Plasma concentrations of fenbendazole (FBZ) and oxfendazole in alpacas (Lama pacos) after single intravenous and oral dosing of FBZ

Abstract

The objective of this study was to determine plasma pharmacokinetics and bioavailability of fenbendazole (FBZ) and oxfendazole (OFZ) after intravenous (iv) and oral administrations of FBZ (5 mg/kg) to alpacas. Plasma concentrations of FBZ and OFZ after administration of FBZ iv and orally (5 mg/kg) were determined by high-performance liquid chromatography with ultraviolet detection. Total clearance (CL) of FBZ was 16.5±4 mL/kg/min (range: 4–31 mL/kg/min), and steady-state volume of distribution (Vd_ss) was 3.3±1 L/kg (range: 1.7–7.4 L/kg). The terminal phase half-life of FBZ after iv administration was 5.9±3.8 hours (range: 0.8–20 hours). After oral administration, the FBZ terminal phase half-life was 23±5 hours (range: 9–37 hours) and the systemic bioavailability of FBZ was 16%±6% (range: 1%–41%). Peak FBZ concentrations after oral administration were 0.13±0.05 µg/mL (range: 0.05–0.28 µg/mL) at 10 hours (range: 8–12 hours). Peak plasma OFZ concentrations after oral dosing with FBZ (5 mg/kg) were 0.14±0.05 µg/mL (0.05–0.3 µg/mL) at 24±7 hours (range: 12–48 hours). FBZ clearance is lower in comparison to that of other species. Systemic availability of FBZ after oral administration is low after oral dosing. Metabolites of FBZ produced by alpacas are similar to those observed in other species.

Keywords:

• bioavailability
• benzimidazoles
• camelid
• pharmacokinetics

Supplementary materials

Apparatus

The LC apparatus consisted of an HPLC pump (Thermo Fisher Scientific, Dionex Bannockburn, IL, USA; 582 solvent delivery module), biocompatible autosampler (Thermo Fisher Scientific; 542 refrigerated), ultraviolet (UV) detector (Thermo Fisher Scientific; 580 dual channel, variable wavelength, UV/visible), thermal control column heater (Thermo Fisher Scientific). The UV detector was set to monitor 292 nm. The column was a C₁₈, 250×4.6 mm, 5 µm particle size, reverse-phase column (Grace Technologies, Deerfield, IL, USA).

Chromatographic conditions

The mobile phase consisted of 0.01 M phosphoric acid (pH 4.0) and acetonitrile (60%:40%, v/v).¹ The vacuum degassed and filtered (0.22 µm pore size filter) mobile phase was delivered at 1.0 mL/min. Column temperature was maintained at 35°C. Data were automatically collected and peaks identified and quantified using Coularray for Windows software (Version 2.0; Thermo Fisher Scientific).

Preparation of fenbendazole and oxfendazole stock solutions

Standards consisted of fenbendazole (FBZ; Sigma Chemical Co, St Louis, MO, USA; F5396) and oxfendazole (OFZ; Research Diagnostics Inc, Flanders, NJ, USA; 1483301). LC-grade acetonitrile, methyl-tert-butyl ether (m-tBE), and phosphoric acid were obtained from Thermo Fisher Scientific (St Louis, MO, USA). All other reagents were of HPLC grade. Ten milligrams of each drug was weighed quantitatively into individual 25 mL volumetric flasks. The drugs were then dissolved in 5 mL DMSO (100%) and further diluted by addition of acetonitrile to 25 mL. These two solutions (0.4 mg/mL; Standard A) were further diluted by placing 1.0 mL of each standard A in a 25 mL volumetric flask and diluting to 25 mL with acetonitrile (0.016 mg/mL FBZ and OFZ; Standard B). Appropriate volumes of A, B were added to 1.0 mL of blank alpaca plasma for preparation of a plasma FBZ/OFZ standard curve covering the range of 0.025 µg/mL to 8.0 µg/mL.

Standard and sample preparation

Plasma from healthy alpacas (not study animals) that were not treated with FBZ or OFZ was used to prepare spiked plasma for development of standard curves. Plasma samples without added drug were prepared to serve as blank controls. A 200 µL volume of 0.1 N ammonium hydroxide was added to control and spiked plasma to increase pH.¹ The samples were vortexed, and 5 mL of m-tBE was added to each tube, capped, and revortexed for 1 minute. Ether-containing samples were centrifuged at 1,000× g for 10 minutes at 4°C to separate the aqueous layer from ether layers. The m-tBE-containing drug was removed, dried under nitrogen at 40°C and reconstituted with 1 mL mobile phase. The samples were vortexed for 30 seconds, and 500 µL was added to autosampler vials and capped. The samples were placed into autosampler tray and 50 µL injected onto chromatograph. Peak area of FBZ and OFZ in standards were determined and used for regression analysis to determine detector response per microgram of analyte. Samples from animals treated with the intravenous formulation and the 10% oral suspension were thawed, 1 mL aliquots placed into individual tubes, and 200 µL of 0.1 N ammonium hydroxide was added and vortexed. Drug was extracted with m-tBE as described within this standard and sample preparation section. All standards and samples were injected in triplicate. Individual standard curves were prepared daily for animal sample analysis. Peak areas were determined for OFZ and FBZ, and the peak areas were used to determine concentration of drug (compared to standard curve) and each concentration for replicate analysis was averaged. Samples that were below the limit of quantitation (LOQ) on first analysis were reextracted, concentrated five-fold, and reanalyzed by HPLC.

Method validation

Standard curves prepared on each day of analysis were compared by determining the intercept, slope, r, and r² using linear regression on six different days. Analyte recovery percentage was estimated on 1 mL of plasma samples spiked at three different concentrations (2 µg/mL, 0.3 µg/mL, and 0.05 µg/mL; six samples per concentration; on three separate days), alkalinized with 0.1 N NH₄OH, and extracted with m-tBE, dried under nitrogen, reconstituted in mobile phase, and injected onto the chromatograph. Peak areas (extracted) were compared to peak areas of identical samples prepared by direct dilution of standards at the same concentrations in mobile phase and subsequent HPLC analysis. $\text{Extraction efficiency }(\%)=\frac{{\text{Peak Area}}_{\text{extracted}}}{{\text{Peak Area}}_{\text{non-extracted}}}\times 100.$

The ratio was used as a measure of analyte recovery for the extraction method. Determination of the LOQ was performed by triplicate injections of standard curves (n=4 on four different days) over the range of concentrations tested (0.025 µg/mL–8.0 µg/mL) and determining the concentration at which the relative standard deviation from the nominal concentration approximated 20%.² The corresponding concentration was taken as the LOQ. The limit of detection (LOD) was determined as the lowest plasma concentration with a signal-to-noise ratio ≥3:1.²

Method accuracy was assessed by preparing replicates (n=6) of three fortification levels (2.0 µg/mL, 0.3 µg/mL, and 0.05 µg/mL of FBZ or OFZ) in 1 mL blank plasma on three separate days. These samples were alkalinized, extracted, and dried as described for standards and samples. The accuracy of the method on a given day was determined by comparing the difference in concentration found to the nominal concentration added and expressing the result as relative standard deviation (%).³ $\text{Within-day accuracy }(\%)=\frac{{\text{FBZ}}_{\text{found}}-{\text{FBZ}}_{\text{added}}}{{\text{FBZ}}_{\text{added}}}\times 100.$

The “between-day” method accuracy at three fortification levels was determined in a similar fashion, except that the difference between the mean of all concentrations on all days (n=3 days) was determined and used to calculate difference between nominal concentrations of FBZ or OFZ added.³

Method precision between and within days was determined using analysis of variance (one-way design) and the expected between-day and within-day mean squares were determined using the following formulae:³ $\text{WD precision }(\%)=\frac{\sqrt{{\text{MS}}_{\text{within-day}}}}{[\text{FBZ}]}\times 100$ $\text{BD precision }(\%)=\frac{\sqrt{{\text{MS}}_{\text{between-day}}}}{[\text{FBZ}]}\times 100.$

Here, WD represents “within-day,” “BD” indicates “between-day,” “MS” represents mean square from the analysis of variance table, and [FBZ] is the mean fenbendazole concentration.

Linearity of standard curves, method accuracy, precision, extraction efficiency, LOQ, and LOD were determined for OFZ in an identical manner.

HPLC–mass spectrometry identification of unknown compound based on metabolite mass

Plasma samples from animals dosed with single doses of FBZ were used to identify an unknown peak, which was present at 24 hours, 48 hours, and 96 hours postdosing. Extracted samples were repeatedly injected (99 µL), and fractions containing the FBZ, OFZ, and unknown metabolite were collected. Each fraction was pooled and reextracted from the mobile phase using m-tBE after addition of 0.1 N ammonium hydroxide. The ether layer was evaporated to dryness and the analyte was resuspended in 200 µL acetonitrile. Reevaluation of fractions by injections of FBZ, OFZ, and unknown peak on the HPLC demonstrated single peaks, with identical retention time corresponding to that sample’s identity. The concentrated fractions were submitted to the Ohio State University Campus Chemical Instrument Center mass spectrometry core for confirmation of molecular mass of this fraction.

Briefly, a Micromass LC-Tof™ II mass spectrometer equipped with an orthogonal electrospray source (Z-spray) was operated in positive ion mode (Micromass, LC-Tof; Waters Corporation, Milford, MA, USA). Sodium iodide was used for mass calibration over a calibration range of m/z 100–2,000. Optimal electrospray ionization (ESI) conditions were capillary voltage 3,000 V, source temperature 110°C, and a cone voltage of 55 V. The ESI gas was nitrogen. All ions transmitted into the pusher region of the time-of-flight analyzer were scanned over m/z 280–600, with a 1-second integration time. Data were acquired in continuum mode during the LC run.

HPLC analysis

The LC/auotsampler system consisted of a Waters Alliance 2690 Separations Module (Waters, Milford, MA, USA). The column used was C₁₈, 250×4.6 mm, 5 µm particle size, reverse-phase column. The mobile phase was altered to 10 mM ammonium acetate, pH 4.0 (35%), and 65% acetonitrile; flow rate was maintained at 1 mL/min and was split postcolumn using a microsplitter valve (Upchurch Scientific, Oak Harbor, WA, USA) to ∼20 µL/min for introduction to the ESI source.

Pharmacokinetic analysis

Zero-time plasma drug concentration intercepts of the biphasic iv disposition curve (A, B) were determined by extrapolation of the plasma fenbendazole concentrations to t=0 for the distribution phase (A) and terminal exponential phase (B). The hybrid rate constants of the biphasic iv disposition curve (α and β) were determined by calculation of the slopes of the distribution and elimination phases, respectively. The first-order elimination rate constant for disappearance of the drug k₁₀ from the central compartment was determined using the formula $k_{10}=\frac{\alpha \times \beta }{k_{21}}$where α and β are the hybrid rate constants for the biphasic distribution curve and k₂₁ is the rate constant for change from the peripheral compartment to central compartment. k₁₂ and k₂₁ are first-order transfer rate constants for drug distribution between the central and peripheral compartments, respectively. These transfer rate constants were calculated from the intercepts of the concentration versus time curve, with the y axis at time =0 for the distribution phase (A) and elimination phase (B), and the hybrid rate constants for the biphasic distribution curve (α, β) were determined using the following relationships: $k_{21}=\frac{(\mathrm{A}\times \beta +\mathrm{B}\times \alpha )}{(\mathrm{A}+\mathrm{B})}$ $k_{12}=\alpha +\beta -k_{21}\times k_{10}.$

The total plasma FBZ clearance was estimated as if it were all from the central compartment using the following relationship: ${\text{CL}}_{\text{tot}}=\frac{{\text{IV}}_{\text{dose}}(\mu \mathrm{g})}{{\text{AUC}}_{0\to \infty }}.$

Here IV_dose is in micrograms and AUC_0→∞ is the area under the concentration versus time curve and calculated from the zero-time concentrations from the distribution and elimination phases of the plasma concentration–time curves (A, B) and the hybrid rate constants for the biphasic distribution curves (α, β) using the following relationship: ${\text{AUC}}_{0\to \infty }=\frac{\mathrm{A}}{\alpha }+\frac{\mathrm{B}}{\beta }.$

The apparent volume of the central (V₁) and peripheral (V₂) compartments and volume of distribution at steady state (V_ss) were estimated from the equation $V_1=\frac{{\text{IV}}_{\text{dose}}(\mu \mathrm{g})}{\mathrm{A}+\mathrm{B}}$where A + B is the zero-time plasma drug concentrations of the disposition phase (A) and elimination phase (B).

Vd_ss was calculated by using the equation $V{\mathrm{d}}_{\text{ss}}=V_1\times \frac{(k_{12}+k_{21})}{k_{21}}$

Distribution half-life (hours) was calculated using the formula $T_{1/2{\lambda }_1}=\frac{\ln 2}{\alpha }$where ln2 =0.693 and α is the slope of the initial log-linear segment of the concentration–time curve. Elimination half-life was calculated as follows: $T_{1/2}=\frac{\ln 2}{\beta }$where ln2 =0.693 and β is the slope of the terminal log-linear segment of the concentration–time curve.

The area under the first moment of the concentration versus time curve (AUMC) from time of dosing to infinity was calculated using the following formula: $\text{AUMC}=\frac{\mathrm{A}}{{\alpha }^2}+\frac{\mathrm{B}}{{\beta }^2}.$

The mean residence time (MRT) was determined from the ratio of AUMC to AUC, as follows: $\text{MRT}=\frac{\text{AUMC}}{\text{AUC}}.$

Oral administration data were analyzed by statistical moment analysis (noncompartmental analysis or NCA; Phoenix WinNonlin Professional (version 6.3); Pharsight Corporation, St Louis MO, USA). Data from the 5 mg/kg dosing (10% oral suspension; n=5) were individually ana-lyzed and compared descriptively. Data were modeled using model NCA 200 with extravascular input. The parent drug (FBZ) was subjected to noncompartmental statistical moment analysis to determine terminal elimination rate constant (λ_z), terminal-phase half life (T_1/2λz), maximum plasma concentration (C_max), time to maximum plasma concentration (T_max), AUC (extrapolated to infinity), AUMC (extrapolated to infinity), and MRT (extrapolated to infinity). The first-order rate constant associated with the terminal log-linear portion of the concentration versus time curve λ_z (h⁻¹) was calculated from individual concentration versus time data using the following formula (minimum of final three data points): ${\lambda }_z=-1({\text{slope}}_{\text{elimination}}).$

Half-life of elimination (hours) was calculated using standard formulae as follows: ${\text{HL}}_{{\lambda }_z}=\frac{\ln 2}{{\lambda }_z}.$

C_max and T_max were taken directly from the concentration versus time data for each animal and time at which maximum concentration was found. AUC was calculated using the following formula: ${\text{AUC}}_{0-\infty }=\delta t\times \frac{C_{\mathrm{n}}+C_{n+1}}{2}.$

Here, n is the number of summed trapezoids formed by n + 1 trapezoids, C reflects corresponding plasma concentrations; (n, n + 1), tn + 1, and δt represent the change in time between these plasma concentrations. AUMC extrapolated to infinity is $\text{AUMC}=\delta t\times \frac{t_1\times C_1+t_2\times C_2}{2}+\frac{t_{\text{last}}\times C_{\text{last}}}{{\lambda }_z}+\frac{C_{\text{last}}}{{\lambda }_z^2}.$

MRT extrapolated to infinity was calculated using the following formula: $\text{MRT}=\frac{\text{AUMC}}{\text{AUC}}.$

Systemic availability of FBZ was determined using the following relationship: $F(\%)=\frac{{\text{AUC}}_{\text{PO}}}{{\text{AUC}}_{\text{IV}}}\times \frac{{\text{Dose}}_{\text{IV}}}{{\text{Dose}}_{\text{PO}}}$where PO stands for oral administration and IV indicates the intravenous mode of administration.

References

• LanduytJDebackereMDelbekeFMcKellarQA high performance liquid chromatographic method for the determination of febantel and its major metabolites in lamb plasmaBiomed. Chromatogr1993778818485378
   PubMed Web of Science ®Google Scholar
• ErmerJValidation in pharmaceutical analysis. Part I an integrated approachJ Pharm Biomed Anal20012475576711248468
   PubMed Web of Science ®Google Scholar
• WernimontGTIntralaboratory development of an analytical processSpendleyWUse of Statistics to Develop and Evaluate Analytical MethodsGaithersburg, MDAOAC International19851130
   Google Scholar

Acknowledgments

Funding for this study was provided by the Morris Animal Foundation, with cosponsorship support of the Llama Association of North America. The authors acknowledge the support of the Department of Veterinary Clinical Sciences and the Camelid Health Program, The Ohio State University. Partial funding for Open Access was provided by The Ohio State University Open Access Fund.

Author contributions

All authors (JL, DL, DEA, and TAS) contributed to the design of this study, which included selection of the animals, instrumenting the animals, administration of drug, and collection of the samples. Two authors (JL and TAS) prepared the samples and analytical equipment for the analysis of drug concentrations, including method validation and analysis of the raw data. All authors (JL, DL, DEA, and TAS) contributed to the final version of the manuscript by reviewing the draft versions, including the final version presented here.

Disclosure

The authors report no conflicts of interest in this work.