Improvement of Albendazole Bioavailability with Menbutone Administration in Sheep

Simple Summary Anthelmintic drugs are among those most widely used in veterinary practice. The development of resistance to these drugs is a widespread problem, especially in small ruminants, and represents a serious threat to animal production. Thus, new possibilities to use the available pharmacological groups in a more efficient way should be explored. The objective of this study was to assess the pharmacokinetic interaction between a benzimidazole (albendazole, ABZ) and a choleretic drug (menbutone, MEN) in sheep, and it may result in greater effectivity of this drug against nematode parasites. Plasma concentrations of ABZSO (ABZ active metabolite) were higher when ABZ was administered with MEN. The proposed interaction is a simple, safe, and inexpensive way of increasing the effectivity of this anthelmintic widely used in livestock. Abstract The pharmacokinetic interaction between a benzimidazole (albendazole, ABZ) and a choleretic drug (menbutone, MEN) was evaluated in sheep. The plasma disposition of albendazole sulfoxide (ABZSO, active metabolite) and albendazole sulfone (ABZSO2, inactive metabolite) was investigated following an oral administration of albendazole (ABZ) (5 mg/kg) alone or with menbutone (MEN) (intramuscular, 10 mg/kg). Blood samples were collected over 3 days post-treatment, and drug plasma concentrations were measured by high performance liquid chromatography (HPLC). ABZSO was measured from 0.5 to 48 h, and ABZSO2 from 2 to 60 h. No parent drug was detected at any sampling time. Mean maximum plasma concentration (Cmax) and the area under the plasma concentration-time curve (AUC) were 12.8% and 21.5% higher for ABZSO when ABZ and MEN were administered together, which indicates a significant increase in the amount absorbed. The rate of absorption was not modified, with similar values for the time to reach Cmax (tmax) (11.5 h with ABZ + MEN and 10.7 h with ABZ treatment), although no significant differences were observed for these latter pharmacokinetic parameters. Regarding ABZSO2, Cmax, AUC and tmax values were similar after both treatments (ABZ or ABZ + MEN). The results obtained indicate that co-administration of ABZ and MEN may be an interesting and practical option to increase the efficacy of this anthelmintic.


Introduction
Albendazole (ABZ) is one of the most extensively used anthelmintic drugs in ruminants as it exhibits high efficacy against a broad spectrum of gastrointestinal and bronchopulmonary worms, little toxicity, and low cost. As with other benzimidazoles, its low aqueous solubility is a major disadvantage, since ABZ is poorly absorbed after oral administration, which limits its clinical use [1,2]. Benzimidazoles bind to parasite β-tubulin, necessary for the formation of microtubules, inhibiting all microtubule-based processes in the parasites, which leads to worm death [3,4].
The development of resistance to the available classes of anthelmintic drugs is a widespread problem, especially in small ruminants, and represents a serious threat to animal production. A rational use of ABZ, based on a better knowledge of its pharmacological properties together with appropriate management strategies in livestock, would be a feasible approach to maintain its therapeutic potential and protect animal health.
The lack of new pharmacological groups to treat parasitosis in veterinary medicine means that the currently available groups should be used in a much more efficient way. The rate of dissolution in the gastrointestinal tract of benzimidazoles is thought to be critical in achieving adequate absorption and, consequently, clinical efficacy. Different approaches have been assessed to increase its oral bioavailability, including alternative drug formulations to improve aqueous solubility [2,21], interferences in liver biotransformation [15,22], and non-chemical strategies, such as management of feed intake [23,24] or prior fasting to administration [25,26]. Although these approaches have improved the knowledge on the pharmacokinetic behavior of ABZ, none of them has been finally translated into the SPC of the medicinal products containing ABZ and thus, to daily clinical practice.
Previous studies have shown that the surfactant effect of the bile salts may be related to a higher dissolution of ABZ in the gastrointestinal tract and, consequently, to an increased absorption of this drug [27][28][29]. Solubility, but not absorption, was the rate limiting step in the bioavailability of this anthelmintic [30,31].
In this context, we hypothesize that ABZ oral bioavailability may be modified by increasing its solubility and intestinal absorption with the coadministration of menbutone (MEN), a choleretic drug commonly used in sheep. MEN, also known as genabilic acid, increases bile, pancreatic, and peptic secretions by 2-5 times baseline. The effect is observed in a few minutes after administration, and remains for 2-3 h [32,33]. Menbutone has been used in veterinary practice for years to treat digestive disorders. This compound is indicated to stimulate hepato-digestive activity in case of digestive disorders and hepatic insufficiency in sheep, cattle, and goats. In the last decade, several veterinary medicines containing this active ingredient have been approved for use in most EU countries (by national authorization or mutual-recognition procedures) [34,35].
Thus, the present study was carried out to evaluate the potential in vivo drug-drug interaction after the co-administration of ABZ and MEN in sheep, and to assess if MEN could affect the pharmacokinetic profile of this anthelmintic and its metabolites. The plasma concentrations of ABZ, ABZSO, and ABZSO 2 were evaluated after administration of albendazole, either alone or concomitantly with menbutone.

Animals, Experimental Design and Sampling Procedures
Twelve healthy adult (4-5 years old) non-pregnant and non-lactating female Churra sheep, weighing 50-55 kg, were used. They were allocated in the Experimental Farm of the University of Leon. Sheep were housed indoors in an adequately ventilated building (temperature 19 ± 2 • C). They were allocated for 15 days before the trial began to allow them to acclimate to their environment and maintained in these housing conditions until the end of the experiment. Animals' health was closely monitored before and throughout the experimental period by a veterinarian. They were maintained on a diet of hay and pelleted feed concentrate twice a day, with water and saltlick ad libitum.
Commercially available formulations of ABZ (Sinvermin ovino ® , Laboratorios Syva S.A.U., Leon, Spain) and MEN (Digestosyva ® , Laboratorios Syva S.A.U., Leon, Spain) were administered to animals. A randomized 2-period crossover design was carried out. Sheep were divided into two groups of six animals each. Group 1 received first ABZ orally at a dose of 5 mg/kg. The drug was administered as oral suspension with a dosing device. After a 2-week washout period, menbutone (MEN) (10 mg/kg) was administered into the deep gluteal muscle of the right hind limb immediately after oral ABZ (5 mg/kg). In group 2, animals received concomitant ABZ (oral) and MEN (intramuscular) treatment first and, after a 2-week washout period, oral ABZ, at the same doses previously described.
Animal procedures and management protocols were authorized in advance by both the Ethics Committee of the University of Leon and the regional authorities (ULE-007-2020). No invasive procedure was involved beyond blood sampling.

Reagents
Pure reference standards of ABZ, ABZSO, ABZSO 2 , and oxibendazol (internal standard, IS) were purchased from Sigma-Aldrich (Merck, Darmstadt, Germany). Acetonitrile and methanol solvents used for drug extraction and analysis were HPLC grade, and obtained from Merck (Darmstadt, Germany). Ultrapure water was produced in our laboratory by using a Millipore Milli-Q Gradient water purification system (Waters Corporation, Mildford, MA, USA).

Analytical Procedures
Extraction of ABZ and metabolites (ABZSO and ABZSO 2 ) from plasma samples was performed within a maximum of 6-8 weeks. Plasma samples (1 mL) were spiked with 40 µg IS (25 µg/mL). Experimental and spiked plasma samples were analyzed by high-performance liquid chromatography (HPLC) according to a method previously validated [30].
A solid-phase extraction (SPE) procedure was carried out to extract ABZ and metabolites from plasma samples. Cartridges (Oasis HLB 1 cc 30 mg, Waters Corporation, Mildford, MA, USA) were conditioned with 1 mL of methanol and 1 mL of water. Then, 1 mL of plasma was added, and cartridges were washed with 3 mL of water, dried with air for 5 min, and eluted with 2 mL of methanol. The eluate was evaporated to dryness under nitrogen stream. The residue was then reconstituted with 0.25 mL of mobile phase, and 50 µL were injected into the chromatographic system. Samples were analyzed for ABZ and metabolites in a HPLC system Waters Alliance e2695 equipped with photodiode array detector (model 2998) (Waters Corporation, Mildford, MA, USA). Chromatographic separation was performed by using an XBridge C 18 column (4.6 mm × 250 mm internal diameter, 5 µm, Waters). The PDA detector was set up at 292 nm. The mobile phase (acetonitrile: ammonium acetate buffer 0.025 M, pH = 6.6) was pumped with variable gradient during the run (12 min). Gradient elution changed from 27:73 to 50:50 in 5 min, maintaining it for 4 min, and returning to 27:73 in 1 min, in which was maintained for 2 min. Flow rate was 1.2 mL/min and injection volume 50 µL. In these conditions, retention times were 8.5 min for ABZ, 3.5 min for ABZSO, 4.9 min for ABZSO 2 , and 7. The following parameters were established according to European Medicines Agency guideline EMA/CHMP/EWP/192217/2009: selectivity, lower limit of quantification (LLOQ), calibration curve, accuracy, precision, and stability [37].
Calibration curves (0.025 to 2 µg/mL) were linear for each analyte, with correlation coefficients in the range of 0.995 to 0.999. Recovery percentages were 100.1%, 99.5%, and 95% for ABZSO, ABZSO 2 , and ABZ, respectively. The limits of quantification (LLOQ) were 0.025 µg/mL for the three compounds. No interferences among analytes were observed under current chromatographic conditions. ABZ and their metabolites in working standard solutions and samples were stable for all temperatures and times analyzed (at room temperature for 24 h, at 4-8 • C for 48 h, and at −20 • C for 7 days), with CV always <15% of the nominal concentration (see Figure S1 and Tables S1 and S2, supplementary file).

Pharmacokinetic Analysis
The pharmacokinetic parameters of albendazole for each sheep individually were performed by non-compartmental analysis using Phoenix WinNonLin 8.3 (Certara, Princeton, NJ, USA), with expressions based on statistical moments theory [38] and standard formulae [39,40]. The elimination rate constant (λ), terminal elimination half-life (t 1/2λ ), area under the curve (AUC), area under the moment curve (AUMC), mean residence time (MRT), and the time of observation prior to the first observation with a measurable concentration (t lag ) were calculated. Plasma elimination rate constant (λ) was estimated by least squares regression of the logarithm of plasma concentration versus time curve over the terminal elimination phase, and t 1/2λ as 0.693/λ. AUC and AUMC were calculated by the trapezoidal rule from the time of treatment administration to the last measurable concentration, and further extrapolated to infinity by dividing the last experimental concentration by the terminal slope (λ). MRT was calculated as AUMC/AUC. Maximum plasma concentration (C max ) and the time to reach C max (t max ) were determined by direct observation of the plasma concentration-time curves.

Statistical Analysis
Pharmacokinetic parameters were calculated for each sheep and reported as mean ± standard deviation (SD). The statistical analysis was performed using the IBM SPSS for Windows software package v. 26 (IBM Corporation, Armonk, NY, USA). Shapiro-Wilk test was used to test for normality. If data were normal, a paired t test was used to evaluate differences between data sets; if not, a Wilcoxon signed-rank test was used. Values were considered significantly different at p ≤ 0.05.

Results
No adverse response was observed in animals for any of the treatments during the study. The parent drug was not detected in any plasma sample after ABZ oral administration to sheep, and only its metabolites (ABZSO and ABZSO 2 ) were recovered from samples.
Mean and individual plasma concentrations of ABZSO and ABZSO 2 as a function of time are shown in Figures 1 and S2, respectively, as well as in Tables S3-S6 (see supplementary file). ABZSO (active metabolite) was detected in plasma between 0.5 and 48 h after ABZ or ABZ + MEN administrations. ABZSO 2 (inactive metabolite) was present for a longer time when ABZ was administered alone (2-60 h) than after ABZ + MEN administration (4-48 h). ABZSO mean concentrations were always higher than those detected for ABSZO 2 until 30 h, falling below those values determined for the inactive metabolite from this sampling time onwards. This trend is also observed in most of the individual plasma concentration-time curves.
ABZSO displayed similar plasma concentration profiles when the parent drug was administered alone or concomitantly with MEN, but higher concentrations were achieved when both drugs (anthelmintic and choleretic) were associated, as shown in mean and most individual plasma concentration-time curves (Figures 1 and S2). ABZSO (active metabolite) was detected in plasma between 0.5 and 48 h after AB or ABZ + MEN administrations. ABZSO2 (inactive metabolite) was present for a long time when ABZ was administered alone (2-60 h) than after ABZ + MEN administratio (4-48 h). ABZSO mean concentrations were always higher than those detected for ABSZO until 30 h, falling below those values determined for the inactive metabolite from th sampling time onwards. This trend is also observed in most of the individual plasm concentration-time curves.
ABZSO displayed similar plasma concentration profiles when the parent drug w administered alone or concomitantly with MEN, but higher concentrations were achieve when both drugs (anthelmintic and choleretic) were associated, as shown in mean an most individual plasma concentration-time curves (Figures 1 and S2).
The mean non-compartmental pharmacokinetic parameters calculated for ABZS after administration of ABZ or ABZ + MEN are summarized in Table 1. There we significant differences in the amount of ABZSO generated following the administration ABZ + MEN versus ABZ alone. When the parent compound was administered with t choleretic drug, Cmax increased significantly (1.48 µ g/mL with ABZ alone vs. 1.67 µ g/m with ABZ + MEN). This concomitant administration also produced significantly high values of AUClast (34.0 µ g·h/mL with ABZ alone vs. 41.3 µ g·h/mL with ABZ + MEN). T same behavior was observed in most of the animals, Cmax increased in 10 anima diminished in 1 animal, and did not change in 1 sheep; and AUClast increased in 9 anima and was lower in 3 sheep. The mean non-compartmental pharmacokinetic parameters calculated for ABZSO after administration of ABZ or ABZ + MEN are summarized in Table 1. There were significant differences in the amount of ABZSO generated following the administration of ABZ + MEN versus ABZ alone. When the parent compound was administered with the choleretic drug, C max increased significantly (1.48 µg/mL with ABZ alone vs. 1.67 µg/mL with ABZ + MEN). This concomitant administration also produced significantly higher values of AUC last (34.0 µg·h/mL with ABZ alone vs. 41.3 µg·h/mL with ABZ + MEN). The same behavior was observed in most of the animals, C max increased in 10 animals, diminished in 1 animal, and did not change in 1 sheep; and AUC last increased in 9 animals, and was lower in 3 sheep. Table 1. Non-compartmental pharmacokinetic parameters (mean ± SD) of albendazole sulfoxide (ABZSO) obtained after oral ABZ administration (5 mg/kg) and oral ABZ (5 mg/kg) + intramuscular MEN (10 mg/kg) administration to 12 sheep. Regarding absorption rate, it did not change as no significant differences were observed in t max mean values (10.7 h vs. 11.5 h). The same situation was also observed when individual plasma concentration-time curves were considered, in which the same number of animals showed a slight increase or decrease in t max values.

Parameters
For ABZSO 2 (inactive metabolite), plasma concentration profiles were similar when ABZ was administered alone or with MEN (Figure 1), and no significant differences were observed for C max , AUC last , and t max ( Table 2). Interindividual variations can be seen in Figure S2: in approximately half of the animals, there is an increase, and in the other half, a decrease in both C max and AUC last . t max values remain unchanged in most animals, with slight increases or decreases in the others. Table 2. Non-compartmental pharmacokinetic parameters (mean ± SD) of albendazole sulfone (ABZSO 2 ) obtained after oral ABZ administration (5 mg/kg) and oral ABZ (5 mg/kg) + intramuscular MEN (10 mg/kg) administration to 12 sheep.

Discussion
ABZ was not detected in any plasma sample. Its extensive metabolization to ABZSO and ABZSO 2 , and the plasma profiles obtained in this study for both metabolites after oral administration to sheep are consistent with those previously published in other studies [11,14,21,[41][42][43][44][45].
After MEN administration, plasma concentration profiles and pharmacokinetic parameters obtained for the active metabolite (ABZSO) indicate that the amount absorbed increases significantly (C max and AUC last ) without modifying its rate (t max ). MEN increased ABZSO C max by 12.8% and AUC last by 21.5%.
Pharmacokinetic parameters obtained in our study are compared only with those indicated by other authors after ABZ administration by the oral route to sheep at the same dose (5 mg/kg), as a lack of proportionality was described for some ABZSO parameters (C max and AUC), with increasing dosage in this animal species [45].
Regarding ABZSO pharmacokinetic parameters, mean values calculated in this study for AUC last after ABZ administration alone were slightly higher than that reported in adult animals (32.7 µg·h/mL) [46], and larger than those obtained in lambs (23.2-24.2 µg·h/mL) [9]. C max values are between those calculated by other authors (1.30-2.03 µg/mL) [43,46,47] in adult sheep, but higher than in lambs (1.27-1.35 µg/mL) [9,47,48]. As for t max , our values were always within the range indicated by other authors in both adult sheep and lambs (8-12.5 h) [9,43,46,47]. ABZSO appeared before in plasma in our study, with a t lag (0.27 h) shorter than that indicated by Lanusse et al. [43] in adult sheep (0.8 h) after ABZ administration. As for MRT last , our values were also similar to those calculated in this animal species (13.2-18.1 h) [9,46].
The greater amount of ABZSO after ABZ + MEN administration appeared to be related to a higher absorption of ABZ, and not due to a delayed elimination, as MRT 0-∞ and elimination half-life were not significantly different from values obtained when ABZ was administered alone. C max significantly increased by 12.8% when MEN was co-administered with ABZ, although further studies should be developed to confirm its clinical significance.
MEN may positively affect the absorption of ABZSO from the intestinal lumen due to its choleretic properties, increasing bile secretion to intestine and thus, the extent of ABZSO absorbed due to its surfactant capacity. On the other hand, taking into account that this metabolite undergoes enterohepatic recirculation, it would lead to greater and sustained intestinal and plasma concentrations of ABZSO. These higher levels will improve its antihelmintic activity against gastrointestinal worms, where some of the most pathogenic parasites of ruminants are located [49,50], and against lung helminths, as diffusion through external surface is the main mechanism of drug entry to parasites.
Regarding ABZSO 2 , as mentioned above, its concentration-time pattern is also similar to those obtained in previous studies [11,21,25,[41][42][43]. In our study, ABZSO 2 pharmacokinetic parameters show no significant variation in either the amount or the rate of incorporation of this metabolite into the blood.
AUC last mean value calculated in this study was clearly larger as those indicated in both adult sheep and lambs (8.6-10.5 µg·h/mL) [9,46]. In the same way, C max was also higher than those obtained in adult sheep (0.42-0.56 µg/mL) [43,46] and lambs (0.35-0.48 µg/mL) [9,47,48]. t max was also longer in our study than in others carried out in adult sheep (15-24 h) [43,46] and similar to that observed in lambs (30 h) [48]. Mean t lag (2.0 h) for ABZSO 2 was less than half of that indicated by Lanusse et al. [43] in adult sheep (4.9 h) after ABZ administration. Our MRT values were similar to that obtained in adult sheep (25.3 h) [46], and longer than in lambs (18.6-19.9 h) [9].
At present there is a need to find new pharmacological tools to ensure an efficient control of parasites in domestic animals. Assessment of drug interactions are increasing in recent years to explore their possibilities as a way to control parasites in livestock. Manipulation of ABZ pharmacokinetic behavior to extend its systemic availability has already assayed with variable results. Coadministration of thymol with ABZ has revealed a negative pharmacokinetic interaction in sheep [51]. Other authors have reported higher values of AUC and C max than ours for ABZSO when piperonyl butoxide was dosed with ABZ [15]. Nevertheless, in this latter interaction, other factors, such as the toxicity for the host or the cost of treatment, limited the use of this potential combination [15]. Other strategies, designed to improve ABZ dissolution rate have not been introduced into veterinary clinical practice to date.
Little information about the influence of tensoactive agents on the pharmacokinetics of ABZ is available. A higher gastrointestinal absorption of ABZ was documented with surfactants in rats [52,53] and cattle for sodium lauryl sulphate [7]. However, these latter authors did not find that absorption improved with sodium taurocholate. Surfactants may augment the permeability of biological membranes and enhance the dissolution rate of ABZ. MEN could participate in the mechanisms proposed. More recently, Ochoa et al. [28] reported that a fatty meal enhances ABZ oral bioavailability in healthy human subjects, attributing this higher absorption to food-induced stimulation of bile secretion, increasing the drug solubility.
MEN is a compound indicated as choleretic and, thus, stimulates the function of digestive tract. Regarding its safety, the EU has established that no MRL is required for this drug in food-producing animals [54].
Although the commercial formulation used in this study already contained surfactant agents, MEN improved the absorption of ABZ, with significant increases in C max and AUC last for ABZSO. The results obtained here show that MEN, when administered with ABZ, was able to increase plasma concentrations of ABZSO and, consequently, its anthelmintic effect, but we have evaluated the interaction between ABZ and MEN with the lowest dose (5 mg/kg). It would be interesting to assess if a proportional increase in concentrations would be achieved when the highest dose of ABZ is assayed (7.5 mg/kg).

Conclusions
The pharmacokinetic behavior of albendazole metabolites was characterized after oral administration of the anthelmintic albendazole (5 mg/kg) with the choleretic agent menbu-tone (intramuscular, 10 mg/kg) in sheep. We demonstrated that menbutone augmented the amount in blood of ABZSO (active metabolite), significantly increasing C max and AUC last , which may contribute to a higher anthelmintic activity in this ruminant species. The association proposed is a simple, safe, and inexpensive way of increasing the effectivity of this anthelmintic widely used in livestock. Further studies should be carried out to assess the practical options of this interaction.
Data Availability Statement: Data are available on request the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.