Comparative pharmacokinetics of levamisole-oxyclozanide combination in sheep and goats following per os administration

Experimental animals

Eight goats [mean body weight (BW): 23.1 ± 4.2 kg] and 8 sheep (mean BW: 24.8 ± 2.9 kg) that were 12- to 16-months old were used in this investigation. They were housed and fed with wheat straw, fodder, and concentrate feed. Water was supplied ad libitum. This study was approved by Animal Ethic Committee of University of Adnan Menderes.

Treatment and sampling

The animals received tablet formulation of LVM and OXZ combination (Zelensin, 375 mg LVM HCl + 750 mg OXZ, Sanovel, Istanbul, Turkey) orally at a dose of 7.5 mg/kg BW and 15 mg/kg BW, respectively. Heparinized blood samples (5 mL) were collected by jugular venipuncture prior to drug administration then at 5, 10, 15, and 30 min, 1, 1.5, 2, 4, 8, 12, 16, 24, 32, 48 h and 3, 4, 5, 6, and 8 days.

Analytical procedures

The plasma concentrations of LVM and OXZ were analyzed by high-performance liquid chromatography (HPLC) following liquid-liquid phase extraction. Stock solutions (100 μg/mL) of pure standard of LVM hydrochloride and OXZ (Sigma, St. Louis, Missouri, USA) were prepared using acetonitrile and water (50:50) as the solvent. These were diluted to give 0.01, 0.05, 0.1, 0.5, 1, 5, 10, and 20 μg/mL standard solutions for plasma for calibration as standard curves and to add to drug-free plasma samples to determine the recovery of both molecules.

Extraction from plasma

Plasma concentrations of LVM and OXZ were determined by HPLC with ultraviolet detection according to methods previously described by Garcia et al (19) and Jo et al (20) with some modifications, respectively.

For LVM analysis, drug-free plasma samples (1 mL) were spiked with standard of LVM to reach the following final concentrations: 0.025, 0.05, 0.1, 0.5, 1 and 5 μg/mL. Water (1 mL) and 0.5 mL of 10 N sodium hydroxide were added to 10 mL-ground glass tubes containing 1 mL spiked or experimental plasma samples. After mixing by vortex for 15 seconds, 6 mL ethyl ether:nhexane (80:20, vol/vol), was added. The sample tubes were stoppered and shaken for 10 min on a rotary mixer. After centrifugation at 3000 × g for 10 min, the upper organic phase was transferred to a thin-walled 10 mL-conical glass tube and evaporated to dryness at 40°C in a rota vapor (Maxi-Dry plus, Heto, Denmark). The dry residue was reconstituted with 250 μL mobile phase. Then, the tubes were placed in an ultrasonic bath and finally, 50 μL of this solution was injected into the chromatographic system.

For OXZ analysis, drug-free plasma samples (1 mL) were spiked with standard of OXZ to reach the following final concentrations: 0.05, 0.1, 0.5, 5, 10, and 20 μg/mL. Acetonitrile (2 mL) was added, and the sample was mixed by vortex (10 s). Anhydrous sodium sulphate (1 g) was added, and the sample tubes were stoppered and shaken for 10 min on a rotary mixer. The samples were centrifuged at 3000 × g for 10 min thereafter, and extracted once again with 2 mL of acetonitrile. The organic layers were recovered after 20 min of centrifugation at 3000 × g. Then n-hexane (5 mL) was added to the combined supernatant. After shaking, the upper phase was discarded after 10 min of centrifugation at 3000 × g. The lower phase was transferred to a thin-walled 10 mL-conical glass tube and evaporated to dryness at 40°C in a rota vapor (Maxi-Dry plus). The dry residue was reconstituted with 250 μL mobile phase. Then, the tubes were placed in an ultrasonic bath and finally, 50 μL of this solution was injected into the chromatographic system.

High-performance liquid chromatography system

The mobile phase consisted of acetonitrile and water (2% acetic acid) (20:70, v/v) and was delivered (1100 Series, QuatPump Agilent, Waldron, Germany) at an isocratic flow rate of 1 mL/min. A nucleosil C18 analytical column (Luna, 4 μm, 150 mm × 4.6 mm; Phenomenex, Macclesfield, Cheshire, UK) with nucleosil C18 guard column was used for analysis of the molecules. Ultraviolet detection (1100 Series, Agilent, Waldron, Germany) was at a wavelength of 225 nm for LVM analysis.

Oxyclozanide were determined using same HPLC system and eluted with a mobile phase consisting of a mixture of acetonitrile and 0.1% phosphoric acid (40:60, v/v). The isocratic mode was run at a flow rate of 1 mL/min and an ultraviolet detector was operated at 300 nm.

Method validation

The analytical methods used for LVM and OXZ in plasma samples were validated before analysis of the experimental samples. The analytes were identified with the retention time of pure reference standards. Recoveries of the molecules under study were measured by comparison of the peak areas from spiked plasma samples with the areas resulting from direct injections of standard solutions. The inter- and intra-assay precisions of the extraction and chromatography procedures were evaluated by processing replicate aliquots of previously drug-free goat and sheep plasma samples containing known amounts of the drugs on different days.

The calibration graphs for LVM and OXZ were prepared (linear range 0.025 to 10 μg/mL). The slopes of the lines between peak areas and drug concentrations were determined by least squares linear regression and correlation coefficient (r) and coefficient of variations (CV) calculated. Linearity was established to determine the drug concentration/detector response relationship. The detection limits of the both LVM and OXZ were established with HPLC analysis of blank plasma fortified with the standard, measuring the baseline noise at the retention time of the peak. The mean baseline noise at the peak retention time plus 3 standard deviations was defined as the detection limit (LOD). The mean baseline noise plus 6 standard deviations was defined as the limit of quantification (LOQ).

Pharmacokinetic and statistical analysis of data

The plasma concentration versus time curves obtained after each treatment in individual animals, were fitted with the WinNonlin software program (Version 5.2; Pharsight Corporation, Mountain View, California, USA). Pharmacokinetic parameters for each animal were analyzed using non-compartmental model analysis with extravascular input for LVM and OXZ. The maximum plasma concentration (Cmax) and time to reach maximum concentration (tmax) were obtained from the plotted concentration-time curve of each drug in each animal. The trapezoidal rule was used to calculate the area under the plasma concentration time curve (AUC). The mean residence time (MRT) was calculated as:

MRTlast = AUMClast/AUClast

Terminal half-life (t1/2λz) was calculated as:

t1/2λz =  - ln(2)/λz

Where: λz represents the first-order rate constant associated with the terminal (log linear) portion of the curve.

The pharmacokinetic parameters obtained from each animal were reported as a median with interquartile ranges (Q1–Q3) and statistically compared by the Mann-Whitney U-test between sheep and goats. The values were considered significantly different at P < 0.05.

Results

The analytic procedures and HPLC analysis of LVM and OXZ were validated before analysis of experimental samples. Mean recoveries of LVM and OXZ from plasma were 86.31% and 74.12% with a relative SD < 10%, respectively. The limit of detections and limit of quantification for LVM and OXZ were 0.014 to 0.043 μg/mL and 0.011 to 0.032, respectively. The inter-assay and intra-assay precisions of the extraction and chromatography procedures were evaluated by processing on different days 6 replicate aliquots of drug-free sheep or goat plasma samples that contained known amounts of LVM and OXZ. The precision determined at each concentration was < 15% of the coefficient of variation, and accuracy ranged from 92% to 106% for both molecules.

Median (with Q1–Q3 ranges) pharmacokinetic parameters of LVM and OXZ combination in goats and sheep are shown in Table I with the plasma concentration versus time curves (Figures 1 and ​and2,2, respectively). This study indicated that the plasma dispositions of both LVM and OXZ in goats were significantly different to those observed in sheep following oral administration at same dose rates. Although no significant difference was found for maximum plasma concentration (Cmax: 1.63 μg/mL versus 1.07 μg/mL), significantly larger area under the concentration vs. time curve (AUC: 8.75 μg.h/mL versus 2.09 μg.h/mL), longer terminal half-life (t1/2λz: 7.91 h versus 3.81 h) and mean residence time (MRT: 7.11 h versus 2.69 h) were observed in sheep as compared with those observed in goats for LVM, respectively. In addition, Cmax (11.01 μg/mL versus 6.68 μg/mL), AUC (488.70 μg.h/mL versus 309.33 μg.h/mL) and t1/2λz (21.74 h versus 18.71 h) values of OXZ in sheep were significantly higher and longer compared with those values observed in goats, respectively.

Figure 1