined by spiking concentrations of orbifloxacin between 0.0625 µg/mL and 20 µg/mL. The mean correlation coefficient (R 2 ) was 0.999. Standard curves were obtained by orbifloxacin peak area and known concentrations. The retention time of orbifloxacin was 2.43 min [20]. The detection limit and quantification limit were determined by analysis of spiked samples at low orbifloxacin concentration.

Pharmacokinetic analysis
Pharmacokinetic parameters were calculated from plasma concentration of orbifloxacin by software PK solution (version 2.0), Summit research services, USA. This program uses non-compartmental model of pharmacokinetic analysis of orbifloxacin.

Results
No local or system adverse reaction was observed after intravenous or intramuscular injection of orbifloxacin. A semi-logarithmic plot of the mean concentration of orbifloxacin in the plasma following intravenous and intramuscular administration of 2.5 mg/kg is shown in Figure 1.
A summary of the pharmacokinetic parameters following intravenous and intramuscular administrations is listed in Table 2. In goats, orbifloxacin showed a range of Vd ss in between 2.85 to 3.09 L/kg and a range of Cl B in between 0.181 to 0.194 L/kg/h after intravenous administration. Orbifloxacin was absorbed rapidly after intramuscular administration and the C max range was (1.72 to 1.79 µg/mL) attained after 1.00 ± 0.00 h (T max ) after injection (Figure 1). The MAT range was 5.21 to 9.13 h. The systemic bioavailability range after intramuscular administration was 147.3 to 165.2%.
In contrast, a much shorter half-life of elimination of orbifloxacin was recorded in Japanese quail (1.71 h); [17].
The elimination half-life after intramuscular administration (17.77 h) was much longer than after intravenous administration (8.63 h) and the slower elimination of orbifloxacin from plasma after intramuscular treatment suggested a 'flip-flop' effect [22]. This would mean that the terminal phase of the plasma concentration-time curve was determined by the absorption rate constant and not by the apparent elimination constant.

Conclusions
Orbifloxacin showed favorable pharmacokinetic properties, such as a long half-life and high bioavailability, with no obvious adverse reactions. This drug could therefore be an effective treatment in goats for a number of bacterial infections. However, further studies are needed to establish a multiple dosage regimen and clinical efficacy against susceptible organisms that infect goats.

Conflict of Interest Statement
None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of this paper.