Plasma Disposition and Faecal Excretion of Netobimin Metabolites and Enantiospecific Disposition of Albendazole Sulphoxide Produced in Ewes

Veterinary Research Communications, 30 (2006) 791–805 DOI: 10.1007/s11259-006-3336-y

 C Springer 2006

Plasma Disposition and Faecal Excretion of Netobimin Metabolites and Enantiospecific Disposition of Albendazole Sulphoxide Produced in Ewes C. Gokbulut1,∗ , V.Y. Cirak2 and B. Senlik2 1 Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine and Research and Development Laboratory, University of Adnan Menderes, Aydin; 2 Department of Parasitology, Faculty of Veterinary Medicine, University of Uludag, Bursa, Turkey ∗ Correspondence: E-mail: [email protected]

Gokbulut, C., Cirak, V.Y. and Senlik, B., 2006. Plasma disposition and faecal excretion of netobimin metabolites and enantiospecific disposition of albendazole sulphoxide produced in ewes. Veterinary Research Communications, 30(7), 791–805

ABSTRACT Netobimin (NTB) was administered orally to ewes at 20 mg/kg bodyweight. Blood and faecal samples were collected from 1 to 120 h post-treatment and analysed by high-performance liquid chromatography (HPLC). Using a chiral phase-based HPLC, plasma disposition of albendazole sulphoxide (ABZSO) enantiomers produced was also determined. Neither NTB nor albendazole (ABZ) was present and only ABZSO and albendazole sulphone (ABZSO2 ) metabolites were detected in the plasma samples. Maximum plasma concentrations (Cmax ) of ABZSO (4.1 ± 0.7 μg/ml) and ABZSO2 (1.1 ± 0.4 μg/ml) were detected at (tmax ) 14.7 and 23.8 h, respectively following oral administration of netobimin. The area under the curve (AUC) of ABZSO (103.8 ± 22.8 (μg h)/ml) was significantly higher than that ABZSO2 (26.3 ± 10.1 (μg h)/ml) ( p < 0.01). (−)-ABZSO and (+)-ABZSO enantiomers were never in racemate proportions in plasma. The AUC of (+)-ABZSO (87.8±20.3 (μg h)/ml) was almost 6 times larger than that of (−)-ABZSO (15.5 ± 5.1 (μg h)/ml) ( p < 0.001). Netobimin was not detected, and ABZ was predominant and its AUC was significantly higher than that of ABZSO and ABZSO2 , following NTB administration in faecal samples ( p < 0.01). Unlike in the plasma samples, the proportions of the enantiomers of ABZSO were close to racemic and the ratio of the faecal AUC of (−)-ABZSO (172.22 ± 57.6 (μg h)/g) and (+)-ABZSO (187.19 ± 63.4 (μg h)/g) was 0.92. It is concluded that NTB is completely converted to ABZ by the gastrointestinal flora and absorbed ABZ is completely metabolized to its sulphoxide and sulphone metabolites by first-pass effects. The specific behaviour of the two enantiomers probably reflects different enantioselectivity of the enzymatic systems of the liver that are responsible for sulphoxidation and sulphonation of ABZ. Keywords: albendazole, albendazole sulphoxide, enantiomers, ewes, netobimin, pharmacokinetics Abbreviations: ABZ, albendazole; ABZSO, albendazole sulphoxide; ABZSO2 , albendazole sulphone; AUC, area under the plasma concentration–time curve; AUMC, area under the first moment curve; DMSO, dimethyl sulphoxide; FBZSO, oxfendazole; FMO, flavin-containing monooxygenase; HPLC, high-performance liquid chromatography; MRT, mean residence time; NTB, netobimin

INTRODUCTION Netobimin (NTB) is a pro-drug of the benzimidazole anthelmintic group used against liver flukes, tapeworms and gastrointestinal and lung nematodes in ruminants. Netobimin is converted into albendazole (ABZ) by gastrointestinal flora following oral and intraruminal

791

792

Figure 1. Metabolic pathways of netobimin (NTB), albendazole (ABZ) and albendazole sulphoxide (ABZSO). ABZSO2 = albendazole sulphone

administration to sheep and cattle. Almost all of the ABZ absorbed from the intestine is rapidly metabolized into its anthelmentically active albendazole sulphoxide (ABZSO) and inactive sulphone (ABZSO2 ) metabolites by the liver (Delatour et al., 1986; Galtier et al., 1991; Figure 1). It is thought that the flavine-containing monooxygenase (FMO) is mainly responsible for sulphoxidation, whereas cytochrome-dependent monooxygenase is responsible for sulphonation (Benoit et al., 1992). The pharmacokinetics of netobimin and its metabolites has been studied in sheep and only the sulphoxide and sulphone metabolites of ABZ were detected in plasma following oral administration of NTB (Lanusse and Prichard, 1990; Cristofol et al., 1997). Moreover, the reduction of NTB to ABZ and oxidation of ABZ into ABZSO were demonstrated in an artificial rumen (Capece et al., 2001) and in the in vitro ruminal and intestinal fluids of sheep and cattle (Lanusse et al., 1992). Sulphoxide benzimidazoles (ABZSO and oxfendazole (FBZSO)), which have a chiral centre about the sulphur atom, are formed as metabolites of sulphides and are metabolized to sulphones (Figure 1). The sulphones are anthelmintically inactive, whereas the sulphides

793

and sulphoxides are both active (Lacey et al., 1987). Although the plasma dispositions of two enantiomers of ABZSO and FBZSO have been investigated in many species after oral administration of pro-chiral ABZ and fenbendazole (Delatour et al., 1990; 1991a,b; Benoit et al., 1992; McKellar et al., 2002; Goudah, 2003), there is no information available on the stereospecific plasma behaviour of the enantiomers produced following pro-drug, NTB administration. Although the in vitro enantioselectivity of ruminal and intestinal flora in sulphoxidation of ABZ and sulphonation and sulphoreduction of ABZSO has been shown using the ruminal and intestinal fluids of sheep and cattle (Lanusse et al., 1992; Virkel et al., 2002), no data were available on the enantiospecific faecal excretion of the enantiomers following NTB administration. The aim of this study was to determine the plasma disposition and faecal excretion of NTB, ABZ, ABZSO and ABZSO2 , and enantiospecific production of ABZSO following oral NTB administration to ewes. MATERIALS AND METHODS Experimental animals Ten crossbred ewes (Karacabey Merino) with a mean weight of 36 (±3.1) kg were used in the study. The animals were kept indoors and fed with hay and concentrated sheep feed. Water was provided ad libitum throughout the course of the study. The study was approved by the Animal Ethic Committee of the Faculty of Veterinary Medicine, University of Uludag. Treatments and sampling Commercially available formulations of NTB (15% suspension, Schering-Plough Ltd, Welwyn Garden City, UK) were administered orally to animals at a dose rate of 20 mg/kg bodyweight. Heparinized blood samples were collected by jugular venepuncture prior to drug administration and 1, 2, 4, 8, 12, 20, 24, 30, 46, 54, 70, 94 and 120 h thereafter. Faecal samples (>5 g) were collected by anal stimulation throughout the blood-sampling period, before drug administration, and then at 8, 12, 20, 24, 30, 46, 70, 94 and 120 h in order to determine faecal excretion of the NTB and its respective metabolites under study. Blood samples were centrifuged at 3000g for 20 min and plasma was transferred to plastic tubes. All the plasma and faecal samples were stored at −20◦ C until estimation of drug concentration. Analytical procedures Plasma concentrations of NTB, ABZ, ABZSO and ABZSO2 were estimated by highperformance liquid chromatography (HPLC) with a liquid-phase extraction procedure adapted from that described by Marriner and Bogan (1980). Extraction from plasma Pure standard compounds of NTB (Schering Plough Ltd., UK), ABZ, rac-ABZSO and ABZSO2 (SmithKline Beecham, West Sussex, UK) and internal standard oxfendazole

794

(FBZSO) were obtained from Hoechst (Frankfurt, Germany). These were diluted with acetonitrile to give 0.5, 1, 2.5, 5 and 10 μg/ml and 10, 50 and 500 μg/ml standard solutions for plasma and faecal samples, respectively, for calibration as standard curves and to add to drug-free plasma and faecal samples for determination of the recovery. Drug-free plasma samples (1 ml) were spiked with standards of NTB, ABZ, rac-ABZSO and ABZSO2 to reach final concentrations of 0.05, 0.1, 0.5, 1 and 5 μg/ml. Ammonium hydroxide (100 μl, 0.1 mol/L, pH 10) was added to 10 ml ground-glass tubes containing 1 ml spiked or experimental plasma samples. Oxfendazole (0.5 μg/ml) was used as an internal standard. After mixing for 15 s, 6 ml ethyl acetate was added. The sample tubes were stoppered and shaken for 10 min on a slow rotary mixer. After centrifugation at 3000g for 10 min, the upper organic phase (4 ml) was transferred to a thin-walled 10 ml conical glass tube and evaporated to dryness at 40◦ C in a rotavapour (Maxi-Dry plus, Heto, Denmark). The dry residue was reconstituted with 400 μl mobile phase. The tubes were then placed in an ultrasonic bath and, finally, 20 μl of this solution was injected into the chromatographic system. Extraction from faeces Wet-faecal concentrations of NTB, ABZ, ABZSO and ABZSO2 were estimated by HPLC with a liquid-phase extraction procedure adapted from that described by Gokbulut and colleagues (2002). Briefly, wet-faecal samples were mixed finely with a spatula to obtain homogeneous concentrations. Drug-free wet faeces samples (0.5 g) were spiked with benzimidazole standards to reach the following final concentrations: 1, 5, 50, 10 and 20 μg/g. Oxfendazole (10 μg/ml) was used as an internal standard. Sodium hydroxide buffer (200 μl, 0.4 mol/L, pH 10) and 1 ml acetonitrile were added to 10 ml ground-glass tubes containing 0.5 g spiked or experimental wet-faecal samples. After mixing, for 15 s, 8 ml ethyl acetate was added. The tubes were shaken on a slow rotary mixer for 15 min. After centrifugation at 3000g for 10 min, 4 ml upper organic phase was transferred to a thin-walled 10 ml conical glass tube and evaporated to dryness at 40◦ C in the sample concentrator. The dry residue was resuspended with 500 μl dimethyl sulphoxide (DMSO).After ultrasonication for 1 min, the samples were filtered with GF/C glass microfibre filter (Whatman International Ltd., Maidstone, UK). Finally, 5 μl of this solution was injected into the chromatographic system. Chromatographic conditions The mobile phase was a mixture of acetonitrile–water to which glacial acetic acid was added (0.5%, v/v). It was pumped through the column (Nucleosil C18 4 μm, 250 mm ×4.6 mm, Macherey-Nagel, Duren, Germany) with Nucleosil C18 guard column (Phenomenex, Cheshire, UK) in a linear gradient fashion changing from 10:90 (acetonitrile–water) to 85:15 for 8 min then 80:20 to 10:90 for 1 min, and the last ratio being maintained for 3 min. The flow rate was 1 ml/min. Samples were processed on a computerized gradient HPLC system (1100 series, Agilent Technologies, Germany) comprising a degasser, a quaternary pump (G1354A), an autosampler (G1313), a column oven (G1316A) and a diode-array

795

detector (G1315B) set at 360 nm for NTB and 292 nm for ABZ, ABZSO and ABZSO2 . The retention times were 6.29 min (ABZSO) 7.10 min (FBZSO), 7.50 min (NTB), 7.81 min (ABZSO2 ) and 8.56 min (ABZ). The extracted samples were re-analysed with a chiral stationary phase to determine the concentration of ABZSO enantiomers produced. The enantiomers were estimated using chiral chromatography adapted from that previously described by Delatour and colleagues (1990). Briefly, a mobile phase of acetonitrile–water (7:93) was pumped at a flow rate of 1 ml/min through a Chiral-AGP column (5 μm, 150 × 40 mm, ChromTech, Cheshire, UK) with ultraviolet detection at 292 nm. Retention times were 3.27 min for (−)-ABZSO and 4.57 min for (+)-ABZSO.

Method of calibration Calibration graphs for the parent drug and the metabolites in the ranges 0.05–5 μg/ml (plasma) and 1–20 μg/g (faeces) were prepared using drug-free plasma and faeces from ewes. Recovery of the parent molecules and the metabolites under study was measured by comparison of the peak areas from spiked plasma samples with the areas resulting from direct injections of standards. The inter-assay precision of the extraction and chromatography procedures was evaluated by processing replicate aliquots of drug-free ewe plasma and faecal samples containing known amounts of the drugs on different days. The slope of the curves of peak area vs drug concentration was determined by least-squares linear regression and showed correlation coefficients between 0.996 and 0.999. The limit of quantification (LOQ) was 0.06 and 0.4 μg/g for plasma and faeces, respectively, these values being the lowest concentrations detected with a coefficient of variation (CV) below 20%. The mean extraction recoveries were 78.5% (CV 8.27%) for NTB, 82.5% (CV 6.83%) for ABZ, 92.4% (CV 8.13%) for ABZSO and 96.5% (CV 4.45%) for ABZSO2 . To determine the dry proportion of wet faecal samples, 2.0 g of wet faeces from each sample were weighed exactly into an evaporating bowl and heated in an oven at 75◦ C for 10 h. The weight of each was determined and the percentage of each dry sample was calculated.

Pharmacokinetic and statistical analysis of data The plasma or faecal concentration vs time curves obtained after each treatment in individual animals were fitted with the WinNonlin software program (Scientific Consulting Inc., North Carolina, USA). Pharmacokinetic parameters for each animal were analysed using non-compartmental model analysis with extravascular input. The maximum plasma concentration (Cmax ) and time to reach maximum concentration (tmax ) were obtained from the plotted concentration-time curves of each drug in each animal. The linear trapezoidal rule was used to calculate the area under the plasma concentration time curve (AUC): AUC0−last =

n  Ci + Ci−1 (ti − ti−1 ) 2 i=1

796

where C represents the plasma concentration and i − 1 and i are adjacent data point times. The area under the first movement curve (AUMC) was calculated using the equation: n  Ci ti + Ci−1 ti−1 AUMC0−last = (ti − ti−1 ) 2 i=2 Thus, the mean residence time (MRT) was calculated as AUMC0−last MRT0−last = AUC0−last Terminal half-life (t 12 λz ) was calculated as − ln(2) t 12 λz = λz where λz represent the first-order rate constant associated with the terminal (log linear) portion of the curve. The pharmacokinetic parameters are reported as mean ± SD. Mean pharmacokinetic parameters were statistically compared by analysis of variance (ANOVA). Mean values were considered significantly different at p < 0.05. RESULTS Neither NTB nor ABZ was detected in any plasma samples analysed. Figure 2 shows the mean (±SD) plasma concentration versus time curves and Table I shows the mean (±SD)

Plasma concentration (μg/ml)

Plasma concentration (μg/ml)

5

4

3

ABZSO ABZSO2

1

0.1

0.01

0

10

20

30

40

50

60

70

Time (h)

2

1

ABZSO ABZSO2 0

10

20

30

40

50

60

70

Time (h) Figure 2. Mean (±SD) plasma concentrations (μg/ml) of albendazole sulphoxide (ABZSO) and albendazole sulphone (ABZSO2 ) following oral administration of netobimin (20 mg/kg) to ewes (n = 10) (Inset graph: semi-log plot of mean ± SD plasma concentration vs time curve)

797

TABLE I Mean ± SD pharmacokinetic parameters of total albendazole sulphoxide (ABZSO), (+)-ABZSO, (−)-ABZSO and albendazole sulphone (ABZSO2 ) following oral administration of netobimin (20 mg/kg) to ewes (n = 10) ABZSO Parameter

Total

(−)-ABZSO

(+)-ABZSO

ABZSO2

tmax (h) Cmax (μ g/ml) tlast (h) AUClast (μ g h/ml) t 1 λz (h)a 2 AUMClast (μg h2 )/ml) MRTlast (h)

14.7 ± 4.2∗ 4.1 ± 0.7∗ 44.2 ± 8.7 103.8 ± 22.8∗ 6.4 ± 3.9 1788.3 ± 502.0∗ 17.0 ± 2.1

9.9 ± 2.8 0.8 ± 0.3∗∗ 37.1 ± 8.4 15.5 ± 5.1∗∗ 5.6 ± 2.1 211.6 ± 72.0∗∗ 13.6 ± 1.2

15.1 ± 3.8 3.4 ± 0.7 44.2 ± 8.7 87.8 ± 20.3 6.2 ± 3.2 1563.2 ± 459.0 17.6 ± 2.2

23.8 ± 6.1 1.1 ± 0.4 42.4 ± 9.9 26.3 ± 10.1 4.3 ± 2.5 603.9 ± 310.6 21.6 ± 5.1

Cmax , peak plasma concentration; tmax , time to reach peak plasma concentration; tlast , time to the last detectable plasma concentration; AUClast , area under the (zero moment) curve from time 0 to the last detectable concentration; AUMClast , area under the moment curve from time 0 to time of last detectable concentration; MRTlast , mean residence time; t 1 λz , terminal half-life 2 a Values represent the harmonic mean for t λ . 1 z 2 ∗ Mean parameters of ABZSO are significantly different from those obtained for ABZSO ( p < 0.001) 2 ∗∗ Mean parameters of (−)-ABZSO are significantly different from those obtained for (+)-ABZSO ( p < 0.001)

pharmacokinetic parameters of ABZSO and ABZSO2 . The AUC of ABZSO (103.8 ± 22.8 (μg h)/ml) was significantly higher than that of ABZSO2 (26.3 ± 10.1 (μg h)/ml) ( p < 0.001). The mean plasma concentration vs time curves and enantiospecific percentage of (−)-ABZSO and (+)-ABZSO are presented and Figures 3 and 4, respectively. The plasma concentrations of the enantiomers were never in racemic proportion. The AUC of the (+) enantiomer was almost 6 times larger than that of the (−) enantiomer ( p < 0.001). Albendazole, ABZSO and ABZSO2 were present between 8 and 120 h, but NTB was not detected in any faecal samples. Mean (± SD) dry-faecal concentrations vs time curves are presented in Figure 5 and mean faecal kinetic parameters of ABZ, ABZSO and ABZSO2 are shown in Table II. Albendazole was predominant and its AUC was significantly higher than that of ABZSO and ABZSO2 in the faecal samples following NTB administration. The mean faecal concentration of the enantiomers vs time curves are shown in Figure 6. The proportions of the enantiomers of ABZSO were close to racemic in the faecal samples analyzed. The AUCs of (−)-ABZSO and (+)-ABZSO were 172.22±57.6 and 187.19±63.4 (μg h)/g, respectively. DISCUSSION Benzimidazole anthelmintics are extensively metabolized in all animal species. Generally, the plasma elimination half-lives of the parent drugs are short and the metabolic moieties predominate in plasma and tissues and in excreta of the host as well as in parasites

798

4.0

Plasma concentration (μg/ml)

3.5 3.0

Total ABZSO (-)ABZSO (+)ABZSO

2.5 2.0 1.5 1.0 0.5 0.0 0

10

20

30

40

50

60

70

Time (h) Figure 3. Mean (±SD) plasma concentrations (μg/ml) of total and (−) and (+) enantiomers of albendazole sulphoxide (ABZSO) following oral adminstration of netobimim (20 mg/kg) to ewes (n = 10)

100

(+)ABZSO

Percentage

80

60

40

20

(-)ABZSO

0 0

10

20

30

40

50

60

Time (h) Figure 4. Mean percentage of (−) and (+) enantiomers of albendazole sulphoxide (ABZSO) following oral adminstration of netobimim (20 mg/kg) to ewes (n = 10)

799

TABLE II Mean ± SD faecal kinetic parameters of albendazole (ABZ), albendazole sulphoxide (ABZSO) and albendazole sulphone (ABZSO2 ) following oral administration of netobimin (20 mg/kg) to ewes (n = 10)

tmax (h) Cmax (μg/g) tlast (h) AUClast (μg h/g) MRTlast (h)

ABZ

ABZSO

ABZSO2

29.7 ± 8.3 27.0 ± 8.5∗ 81.4 ± 21.6 942.9 ± 311.9∗ 36.0 ± 4.8

28.3 ± 11.2 9.3 ± 2.7 64.3 ± 15.1 348.2 ± 119.8 32.0 ± 5.6

12.0 ± 5.7 13.7 ± 8.0 57.4 ± 9.1 355.6 ± 156.1 25.1 ± 3.8

Cmax , peak faecal concentration; tmax , time to reach peak faecal concentration; tlast , time to the last detectable faecal concentration; AUClast , area under the (zero moment) curve from time 0 to the last detectable concentration; MRTlast , mean residence time ∗ Mean parameters of ABZ are significantly different from those obtained for parent ABZSO and ABZSO2 ( p < 0.01)

Dry-faecal concentration ((μg/g)

35 30 25

ABZ ABZSO ABZSO2

20 15 10 5 0 0

20

40

60

80

100

120

Time (h) Figure 5. Mean (±SD) dry-faced concentrations (μg/ml) of albendazole (ABZ), albendazole sulphoxide (ABZSO) albendazole sulphone (ABZSO2 ) following oral adminstration of netobimim (20 mg/kg) to ewes (n = 10)

800

Dry-faecal concentration (μg/g)

10

Total ABZSO (-)ABZSO (+)ABZSO

8

6

4

2

0 0

20

40

60

80

100

Time (h) Figure 6. Mean (± SD) dry-faced concentrations and (μg/ml) of total (−) and (+) enantiomers of albendazole sulphoxide (ABZSO) following oral administration of netobimim (20 mg/kg) to ewes (n = 10)

recovered from benzimidazole-treated animals (Delatour and Parish, 1986; Lanusse and Prichard, 1993). Sulphide and sulphoxide benzimidazoles are known to bind nematode tubulin (Lacey et al., 1987) and therefore have activity against nematodes, although sulphides exert inhibitory activity on tubulin at lower concentrations than sulphoxides (McKellar and Scott, 1990). Absorption of NTB from gastrointestinal tract has been reported in sheep following intraruminal administration at dose rate of 20 mg/kg (Lanusse and Prichard, 1990). However, pro-drug was not detectable at dose rate of 20 mg/kg in ewes and cattle (Lanusse and Prichard, 1992; Cristofol et al., 1995, 1997) following oral administration. In the present study, NTB was not detected in any plasma and faecal samples. However, ABZ was predominant in the faeces following oral administration. This suggests an efficient conversion of the pro-drug NTB in to the active form ABZ by the reductive environment of the gastrointestinal tract, since it has been shown that, following intraruminal or oral administration, NTB is reduced to ABZ in the gastrointestinal tract of cattle and sheep (Lanusse and Prichard, 1990, 1992). Recent in vitro studies with ruminal fluids also support this (Capece et al., 2001; Virkel et al., 2002, 2004b). Albendazole was also not detected in any plasma samples analysed following oral administration of NTB. Although other tissues such as the gastrointestinal (GI) mucosa and lung may be involved (Virkel et al., 2004a), a higher metabolic capacity and first-pass oxidation in the liver is the likely explanation for the absence of the ABZ in the systemic

801

circulation. The pharmacokinetic profiles of ABZSO and ABZSO2 obtained in this study were similar to data previously reported in the literature (McKellar et al., 1993; Cristofol et al., 1997; Merino et al., 1999). In the present study, the Cmax of ABZSO (4.1 μg/ml) was almost four times higher and reached tmax (14.7 h) significantly earlier than with ABZSO2 (Cmax = 1.1, tmax = 23.8 h). In addition, the AUC of ABZSO was 4 times that of ABZSO2 . These differences could be due to relatively slow oxidation of ABZSO to ABZSO2 , as described earlier by Galtier and colleagues (1986). Pharmacodynamic and pharmacokinetic properties of enantiospecific pairs are commonly different and are of major importance for their effective and safe therapeutic use (Landoni et al., 1997; Delatour et al., 1994). In the present study the enantiospecific ratio (+/-) of plasma concentrations of ABZSO changed over time in favour of (+), in agreement with previous observations following administration of ABZ (Delatour et al., 1990, 1991a; Benoit et al., 1992; Goudah, 2003). At t0 , the ratio (+)-ABZSO/(−)-ABZSO was 3, and subsequently the proportion of the (+) enantiomer increased linearly during the course of the kinetics. The AUC of the (+) enantiomer was almost 6 times larger than that of the (−) enantiomer, and this may contribute to the anthelmintic effect of the (+)-ABZSO enantiomer, since it has been shown that (−)-ABZSO is quickly eliminated as the inactive sulphone metabolite (Goudah, 2003). In addition, Alvarez and colleagues (1999, 2000) have reported that the main enantiomer taken up by Fasciola hepatica was (+)-ABZSO following ABZ administration in infected sheep. The enantiospecific disposition of ABZSO produced has been described previously in human and various animal species following ABZ administration of pro-chiral. The stereospecific dispositions of the two enantiomers were different between monogastrics and ruminants (Delatour et al., 1990, 1991a,b). The metabolism of sulphide to sulphoxide benzimidazoles has been thought to be catalysed principally by the flavin monooxygenase (FMO) system (Galtier et al., 1986). More recently, Rawden and colleagues (2000) suggested that both cytochrome P450 (CYP450) and FMO systems are responsible for the sulphoxidation, whereas metabolism of sulphoxide to sulphone is thought to be catalysed by hepatic cytochrome P450 (Souhaili-el-Amri et al., 1988). It has also been demonstrated that the FMO system is enantioselective in favour of production of the (+) sulphoxide of ABZ, whereas only cytochrome P450 systems specifically produce (−)-ABZSO, which was shown to be the main substrate for the formation of the inactive sulphone metabolite (Delatour et al., 1991b; Morani et al., 1995). Differences in the interspecies enantioselectivity could be explained by different metabolic enzyme contributions since the FMO and cytochrome P450 systems act equally in monogastrics, whereas the FMO system may be predominant in ruminants (Delatour et al., 1994). The eudismic (potency) ratios of the enantiospecific pair of benzimidazole sulphoxides have not been determined yet, and their anthelmintic activity is still unclear (Landoni et al., 1997). Netobimin was not detected and ABZ was predominant and in significantly higher concentrations than ABZSO and ABZSO2 in the faecal samples analysed. The results of the faecal excretion confirm the in vitro studies carried out by Capece and colleagues (2001) and Lanusse and colleagues (1992), who demonstrated the fast reduction of NTB to ABZ in an artificial rumen and in the in vitro ruminal and intestinal fluids of sheep and cattle. The great reductive capacity of gastrointestinal environment is probably responsible for this

802

process. This result suggests that the relatively higher faecal concentration of that in ABZ compared to that in plasma could be effective against gastrointestinal parasites, since ABZ is anthelmintically much more active than ABZSO and ABZSO2 has no activity (Lacey et al., 1987). A reversible exchange of benzimidazoles takes place between plasma and the GI tract and this process is mediated by pH gradient (Lanusse and Prichard, 1993). Moreover, the presence of extensive exchange between plasma and abomasum has been shown following subcutaneous ABZSO administration in cattle (Lanusse et al., 1993). Intestinal metabolism and extensive secretion of ABZSO into the intestinal lumen have also been shown using isolated intestinal loops (Redondo et al., 1999). In the present study, ABZ was the predominant metabolite in the faeces, whereas ABZSO and ABZSO2 were predominant in the plasma. This could suggest the formation of ABZ from ABZSO distributed from the plasma into the GI tract, allowing the sulphoreduction of ABZSO into ABZ by GI microflora, since it has been shown that ABZSO can be reduced to ABZ by ruminal and intestinal flora (Lanusse et al., 1992, 1993; Virkel et al., 2002). Unlike the plasma samples, the proportions of the enantiomers of ABZSO were close to racemic in faecal samples analysed in agreement with study carried out by Capece and colleagues (2001), who suggested that the ruminal metabolism was not enantioselective following ruminal microbial fermentation using an artificial rumen model. On the other hand, an in vitro study carried out by Virkel and colleagues (2002) has shown that (+)-ABZSO was the main substrate for ABZ production in the ruminal fluid following incubation of both enantiomers separately. In the previous in vitro studies carried out by Virkel and colleagues (2002) and Capece and colleagues (2001) the substrates were incubated for 6 h and 10 h, respectively, whereas in the present study the faecal excretion of ABZ and the enantiomers was determined between 8 h and 120 h, and Cmax of ABZSO was obtained at 28.3 ± 11.2 h following oral NTB administration. In addition, the enantiospecific proportions of ABZSO in the ruminal fluid may not reflect the faecal excretion profiles since the consequence of the enantiomers in the rest of the GI tract could be different. It was reported that both passive diffusion and active transport could be involved in the intestinal elimination of ABZSO (Merino et al., 2003). Although interaction between parent ABZ and P-glycoprotein does not occur (Merino et al., 2002), enantioselective transport of ofloxacin and talinolol by P-glycoprotein has been shown in the process of intestinal elimination (Rabbaa et al., 1996; Hanafy et al., 2001). Thus, different intestinal metabolism, interactions and transport of ABZSO could determine the proportions of the enantiomers in faeces and this could explain the different enantiospecific dispositions between plasma and faeces. The excretion of anthelmintics in the faeces of livestock has given rise to concern since it was observed that the avermectins have adverse effects on some useful arthropods such as dipteran flies and coleopteran beetles that inhabit and feed in dung (Wall and Strong, 1987). ABZ is mainly eliminated via the urine in ruminants (Blume et al., 1976; Hennessey et al., 1993a), whereas oxfendazole and fenbendazole are mainly eliminated via the faeces (Hennessey et al., 1993c; Strong et al., 1996). It has been estimated that up to 50% of an administered dose of fenbendazole is excreted as the sulphide in cattle, sheep and goats (Duwel, 1977; Short et al., 1987a, b). Following administration of oxfendazole to sheep and goat, approximately 65–80% of the dose was excreted in faeces and 17–25% in

803

urine (Hennessey et al., 1993b). While it is recognized that benzimidazoles are unlikely to affect dung-dwelling arthropods (McKellar, 1997), their excretion in the faeces of sheep has not been characterized and consequently the associated environmental impact is not known.

REFERENCES Alvarez, L., Sanchez, S. and Lanusse, C., 1999. In vivo and ex vivo uptake of albendazole and its sulphoxide metabolite by cestode parasites: relationship with their kinetics behaviour in sheep. Journal of Veterinary Pharmacology and Therapeutics, 22, 77–86 Alvarez, L., Imperiale, F., Sanchez, S., Murno, G. and Lanusse, C., 2000. Uptake of albendazole and albendazole sulphoxide by Haemonchus contortus and Fasciola hepatica in sheep. Veterinary Parasitology, 94, 75– 89 Benoit, E., Besse, S. and Delatour, P., 1992. Effect of repeated doses of albendazole on enantiomerism of its sulphoxide metabolite in goats. American Journal of Veterinary Research, 53, 1663–1665. Blume, R.R., Younger, R.L., Aga, A. and Myers, C.J., 1976. Effects of residues of certain anthelmintics in bovine manure on Onthophagus gazella, a non-target organism. Southwest Entomologist, 1, 100–103 Capece, B.P., Calsamiglia, S., Castells, G., Arboix, M. and Cristofol, C., 2001. Effect of ruminal microflora on the biotransformation of netobimin, albendazole, albendazole sulfoxide, and albendazole sulfoxide enantiomers in an artificial rumen. Journal of Animal Science, 79, 1288–1294 Cristofol, C., Carretero, A., Fernandez, M., Navarro, M., Sautet, J., Ruberte, J. and Arboix, M., 1995. Transplacental transport of netobimin metabolites in ewes. European Journal of Drug Metabolism and Pharmacokinetics, 20, 167–171 Cristofol, C., Franquelo, C., Navarro, M., Carretero, A., Ruberte, J. and Arboix, M., 1997. Comparative pharmacokinetics of netobimin metabolites in pregnant ewes. Research in Veterinary Science, 62, 117–120 Delatour, P. and Parish, R., 1986. Benzimidazole anthelmintics and related compounds: toxicity and evaluation of residues. In: A.G. Rico (ed.), Drug Residues in Animals, (Academic Press, New York), 175–203 Delatour, P., Cure, M.C., Benoit, E. and Garnier, F., 1986. Netobimin (Totabin-SCH): preliminary investigation on metabolism and pharmacology. Journal of Veterinary Pharmacology and Therapeutics, 9, 230– 234 Delatour, P., Benoit, E., Garnier, F. and Besse, S., 1990. Chirality of the sulphoxide metabolites of fenbendazole and albendazole in sheep. Journal of Veterinary Pharmacology and Therapeutics, 13, 361–366 Delatour, P., Garnier, F., Benoit, E. and Caude, I., 1991a. Chiral behaviour of the metabolite albendazole sulphoxide in sheep, goats and cattle. Research in Veterinary Science, 50, 134–138 Delatour, P., Benoit, E., Besse, S. and Boukraa, A., 1991b. Comparative enantioselectivity in the sulphoxidation of albendazole in man, dogs and rats. Xenobiotica, 21, 217–221 Delatour, P., Benoit, E. and Soraci, A., 1994. Reckebusch Memorial Lecture: Drug chirality: its significance in veterinary pharmacology and therapeutics. In: P. Lees (ed.), Proceedings of the 6th International Congress of the European Association for Veterinary Pharmacology and Toxicology, (Blackwell Scientific, Edinburgh) 6–9 Duwel, D., 1977. Fenbendazole II, biological properties and activity. Pesticide Science, 8, 550–555 Galtier, P., Alvinerie, M. and Delatour, P., 1986. In vitro sulphoxidation of albendazole by ovine liver microsomes: assay and frequency of various xenobiotics. American Journal of Veterinary Research, 47, 447–450 Galtier, P., Alvinerie, M., Steimer, J.L., Francheteau, P., Plusquellec, Y. and Houin, G., 1991. Simultaneous pharmacokinetic modeling of a drug and two metabolites: application to albendazole in sheep. Journal of Pharmacological Science, 80, 3–10 Gokbulut, C., Nolan, A.M. and McKellar, Q.A., 2002. Pharmacokinetic disposition, faecal excretion and in vitro metabolism of oxibendazole following oral administration in horses. Research in Veterinary Science, 72, 11– 15 Goudah, A., 2003. Aspects of the pharmacokinetics of albendazole sulphoxide in sheep. Veterinary Research Communications, 27, 555–566 Hanafy, A., Langguth, P. and Spahn-Langguth, H., 2001. Pretreatment with potent P-glycoprotein ligands may increase intestinal secretion in rats. European Journal of Pharmacological Science, 12, 405–415 Hennessy, D.R., Sangster, N.C., Steel, J.W. and Collins, G.W., 1993a. Comparative pharmacokinetic behaviour of albendazole in sheep and goats. International Journal of Parasitoloji, 23, 321–325

804

Hennessy, D.R., Sangster, N.C., Steel, J.W. and Collins, G.W., 1993b. Comparative kinetic disposition of oxfendazole in sheep and goat before and during infection with Haemonchus contortus and Trichostrongylus colubriformis. Journal of Veterinary Pharmacology and Therapeutics, 16, 245–250 Hennessy, D.R., Steel, J.W. and Prichard, R.K., 1993c. Biliary secretion and enterohepatic recycling of fenbendazole metabolites in sheep. Journal of Veterinary Pharmacology and Therapeutics, 16, 132–140 Lacey, E., Brady, R.L., Prichard, R.K. and Watson, T.R., 1987. Comparison of inhibition of polymerisation of mammalian tubulin and helminth ooicidal activity by benzimidazole carbonates. Veterinary Parasitology, 23, 105–119 Landoni, M.F., Soraci, A.L., Delatour, P. and Lees, P., 1997. Enantioselective behaviour of drugs used in domestic animals, a review. Journal of Veterinary Pharmacology and Therapeutics, 20, 1–16 Lanusse, C.E. and Prichard, R.K., 1990. Pharmacokinetic behaviour of netobimin and its metabolites in sheep. Journal of Veterinary Pharmacology and Therapeutics, 13, 170–178 Lanusse, C.E. and Prichard, R.K., 1992. Effects of methimazole on the kinetics of netobimin metabolites in cattle. Xenobiotica, 22, 115–123 Lanusse, C.E. and Prichard, R.K., 1993. Relationship between pharmacological properties and clinical efficacy of ruminant anthelmintics. Veterinary Parasitology, 49, 123–158 Lanusse, C.E., Nare, B., Gascon, L.H. and Prichard, R.K., 1992. Metabolism of albendazole and albendazole sulphoxide by ruminal and intestinal fluids of sheep and cattle. Xenobiotica, 22, 419–426 Lanusse, C.E., Gascon, L.H., and Prichard, R.K., 1993. Gastrointestinal distribution of albendazole metabolites following netobimin administration to cattle: relationship with plasma disposition kinetics. Journal of Veterinary Pharmacology and Therapeutics, 16, 38–47 Marriner, S.E. and Bogan, J.A., 1980. Pharmacokinetics of albendazole in sheep. American Journal of Veterinary Research, 41, 483–491 McKellar, Q.A., 1997. Ecotoxicology and residues of anthelmintic compounds. Veterinary Parasitology, 72, 413– 435 McKellar, Q.A. and Scott, E.W., 1990. The benzimidazole anthelmintic agents: a review. Journal of Veterinary Pharmacology and Therapeutics, 13, 223–247 McKellar, Q.A., Jackson, F., Coop, R.L. and Baggot, J.D., 1993. Plasma profiles of albendazole metabolites after administration of netobimin and albendazole in sheep, effects of parasitism and age. British Veterinary Journal, 149, 101–113 McKellar, Q.A., Gokbulut, C., Muzandu, K. and Benchaoui, H., 2002. Fenbendazole pharmacokinetics, metabolism, and potentiation in horses. Drug Metabolism and Disposition. 30, 1230–1239 Merino, G., Alvarez, A.I., Redondo, P.A., Garcia, J.L., Larrode, O.M. and Prieto, J.G., 1999. Bioavailability of albendazole sulphoxide after netobimin administration in sheep, effects of fenbendazole coadministration. Research in Veterinary Science, 66, 281–283 Merino, G., Alvarez, A.I., Prieto, J.G. and Kim, R.B., 2002. The anthelmintic agent albendazole does not interact with P-glycoprotein. Drug Metabolism and Disposition, 30, 365–369 Merino, G., Molina, A.J., Garcia, J.L., Pulido, M.M., Prieto, J.G. and Alvarez, A.I., 2003. Intestinal elimination of albendazole sulfoxide: pharmacokinetic effects of inhibitors. International Journal of Pharmacology, 263, 123–132 Morani, P., Buronfosse, T., Longin-Sauvageon, C., Delatour, P. and Benoit, E., 1995. Chiral sulphoxidation of albendazole by the flavin adenine dinucleotide-containing and cytochrome P450-dependent mono-oxygenases from rat liver microsomes. Drug Metabolism and Disposition, 23, 160–165 Rabbaa, L., Dautrey, S., Colas-Linhart, N., Carbon, C. and Farinotti, R., 1996. Intestinal elimination of ofloxacin enantiomers in the rat: evidence of a carrier-mediated process. Antimicrobial Agents and Chemotherapy, 36, 2506–2511. Rawden, H.L., Kokwaro, G.O., Ward, S.A. and Edwards, G., 2000. Relative contribution of cytochromes P450 and flavin-containing monooxygenases to the metabolism of albendazole by human liver microsomes. British Journal of Clinical Pharmacology. 49, 313–322 Redondo, P.A., Alvarez, A.I., Garcia, J.L., Larrode, O.M., Merino, G., and Prieto, J.G., 1999. resystemic metabolism of albendazole: experimental evidence of an efflux process of albendazole sulfoxide to intestinal lumen. Drug Metabolism and Disposition, 27, 736–740 Short, C.R., Barker, S.A. and Hsieh, L.C., 1987a. Disposition of fenbendazole in the goat. American Journal of Veterinary Research, 48, 811–815 Short, C.R., Barker, S.A. and Hsieh, L.C., 1987b. Disposition of fenbendazole in cattle. American Journal of Veterinary Research, 48, 958–961 Souhaili-El Amri, H., Mothe, O., Totos, M., Masson, C., Batt, A., Delatour, P. and Siest. G., 1988. Albendazole sulphonation by rat liver cytochrome P459c. Journal of Veterinary Pharmacology and Therapeutics, 246, 758–764

805

Strong, L., Wall, R., Woolford, A. and Djeddour, D., 1996. The effect of faecally excreted ivermectin and fenbendazole on the insect colonisation of cattle dung following the oral administration of sustained-release boluses. Veterinary Parasitology, 62, 253–266 Virkel, G., Lifschitz, A., Pis, A. and Lanusse, C., 2002. In vitro ruminal biotransformation of benzimidazole sulphoxide anthelmintics: enantioselective sulphoreduction in sheep and cattle. Journal of Veterinary Pharmacology and Therapeutics, 25, 15–13 Virkel, G., Lifschitz, A., Pis, A., Sallovitz, J. and Lanusse, C., 2004a. Comparative hepatic and extrahepatic enantioselective sulfoxidation of albendazole and fenbendazole in sheep and cattle. Drug Metabolism and Disposition, 32, 536–544 Virkel, G., Lifschitz, A., Sallovitz, J., Inza, G. and Lanusse, C., 2004b. Effect of the ionophore antibiotic monensin on the ruminal biotransformation of benzimidazole anthelmintics. The Veterinary Journal, 167, 265–271 Wall, R. and Strong, L., 1987. Environmental consequences of treating cattle with the antiparasitic drug ivermectin. Nature, 324, 418–420 (Accepted: 15 March 2005)