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Plasma disposition and faecal excretion of oxfendazole, fenbendazole and albendazole following oral administration to donkeys
The Veterinary Journal The Veterinary Journal 172 (2006) 166–172 www.elsevier.com/locate/tvjl
Plasma disposition and faecal excretion of oxfendazole, fenbendazole and albendazole following oral administration to donkeys Cengiz Gokbulut a
a,*
, Ferda Akar a, Quintin A. McKellar
b
Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Adnan Menderes, Isikli Koyu, Aydin, Turkey b The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Hertfordshire AL9 7TA, UK Accepted 10 February 2005
Abstract Fenbendazole (FBZ), oxfendazole (fenbendazole sulphoxide, FBZSO), and albendazole (ABZ) were administered orally to donkeys at 10 mg/kg bodyweight. Blood and faecal samples were collected from 1 to 120 h post-treatment. The plasma and faecal samples were analysed by high performance liquid chromatography (HPLC). The parent molecule and its sulphoxide and sulphone (FBZSO2) metabolites did not reach detectable concentrations in any plasma samples following FBZ administration. ABZ was also not detected in any plasma samples, but its sulphoxide and sulphone metabolites were detected, demonstrating that ABZ was completely metabolised by first-pass mechanisms in donkeys. Maximum plasma concentrations (Cmax) of FBZSO (0.49 lg/mL) and FBZSO2 (0.60 lg/mL) were detected at (tmax) 5.67 and 8.00 h, respectively, following administration of FBZSO. The area under the curve (AUC) of the sulphone metabolite (10.33 lg h/mL) was significantly higher than that of the parent drug FBZSO (5.17 lg h/mL). Cmax of albendazole sulphoxide (ABZSO) (0.08 g/mL) and albendazole sulphone (ABZSO2) (0.04 lg/mL) were obtained at 5.71 and 8.00 h, respectively, following ABZ administration. The AUC of the sulphoxide metabolite (0.84 lg h/mL) of ABZ was significantly higher than that of the sulphone metabolite (0.50 lg h/mL). The highest dry-faecal concentrations of parent molecules were detected at 32, 34 and 30 h for FBZSO, FBZ and ABZ, respectively. The sulphide metabolite was significantly higher than the parent molecule after FBZSO administration. The parent molecule was predominant in the faecal samples following FBZ administration. After ABZ administration, the parent molecule was significantly metabolised, probably by gastrointestinal microflora, to its sulphoxide metabolite (ABZSO) that showed a similar excretion profile to the parent molecule in the faecal samples. The AUC of the parent FBZ was significantly higher than that of FBZSO and ABZ in faeces. It is concluded that the plasma concentration of FBZSO was significantly higher than that of FBZ and ABZ. Although ABZ is not licensed for use in Equidae, its metabolites presented a greater plasma kinetic profile than FBZ which is licensed for use in horses. A higher metabolic capacity, first-pass effects and lower absorption of benzimidazoles in donkeys decrease bioavailability and efficacy compared to ruminants. 2005 Elsevier Ltd. All rights reserved. Keywords: Donkey; Oxfendazole; Fenbendazole; Albendazole; Pharmacokinetics; Anthelmintics
1. Introduction
*
Corresponding author. Tel.: +90 256 247 0340; fax: + 90 256 247 0700. E-mail address: [email protected] (C. Gokbulut). 1090-0233/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2005.02.022
Benzimidazole anthelmintics have a broad spectrum of activity against gastrointestinal helminths, including migrating strongyle larvae and lungworm infections, and are well tolerated by mammals (McKellar and
C. Gokbulut et al. / The Veterinary Journal 172 (2006) 166–172
sion of those compounds appears to differ substantially from ruminants. In the horse, the bioavailability and residence time of the tested benzimidazoles (and metabolites) were lower and shorter, respectively, than in ruminants (McKellar et al., 2002; Gokbulut, 2000; Marriner and Bogan, 1985, 1981). Sulphide and sulphoxide benzimidazoles are known to bind nematode tubulin (Lacey et al., 1987) and therefore have activity against nematodes although sulphides exert an inhibitory activity on tubulin at lower concentrations than sulphoxides. In most species examined, the sulphoxide moiety predominates in plasma and is thought to confer activity against gut dwelling nematodes following secretion across the gastrointestinal wall into the gut lumen where it may undergo sulphoreduction (McKellar and Scott, 1990). Albendazole is extensively used in ruminants to treat gastrointestinal helminths but there is a paucity of data available in the literature on the toxic or side effects in equines and ABZ is not used in these species. Donkeys are widely used for transportation especially in rural and mountainous areas of Turkey. Because of the lack of registered those for donkeys, anthelmintics registered for horses and ruminants are commonly used to treat gastrointestinal helminths in this species. In the present study, the comparative pharmacokinetics and faecal excretions of FBZ, FBZSO and ABZ following oral administration in donkeys were investigated.
Scott, 1990). They are extensively metabolised 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 the excreta of the host as well as in parasites recovered from benzimidazole-treated animals (Lanusse and Prichard, 1993; Delatour and Parish, 1986). The less soluble benzimidazole compounds have a longer residence in the forestomachs of ruminants from which they are absorbed over prolonged periods and thus remain in the plasma for a relatively long time. Since an equilibrium exists between the plasma and the gastrointestinal tract, the duration of exposure of gut parasites to an effective concentration of the drug is extended (McKellar and Scott, 1990). Extremely insoluble anthelmintics may be less effective, since they may not be absorbed and are excreted unchanged in the faeces. This may explain the difference between the plasma concentrations of oxfendazole (fenbendazole sulphoxide, FBZSO) following oral administration and its inter-convertible metabolite fenbendazole (FBZ) (Ngomuo et al., 1984). A large proportion of the less soluble FBZ is known to be excreted in the faeces of ruminants (Duwel, 1977). Oxfendazole, fenbendazole and albendazole (ABZ) are commercially available sulphur-containing benzimidazoles that commonly undergo microsomal oxidation in liver. Sulphide benzimidazoles (FBZ and ABZ) are reversibly metabolised to their sulphoxide derivatives (Marriner and Bogan, 1981, 1980; Gyurik et al., 1981; Mohammed Ali et al., 1987). Irreversible sulphonation follows sulphoxidation and is a slower oxidative step resulting in a sulphone metabolite (Averkin et al., 1975) (Fig. 1). The pharmacokinetics of FBZ and FBZSO have been studied in horses in which the metabolic inter-conver-
2. Materials and methods 2.1. Animals Seven donkeys (Equus asinus), 88–122 kg body weight, were randomly allocated to three groups
O
O S
N
167
N
S NH-CO2CH3
NH-CO2CH3
N H
N
S
Fenbendazole (FBZ)
NH-CO2CH3
O N H
N H
Fenbendazole sulphoxide
Fenbendazole sulphone (FBZSO2)
(Oxfendazole - FBZSO)
O
CH3CH2CH2-S
CH3CH2CH2-S
N
O N
NH-CO2CH3 N H
Albendazole (ABZ)
CH3CH2CH2-S NH-CO2CH3
N H
Albendazole sulphoxide (ABZSO)
N
O
NH-CO2CH3 N H
Albendazole sulphone (ABZSO2)
Fig. 1. Metabolic pathways of fenbendazole (FBZ) and albendazole (ABZ).
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comprising two groups of two animals and one group of three animals. The animals were kept indoors and had clover hay and water available ad libitum throughout the course of the study. No invasive procedures were involved beyond blood and faecal sampling procedures. The study was approved by Animal Ethics Committee of the University of Adnan Menderes. 2.2. Treatments and sampling Commercially available formulations of oxfendazole (FBZSO; Klozak, 375 mg tablets, Sanovel), fenbendazole (FBZ; Curantel, 4% powder, Bremer Pharma), and albendazole (ABZ; Valbazen, 600 mg tablets, Pfizer) were orally administered to the animals. All tablets were crushed prior to administration and total doses for each animal were mixed in 50 mL distilled water to form a slurry and were then administered by nasogastric intubation at a dose rate of 10 mg/kg bodyweight. The animals were treated early in the morning before feeding. After the treatment the animals were observed continuously for any adverse reactions within the first day. Heparinised blood samples were collected by jugular venipuncture prior to drug administration and 1, 2, 4, 8, 12, 20, 24, 32, 48, 72, 96 and 120 h thereafter. Faecal samples (>10 g) were collected per rectum throughout the blood-sampling period, before drug administration and then at 8, 12, 20, 24, 32, 48, 72, 96 and 120 h in order to determine faecal excretion of the benzimidazoles 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. The experiment was repeated with the groups alternated according to a three-phase cross-over design. A four-week wash out period was allocated between each experiment. 2.3. Analytical procedures High performance liquid chromatography (HPLC) with a liquid–liquid phase extraction procedure (adapted from Marriner and Bogan, 1980) was used to measure plasma concentrations of FBZSO, its sulphide FBZ and sulphone FBZSO2 metabolites for the FBZSO study; FBZ, FBZSO and, FBZSO2 for the FBZ study, and ABZ, albendazole sulphoxide (ABZSO), and albendazole sulphone (ABZSO2) for the ABZ study. 2.3.1. Extraction from plasma Pure FBZSO, FBZ and FBZSO2 were obtained from Hoechst, and ABZ, ABZSO and ABZSO2 were obtained from SmithKline Beecham. These were diluted with acetonitrile to give 0.5, 1, 2.5, 5, 10 and 10, 50, 500 lg/mL standard solutions for plasma and faecal samples, respectively. These were used for calibration
of standard curves and added to drug-free plasma and faecal samples to determine the recovery. Drug-free plasma samples (1 mL) were spiked with standards to reach the following final concentrations: 0.05, 0.1, 0.25, 0.5, and 1 lg/mL. Ammonium hydroxide (100 lL, 0.1 N, pH 10) was added to 10 mL-ground glass tubes containing 1 mL spiked or experimental plasma samples. Albendazole sulphoxide (0.2 lg/mL) was used as an internal standard for the FBZSO and FBZ analysis; and FBZSO (0.2 lg/mL) was used as an internal standard for the ABZ analysis. After mixing for 15 s, 6 mL chloroform (6 mL ethyl acetate for the ABZ study) were added. The tubes were shaken on a slow rotary mixer for 10 min. After centrifugation at 3000g for 10 min, the supernatant was removed with a Pasteur pipette. The organic phase (4 mL) was transferred to a thin-walled 10 mL-conical glass tube and evaporated to dryness at 40 C in a Buchi Rota Vapor R-3000. The dry residue was resuspended with 250 lL dimethyl sulphoxide (DMSO). Then the tubes were placed in an ultrasonic bath and finally, 20 lL of this solution were injected into the chromatographic system. 2.3.2. Extraction from faeces Wet-faecal concentrations of FBZSO, FBZ and ABZ were estimated by HPLC with a liquid–liquid phase extraction procedure adapted from that described by Gokbulut et al. (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, 100, and 200 lg/ g. Albendazole sulphoxide and FBZSO were used as internal standards for FBZSO–FBZ and ABZ studies, respectively. Sodium hydroxide buffer (200 lL, 0.4 M, pH 10) and 1 mL acetonitrile were added to 10 mLground 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 organic phase of the supernatant was transferred to a thinwalled 10 mL-conical glass tube and evaporated to dryness at 45 C in the sample concentrator. The dry residue was resuspended with 500 lL DMSO. After ultrasonication for 1 min, the samples were filtered with GF/C glass microfibre filter (Whatman). Finally, 20 lL of this solution were injected into the chromatographic system. 2.3.3. Chromatographic conditions The mobile phase was a mixture of acetonitrile–water to which glacial acetic acid was added (0.5%, v/v). For FBZSO, FBZ, ABZ and their metabolites, it was pumped through the column (Macherey-Nagel,
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nucleosil C18, 4 lm, 250 mm · 4.6 mm) with a nucleosil C18 guard column (Phenomenex, Cheshire, UK) in a linear gradient fashion changing from 25:75 (acetonitrile– water) to 75:25 for 9 min, 75:25 to 25:75 for 1 min and the last ratio was maintained for 3 min. The flow rate was 1 mL/min. Samples were processed on a computerised gradient HPLC system (SCL-10Avp, Shimadzu) comprising a degasser (DGU-14A), a pump (LC10ATvp) with a mixer (FCV-10ALvp) and an ultraviolet (UV) detector (SP8450, Spectra Physics) set at 292 nm. The retention times were 3.74 (ABZSO) 5.16 (FBZSO), 6.75 (FBZSO2), 7.70 (ABZSO2), 8.87 (ABZ) and 9.39 min (FBZ). 2.3.4. Method of calibration Calibration graphs for the parent drugs and their metabolites in the range 0.05–1 lg/mL (plasma) and 1– 200 g/g (faeces) were prepared using drug-free plasma and faeces from donkeys. Recovery of the three 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 donkey plasma and faecal samples containing known amounts of the drugs on different days. The limits of quantification of assay was defined at the lowest detected concentration that would result in coefficient of variation <20%. The limit of detections of the plasma and faecal assays were 0.005 lg/mL and 0.2 lg/g, respectively, for FBZSO and FBZ studies, and 0.006 lg/mL and 0.4 lg/g, respectively, for the ABZ study. The concentrations of parent molecules and their metabolites in unknown samples were calculated by reference to plasma samples to which known amounts of drugs (and metabolites) had been added and taken through the analytical procedure. To determine the dry proportion of wet faecal samples, 2.0 g of wet faeces from each sample was 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. 2.3.5. Pharmacokinetic and statistical analysis of data The plasma and faecal concentration vs. time curves obtained after each treatment in individual animals were fitted with the WinNonlin software program (Scientific Consulting Inc). Pharmacokinetic parameters for each animal were analysed using non-compartmental model analysis with extravascular input. The maximum plasma and faecal concentration (Cmax) and time to reach maximum concentration (tmax) were obtained from the plotted concentration–time curve of
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each drug in each animal. The area under the plasma and faecal concentration time curve (AUC) and mean residence time (MRT) from time zero to last time with a measurable concentration were calculated by trapezoidal rule. Terminal half life ðt1=2kz Þ was calculated as: t1=2kz ¼ lnð2Þ=kz ; where kz represents the first-order rate constant associated with the terminal (log linear) portion of the curve. The pharmacokinetic parameters are reported as means ± SEM. Mean pharmacokinetic parameters were statistically compared by an analysis of variance (ANOVA). Mean values were considered significantly different at P < 0.05.
3. Results The analytical method used to extract and quantify the plasma and faecal concentration of benzimidazole parent molecules and the respective metabolites by chromatographic analysis using UV detector was validated. The regression lines between peak areas and drug concentration showed correlation coefficients between 0.993 and 0.999. The mean extraction recoveries were 92.3% (FBZSO), 78.4% (FBZ), 94.1% (FBZSO2), 87.5% (ABZ), 85.1% (ABZSO) and 88.7% (ABZSO2) for plasma; and 87.2% (FBZSO), 76.4% (FBZ), 83.2% (FBZSO2), 86.9% (ABZ), 87.8% (ABZSO) and 89.3% (ABZSO2) for faecal samples. The inter-assay precision showed variation coefficients between 3.26% and 6.87% for plasma and faecal analysis. Fenbendazole and its metabolites (FBZSO and FBZSO2) were not detected in plasma at any time following FBZ administration. The plasma concentration time curves for FBZSO, FBZSO2 and ABZSO and ABZSO2 following oral administration of FBZSO and ABZ are shown in Figures 2 and 3, respectively. The pharmacokinetic data associated with each of these drug administrations and respective metabolites are given in Table 1. Following administration of FBZSO and ABZ, the FBZSO2 and ABZSO metabolic moieties predominated in plasma. The parent molecule did not reach detectable concentrations in any plasma samples after ABZ administration. The sulphide metabolite (FBZ) of FBZSO was not detected in any plasma samples. Maximum plasma concentrations (Cmax) of FBZSO (0.49 lg/mL), FBZSO2 (0.60 lg/mL); ABZSO (0.08 lg/mL) and ABZSO2 (0.04 lg/mL) were obtained at (tmax) 5.67, 8.00, 5.71 and 8.00 h, respectively. The AUC of sulphone metabolite FBZSO2 (10.33 lg h/mL) was significantly higher than that of FBZSO (5.17 lg h/mL). The AUC of sulphoxide metabolite (0.84 lg h/mL) of ABZ was significantly higher than
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Dry-faecal concentration (µg/g)
200
0.50 0.45
FBZSO FBZSO2
0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0
10
20
30
40
50
150
FBZSO FBZ
100
50
0 0
Time (h)
20
40
60
80
100
120
Time (h)
Fig. 2. Mean (±SEM) plasma concentrations (lg/mL) of oxfendazole (FBZSO) and its sulphone metabolite (FBZSO2) following oral administration of oxfendazole (FBZSO) at 10 mg/kg bodyweight in donkeys (n = 7).
Plasma concentration (µg/mL)
0.08
0.06
ABZSO ABZSO 2 0.04
0.02
0.00 0
5
10
15
20
25
30
35
40
45
50
Time (h)
Fig. 3. Mean (±SEM) plasma concentrations (lg/mL) of albendazole sulphoxide (ABZSO) and albendazole sulphone (ABZSO2) following oral administration of albendazole (ABZ) at 10 mg/kg bodyweight in donkeys (n = 7).
that of the sulphone metabolite (0.50 lg h/mL). Although residence times for sulphoxide forms of both drugs were similar, MRT of FBZSO2 was significantly longer than that of sulphone metabolite of ABZ.
Fig. 4. Mean (±SEM) dry faecal concentrations (lg/g) of oxfendazole (FBZSO) and its sulphide metabolite, fenbendazole (FBZ) following oral administration of oxfendazole at 10 mg/kg bodyweight in donkeys (n = 7).
The dry-faecal concentration time curves for FBZ, FBZSO, ABZ and ABZSO following oral administration of FBZSO, FBZ and ABZ are shown in Figs. 4– 6, respectively. The faecal kinetic data associated with each of these drug administrations and respective metabolites are given in Table 2. Following each administration no drug could be detected in faeces for at least 8 h and the maximum mean concentration occurred between 28.0 and 34.7 h. Concentrations had declined to below the limit of analytical detection in most samples by 96 h after oral administration. In faeces, the sulphide metabolite or parent was present at significantly higher concentrations than the more oxidised moieties. After ABZ administration, the parent molecule was significantly metabolised to sulphoxide metabolite (ABZSO) and it showed a similar excretion profile to parent molecule in the faecal samples. The Cmax and AUC of parent FBZ (722.2 lg/g, 20 487 lg h/g) molecule were significantly higher and larger, respectively, than those of FBZSO (59.2 lg/g, 1830 lg h/g) and ABZ (221.7 lg/ g, 5876 lg h/g) in faecal samples.
Table 1 Mean ± SEM pharmacokinetic parameters of oxfendazole (FBZSO), fenbendazole sulphone (FBZSO2), albendazole sulphoxide (ABZSO) and albendazole sulphone (ABZSO2) following oral administration of oxfendazole (10 mg/kg) and albendazole (10 mg/kg) to donkeys Pharmacokinetic parameters
tmax (h) Cmax (lg/mL) AUC (lg h/mL) t1=2kz a (h) AUMC (lg h2/mL) MRT (h)
FBZSO administered
ABZ administered
FBZSO
FBZSO2
ABZSO
ABZSO2
5.67 ± 2.89 0.49 ± 0.11 5.17 ± 0.82 4.49 ± 0.74 58.12 ± 14.18 10.95 ± 1.92
8.00 ± 2.53 0.60 ± 0.09b 10.33 ± 0.87b 7.53 ± 0.35 161.00 ± 22.99b 15.38 ± 1.50b
5.71 ± 0.87 0.08 ± 0.01 0.84 ± 0.14 6.65 ± 3.22 8.27 ± 1.86 9.15 ± 1.20
8.00 ± 2.31 0.04 ± 0.00 0.50 ± 0.11 7.44 ± 1.19 5.68 ± 1.93 9.98 ± 1.58
Cmax: peak plasma concentration; tmax: time to reach peak plasma concentration; AUC: area under the (zero moment) curve from time 0 to the last detectable concentration, AUMC: area under the moment curve from time 0 to t last detectable concentration; MRT: mean residence time; t1=2kz : terminal half-life. a Values represent the harmonic mean for t1=2kz . b Mean parameters of FBZSO2 are significantly different from those obtained for ABZSO2.
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Dry-faecal concentration (µ g/g)
1000 900 800 700 600
FBZ FBZSO
500 400 300 200 100 0 0
20
40
60
80
100
120
Time (h)
Fig. 5. Mean (±SEM) dry faecal concentrations (lg/g) of fenbendazole (FBZ) and its sulphoxide metabolites (FBZSO) following oral administration of fenbendazole at 10 mg/kg bodyweight in donkeys (n = 7).
Dry-faecal concentration (µg/g)
350 300
ABZ ABZSO
250 200 150 100 50 0 0
20
40
60
80
100
120
Time (h)
Fig. 6. Mean (±SEM) dry faecal concentrations (lg/g) of albendazole (ABZ) and its sulphoxide metabolites (ABZSO) following oral administration of albendazole at 10 mg/kg bodyweight in donkeys (n = 7).
4. Discussion The pharmacokinetics and activity of benzimidazoles are particularly influenced by the physicochemical properties and metabolic pathways of the active molecules.
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The benzimidazole anthelmintics are only sparingly soluble in water and their absorption and pharmacokinetics are affected by their aqueous solubility (Ngomuo et al., 1984). The results reported here indicate that FBZ and its metabolites (FBZSO and FBZSO2) were not detected in plasma at any time, and the plasma concentration of FBZSO was significantly greater than that of ABZ following oral administration at same dose rate in the donkey. This probably reflects better water solubility and subsequent absorption of FBZSO than FBZ and ABZ since, at least for FBZ, the same metabolites are produced by each compound and would be expected to have similar metabolic and excretory rates. Although the plasma dispositions of FBZSO and its sulphone metabolite showed a similar profile in donkeys to those found in horses (McKellar et al., 2002; Marriner and Bogan, 1985), FBZ and its sulphoxide and sulphone metabolites were not detectable in plasma any time and this differs from horses. It is likely that FBZ absorption from the gastrointestinal track is substantially lower than absorptions of FBZSO and ABZ in donkeys and the larger faecal excretion of FBZ than FBZSO and ABZ supports this. The much higher solubility of FBZSO (3.01 mg/L) than FBZ (0.07 mg/L) (Marriner and Bogan, 1985) may explain its greater absorption in donkeys. It is also apparent that the sulphone and sulphoxide metabolites were predominant in plasma following administration of FBZSO and ABZ administration, respectively. It was demonstrated that horses metabolise FBZ and FBZSO to their sulphone metabolites more quickly than ruminants (McKellar et al., 2002; Marriner and Bogan, 1985). Although bioavailability of ABZ was lower than ruminants, its metabolic pathways displayed a similar profile to that found in ruminants. Plasma disposition of ABZ was different compared to FBZSO and FBZ in donkeys. Although the parent ABZ was not detected in plasma, its sulphoxide and sulphone metabolites appeared in plasma following oral ABZ administration. In addition Cmax and AUC of the
Table 2 Mean ± SEM faecal kinetic parameters of oxfendazole (FBZSO), fenbendazole (FBZ), albendazole (ABZ) and albendazole sulphoxide (ABZSO) following oral administration of oxfendazole, fenbendazole and albendazole to donkeys FBZSO administered
tmax (h) Cmax(lg/g) tlast (h) AUC (lg h/g) MRT(h)
FBZ administered
ABZ administered
FBZSO
FBZ
FBZ
FBZSO
ABZ
ABZSO
32.00 ± 3.32 59.2 ± 19.4 56.00 ± 4.69 1830 ± 579 38.7 ± 3.25
31.71 ± 8.82 133.3 ± 27.4 68.00 ± 3.44 4595 ± 932 40.2 ± 2.87
34.29 ± 3.80 722.2 ± 155.5a 88.00 ± 10.31a 20487 ± 4098a 35.9 ± 3.42
34.67 ± 4.63 20.8 ± 5.0 50.67 ± 7.63 594 ± 15 0 46.2 ± 4.97
29.71 ± 1.48 221.7 ± 92.6b 64.00 ± 4.86 5876 ± 2382b 37.6 ± 3.76
28.00 ± 1.96 176.9 ± 41.6 72.00 ± 5.25 6925 ± 1604 39.5 ± 3.43
Cmax: peak faecal concentration; tmax: time to reach peak faecal concentration; AUClast: area under the (zero moment) curve from time 0 to the last detectable concentration, MRTlast: mean residence time. a Mean parameters of FBZ are significantly different from those obtained for parent FBZSO and ABZ. b Mean parameters of ABZ are significantly different from those obtained for parent FBZSO.
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sulphoxide metabolite (0.08 lg and 0.84 lg h/mL, respectively) which is thought to be anthelmintically active were significantly greater than those of the sulphone metabolite (0.04 lg/mL and 0.5 lg h/mL, respectively) which is thought to be anthelmintically inactive. In the present study, the times to maximum faecal concentrations (32 h for FBZSO, 34 h for FBZ and 30 h for ABZ) reflect gut transit time following oral administration in donkeys and these values were longer than those of horses (24 h for FBZ and FBZSO – McKellar et al., 2002). It is likely that hay-based diet and different anatomical characterisations caused this delay in donkeys compared with horses which were at grass pasture. In sheep and cattle, it has been reported that the reduction of FBZSO to sulphide (FBZ) occurred in ruminal fluid (Beretta et al., 1987). The reductive environment of the gastrointestinal tract of the donkey could be responsible for metabolic reduction. Following FBZ administration, the parent drug was predominant and its sulphoxide metabolite was present in extremely low concentrations in faecal samples. After ABZ administration, its sulphoxide metabolite was present at higher concentrations in faeces compared with the sulphoxide metabolite produced following FBZ administration. In addition, the ratios of faecal AUC of the parent molecule:sulphoxide metabolite were 1:1 and 34:1 after ABZ and FBZ administration, respectively. In addition, following FBZSO administration, the parent molecule was extensively metabolised to its sulphide metabolite (FBZ), which displayed a greater faecal excretion profile than the parent FBZSO. Despite this, the sulphide metabolite was not detected in any plasma samples. It is possible that high first-pass metabolism decreased the bioavailability of the sulphide metabolite by converting it once again into the parent molecule again. This may explain why the plasma concentration of FBZSO was significantly greater than that of FBZ and ABZ following oral administration at same dose rate in donkeys. In conclusion, this study has demonstrated that the plasma concentration of FBZSO was significantly higher than that of ABZ, whereas the plasma concentration of ABZ was very low and that of FBZ did not reach detectable concentrations following oral administration of each compound at the same dose rate (10 mg/kg) in donkeys. High intestinal concentrations could be effective against gastrointestinal nematodes that inhabit the gut lumen, but very low plasma concentrations of ABZ may not be effective against migrating fourth-larval stages of large strongyles or lungworms. Repeated dosage regimes of ABZ or co-administration with metabolic inhibitors could be used to treat migrating larval or tissue stages of strongyles and lungworms in donkeys, although such strategies should be confirmed before they can be recommended.
Acknowledgements The technical assistance of Mr. U. Karademir and Mr. M. Boyacioglu is gratefully acknowledged.
References Averkin, E., Beard, C., Dvorak, C., Edwards, J., Fried, J., Schiltz, R., Kistner, T.P., Drudge, J.H., Lyons, E.T., Sharp, M.L., Corvin, R.M., 1975. Methyl 5(6)-phenylsulfinyl-2-benzimidazole carbamate: a new potent anthelmintic. Journal of Medical Chemistry 19, 1164–1166. Beretta, C., Fadini, L., Malvisi, J., Montesissa, C., 1987. In vitro febantel transformation by sheep and cattle ruminal fluids and metabolism by hepatic subcellular fractions from different animal species. Biochemical Pharmacology 36, 3107–3117. Delatour, P., Parish, R., 1986. Benzimidazole anthelmintics and related compounds: Toxicity and evaluation of residues. In: Rico, A.G. (Ed.), Drug Residues in Animals. Academic Press, New York, pp. 175–203. Duwel, D., 1977. Fenbendazole II, biological properties and activity. Pesticide Science 8, 550–555. Gokbulut, C., Nolan, A.M., 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. Gokbulut, C. 2000. Pharmacokinetic disposition, faecal excretion, metabolism and chirality of anthelmintic drugs in horses. Ph.D. Thesis, University of Glasgow, Faculty of Veterinary Medicine, Department of Pharmacology. Glasgow, Scotland, UK. Gyurik, R.J., Chow, A.W., Zaber, B., Brunner, E.L., Miller, J.A., Villani, A.J., Petra, L.A., Parish, R.C., 1981. The metabolism of albendazole in cattle, sheep, rats and mice. Drug Metabolism and Disposition 9, 503–508. Lacey, E., Brady, R.L., Prichard, R.K., Watson, T.R., 1987. Comparison of inhibition of polymerisation of mammalian tubulin and helminth ovicidal activity by benzimidazole carbonates. Veterinary Parasitology 23, 105–119. Lanusse, C.E., Prichard, R.K., 1993. Relationship between pharmacological properties and clinical efficacy of ruminant anthelmintics. Veterinary Parasitology 49, 123–158. Marriner, S.E., Bogan, J.A., 1980. Pharmacokinetics of albendazole in sheep. American Journal of Veterinary Research 41, 483–491. Marriner, S.E., Bogan, J.A., 1981. Pharmacokinetics of fenbendazole in sheep. American Journal of Veterinary Research 42, 1146– 1148. Marriner, S.E., Bogan, J.A., 1985. Plasma concentration of fenbendazole and oxfendazole in the horse. Equine Veterinary Journal 17, 58–61. McKellar, Q.A., Scott, E.W., 1990. The benzimidazole anthelmintic agents: a review. Journal of Veterinary Pharmacology and Therapeutics 13, 223–247. McKellar, Q.A., Gokbulut, C., Benchaoui, H.A., Muzandu, K.M., 2002. Fenbendazole pharmacokinetics, metabolism and potentiation in horses. Drug Metabolism and Disposition 30, 1230– 1239. Mohammed Ali, N.A.K., Bogan, J.A., Marriner, S.E., Richards, R.J., 1987. Pharmacokinetics of triclabendazole alone or in combination with fenbendazole in sheep. Journal of Veterinary Pharmacology and Therapeutics 9, 442–445. Ngomuo, A.J., Marriner, S.E., Bogan, J.A., 1984. The pharmacokinetics of fenbendazole and oxfendazole in cattle. Veterinary Research Communications 8, 187–193.
Plasma disposition and faecal excretion of oxfendazole, fenbendazole and albendazole following oral administration to donkeys Cengiz Gokbulut a
a,*
, Ferda Akar a, Quintin A. McKellar
b
Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Adnan Menderes, Isikli Koyu, Aydin, Turkey b The Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Hertfordshire AL9 7TA, UK Accepted 10 February 2005
Abstract Fenbendazole (FBZ), oxfendazole (fenbendazole sulphoxide, FBZSO), and albendazole (ABZ) were administered orally to donkeys at 10 mg/kg bodyweight. Blood and faecal samples were collected from 1 to 120 h post-treatment. The plasma and faecal samples were analysed by high performance liquid chromatography (HPLC). The parent molecule and its sulphoxide and sulphone (FBZSO2) metabolites did not reach detectable concentrations in any plasma samples following FBZ administration. ABZ was also not detected in any plasma samples, but its sulphoxide and sulphone metabolites were detected, demonstrating that ABZ was completely metabolised by first-pass mechanisms in donkeys. Maximum plasma concentrations (Cmax) of FBZSO (0.49 lg/mL) and FBZSO2 (0.60 lg/mL) were detected at (tmax) 5.67 and 8.00 h, respectively, following administration of FBZSO. The area under the curve (AUC) of the sulphone metabolite (10.33 lg h/mL) was significantly higher than that of the parent drug FBZSO (5.17 lg h/mL). Cmax of albendazole sulphoxide (ABZSO) (0.08 g/mL) and albendazole sulphone (ABZSO2) (0.04 lg/mL) were obtained at 5.71 and 8.00 h, respectively, following ABZ administration. The AUC of the sulphoxide metabolite (0.84 lg h/mL) of ABZ was significantly higher than that of the sulphone metabolite (0.50 lg h/mL). The highest dry-faecal concentrations of parent molecules were detected at 32, 34 and 30 h for FBZSO, FBZ and ABZ, respectively. The sulphide metabolite was significantly higher than the parent molecule after FBZSO administration. The parent molecule was predominant in the faecal samples following FBZ administration. After ABZ administration, the parent molecule was significantly metabolised, probably by gastrointestinal microflora, to its sulphoxide metabolite (ABZSO) that showed a similar excretion profile to the parent molecule in the faecal samples. The AUC of the parent FBZ was significantly higher than that of FBZSO and ABZ in faeces. It is concluded that the plasma concentration of FBZSO was significantly higher than that of FBZ and ABZ. Although ABZ is not licensed for use in Equidae, its metabolites presented a greater plasma kinetic profile than FBZ which is licensed for use in horses. A higher metabolic capacity, first-pass effects and lower absorption of benzimidazoles in donkeys decrease bioavailability and efficacy compared to ruminants. 2005 Elsevier Ltd. All rights reserved. Keywords: Donkey; Oxfendazole; Fenbendazole; Albendazole; Pharmacokinetics; Anthelmintics
1. Introduction
*
Corresponding author. Tel.: +90 256 247 0340; fax: + 90 256 247 0700. E-mail address: [email protected] (C. Gokbulut). 1090-0233/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2005.02.022
Benzimidazole anthelmintics have a broad spectrum of activity against gastrointestinal helminths, including migrating strongyle larvae and lungworm infections, and are well tolerated by mammals (McKellar and
C. Gokbulut et al. / The Veterinary Journal 172 (2006) 166–172
sion of those compounds appears to differ substantially from ruminants. In the horse, the bioavailability and residence time of the tested benzimidazoles (and metabolites) were lower and shorter, respectively, than in ruminants (McKellar et al., 2002; Gokbulut, 2000; Marriner and Bogan, 1985, 1981). Sulphide and sulphoxide benzimidazoles are known to bind nematode tubulin (Lacey et al., 1987) and therefore have activity against nematodes although sulphides exert an inhibitory activity on tubulin at lower concentrations than sulphoxides. In most species examined, the sulphoxide moiety predominates in plasma and is thought to confer activity against gut dwelling nematodes following secretion across the gastrointestinal wall into the gut lumen where it may undergo sulphoreduction (McKellar and Scott, 1990). Albendazole is extensively used in ruminants to treat gastrointestinal helminths but there is a paucity of data available in the literature on the toxic or side effects in equines and ABZ is not used in these species. Donkeys are widely used for transportation especially in rural and mountainous areas of Turkey. Because of the lack of registered those for donkeys, anthelmintics registered for horses and ruminants are commonly used to treat gastrointestinal helminths in this species. In the present study, the comparative pharmacokinetics and faecal excretions of FBZ, FBZSO and ABZ following oral administration in donkeys were investigated.
Scott, 1990). They are extensively metabolised 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 the excreta of the host as well as in parasites recovered from benzimidazole-treated animals (Lanusse and Prichard, 1993; Delatour and Parish, 1986). The less soluble benzimidazole compounds have a longer residence in the forestomachs of ruminants from which they are absorbed over prolonged periods and thus remain in the plasma for a relatively long time. Since an equilibrium exists between the plasma and the gastrointestinal tract, the duration of exposure of gut parasites to an effective concentration of the drug is extended (McKellar and Scott, 1990). Extremely insoluble anthelmintics may be less effective, since they may not be absorbed and are excreted unchanged in the faeces. This may explain the difference between the plasma concentrations of oxfendazole (fenbendazole sulphoxide, FBZSO) following oral administration and its inter-convertible metabolite fenbendazole (FBZ) (Ngomuo et al., 1984). A large proportion of the less soluble FBZ is known to be excreted in the faeces of ruminants (Duwel, 1977). Oxfendazole, fenbendazole and albendazole (ABZ) are commercially available sulphur-containing benzimidazoles that commonly undergo microsomal oxidation in liver. Sulphide benzimidazoles (FBZ and ABZ) are reversibly metabolised to their sulphoxide derivatives (Marriner and Bogan, 1981, 1980; Gyurik et al., 1981; Mohammed Ali et al., 1987). Irreversible sulphonation follows sulphoxidation and is a slower oxidative step resulting in a sulphone metabolite (Averkin et al., 1975) (Fig. 1). The pharmacokinetics of FBZ and FBZSO have been studied in horses in which the metabolic inter-conver-
2. Materials and methods 2.1. Animals Seven donkeys (Equus asinus), 88–122 kg body weight, were randomly allocated to three groups
O
O S
N
167
N
S NH-CO2CH3
NH-CO2CH3
N H
N
S
Fenbendazole (FBZ)
NH-CO2CH3
O N H
N H
Fenbendazole sulphoxide
Fenbendazole sulphone (FBZSO2)
(Oxfendazole - FBZSO)
O
CH3CH2CH2-S
CH3CH2CH2-S
N
O N
NH-CO2CH3 N H
Albendazole (ABZ)
CH3CH2CH2-S NH-CO2CH3
N H
Albendazole sulphoxide (ABZSO)
N
O
NH-CO2CH3 N H
Albendazole sulphone (ABZSO2)
Fig. 1. Metabolic pathways of fenbendazole (FBZ) and albendazole (ABZ).
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comprising two groups of two animals and one group of three animals. The animals were kept indoors and had clover hay and water available ad libitum throughout the course of the study. No invasive procedures were involved beyond blood and faecal sampling procedures. The study was approved by Animal Ethics Committee of the University of Adnan Menderes. 2.2. Treatments and sampling Commercially available formulations of oxfendazole (FBZSO; Klozak, 375 mg tablets, Sanovel), fenbendazole (FBZ; Curantel, 4% powder, Bremer Pharma), and albendazole (ABZ; Valbazen, 600 mg tablets, Pfizer) were orally administered to the animals. All tablets were crushed prior to administration and total doses for each animal were mixed in 50 mL distilled water to form a slurry and were then administered by nasogastric intubation at a dose rate of 10 mg/kg bodyweight. The animals were treated early in the morning before feeding. After the treatment the animals were observed continuously for any adverse reactions within the first day. Heparinised blood samples were collected by jugular venipuncture prior to drug administration and 1, 2, 4, 8, 12, 20, 24, 32, 48, 72, 96 and 120 h thereafter. Faecal samples (>10 g) were collected per rectum throughout the blood-sampling period, before drug administration and then at 8, 12, 20, 24, 32, 48, 72, 96 and 120 h in order to determine faecal excretion of the benzimidazoles 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. The experiment was repeated with the groups alternated according to a three-phase cross-over design. A four-week wash out period was allocated between each experiment. 2.3. Analytical procedures High performance liquid chromatography (HPLC) with a liquid–liquid phase extraction procedure (adapted from Marriner and Bogan, 1980) was used to measure plasma concentrations of FBZSO, its sulphide FBZ and sulphone FBZSO2 metabolites for the FBZSO study; FBZ, FBZSO and, FBZSO2 for the FBZ study, and ABZ, albendazole sulphoxide (ABZSO), and albendazole sulphone (ABZSO2) for the ABZ study. 2.3.1. Extraction from plasma Pure FBZSO, FBZ and FBZSO2 were obtained from Hoechst, and ABZ, ABZSO and ABZSO2 were obtained from SmithKline Beecham. These were diluted with acetonitrile to give 0.5, 1, 2.5, 5, 10 and 10, 50, 500 lg/mL standard solutions for plasma and faecal samples, respectively. These were used for calibration
of standard curves and added to drug-free plasma and faecal samples to determine the recovery. Drug-free plasma samples (1 mL) were spiked with standards to reach the following final concentrations: 0.05, 0.1, 0.25, 0.5, and 1 lg/mL. Ammonium hydroxide (100 lL, 0.1 N, pH 10) was added to 10 mL-ground glass tubes containing 1 mL spiked or experimental plasma samples. Albendazole sulphoxide (0.2 lg/mL) was used as an internal standard for the FBZSO and FBZ analysis; and FBZSO (0.2 lg/mL) was used as an internal standard for the ABZ analysis. After mixing for 15 s, 6 mL chloroform (6 mL ethyl acetate for the ABZ study) were added. The tubes were shaken on a slow rotary mixer for 10 min. After centrifugation at 3000g for 10 min, the supernatant was removed with a Pasteur pipette. The organic phase (4 mL) was transferred to a thin-walled 10 mL-conical glass tube and evaporated to dryness at 40 C in a Buchi Rota Vapor R-3000. The dry residue was resuspended with 250 lL dimethyl sulphoxide (DMSO). Then the tubes were placed in an ultrasonic bath and finally, 20 lL of this solution were injected into the chromatographic system. 2.3.2. Extraction from faeces Wet-faecal concentrations of FBZSO, FBZ and ABZ were estimated by HPLC with a liquid–liquid phase extraction procedure adapted from that described by Gokbulut et al. (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, 100, and 200 lg/ g. Albendazole sulphoxide and FBZSO were used as internal standards for FBZSO–FBZ and ABZ studies, respectively. Sodium hydroxide buffer (200 lL, 0.4 M, pH 10) and 1 mL acetonitrile were added to 10 mLground 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 organic phase of the supernatant was transferred to a thinwalled 10 mL-conical glass tube and evaporated to dryness at 45 C in the sample concentrator. The dry residue was resuspended with 500 lL DMSO. After ultrasonication for 1 min, the samples were filtered with GF/C glass microfibre filter (Whatman). Finally, 20 lL of this solution were injected into the chromatographic system. 2.3.3. Chromatographic conditions The mobile phase was a mixture of acetonitrile–water to which glacial acetic acid was added (0.5%, v/v). For FBZSO, FBZ, ABZ and their metabolites, it was pumped through the column (Macherey-Nagel,
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nucleosil C18, 4 lm, 250 mm · 4.6 mm) with a nucleosil C18 guard column (Phenomenex, Cheshire, UK) in a linear gradient fashion changing from 25:75 (acetonitrile– water) to 75:25 for 9 min, 75:25 to 25:75 for 1 min and the last ratio was maintained for 3 min. The flow rate was 1 mL/min. Samples were processed on a computerised gradient HPLC system (SCL-10Avp, Shimadzu) comprising a degasser (DGU-14A), a pump (LC10ATvp) with a mixer (FCV-10ALvp) and an ultraviolet (UV) detector (SP8450, Spectra Physics) set at 292 nm. The retention times were 3.74 (ABZSO) 5.16 (FBZSO), 6.75 (FBZSO2), 7.70 (ABZSO2), 8.87 (ABZ) and 9.39 min (FBZ). 2.3.4. Method of calibration Calibration graphs for the parent drugs and their metabolites in the range 0.05–1 lg/mL (plasma) and 1– 200 g/g (faeces) were prepared using drug-free plasma and faeces from donkeys. Recovery of the three 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 donkey plasma and faecal samples containing known amounts of the drugs on different days. The limits of quantification of assay was defined at the lowest detected concentration that would result in coefficient of variation <20%. The limit of detections of the plasma and faecal assays were 0.005 lg/mL and 0.2 lg/g, respectively, for FBZSO and FBZ studies, and 0.006 lg/mL and 0.4 lg/g, respectively, for the ABZ study. The concentrations of parent molecules and their metabolites in unknown samples were calculated by reference to plasma samples to which known amounts of drugs (and metabolites) had been added and taken through the analytical procedure. To determine the dry proportion of wet faecal samples, 2.0 g of wet faeces from each sample was 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. 2.3.5. Pharmacokinetic and statistical analysis of data The plasma and faecal concentration vs. time curves obtained after each treatment in individual animals were fitted with the WinNonlin software program (Scientific Consulting Inc). Pharmacokinetic parameters for each animal were analysed using non-compartmental model analysis with extravascular input. The maximum plasma and faecal concentration (Cmax) and time to reach maximum concentration (tmax) were obtained from the plotted concentration–time curve of
169
each drug in each animal. The area under the plasma and faecal concentration time curve (AUC) and mean residence time (MRT) from time zero to last time with a measurable concentration were calculated by trapezoidal rule. Terminal half life ðt1=2kz Þ was calculated as: t1=2kz ¼ lnð2Þ=kz ; where kz represents the first-order rate constant associated with the terminal (log linear) portion of the curve. The pharmacokinetic parameters are reported as means ± SEM. Mean pharmacokinetic parameters were statistically compared by an analysis of variance (ANOVA). Mean values were considered significantly different at P < 0.05.
3. Results The analytical method used to extract and quantify the plasma and faecal concentration of benzimidazole parent molecules and the respective metabolites by chromatographic analysis using UV detector was validated. The regression lines between peak areas and drug concentration showed correlation coefficients between 0.993 and 0.999. The mean extraction recoveries were 92.3% (FBZSO), 78.4% (FBZ), 94.1% (FBZSO2), 87.5% (ABZ), 85.1% (ABZSO) and 88.7% (ABZSO2) for plasma; and 87.2% (FBZSO), 76.4% (FBZ), 83.2% (FBZSO2), 86.9% (ABZ), 87.8% (ABZSO) and 89.3% (ABZSO2) for faecal samples. The inter-assay precision showed variation coefficients between 3.26% and 6.87% for plasma and faecal analysis. Fenbendazole and its metabolites (FBZSO and FBZSO2) were not detected in plasma at any time following FBZ administration. The plasma concentration time curves for FBZSO, FBZSO2 and ABZSO and ABZSO2 following oral administration of FBZSO and ABZ are shown in Figures 2 and 3, respectively. The pharmacokinetic data associated with each of these drug administrations and respective metabolites are given in Table 1. Following administration of FBZSO and ABZ, the FBZSO2 and ABZSO metabolic moieties predominated in plasma. The parent molecule did not reach detectable concentrations in any plasma samples after ABZ administration. The sulphide metabolite (FBZ) of FBZSO was not detected in any plasma samples. Maximum plasma concentrations (Cmax) of FBZSO (0.49 lg/mL), FBZSO2 (0.60 lg/mL); ABZSO (0.08 lg/mL) and ABZSO2 (0.04 lg/mL) were obtained at (tmax) 5.67, 8.00, 5.71 and 8.00 h, respectively. The AUC of sulphone metabolite FBZSO2 (10.33 lg h/mL) was significantly higher than that of FBZSO (5.17 lg h/mL). The AUC of sulphoxide metabolite (0.84 lg h/mL) of ABZ was significantly higher than
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C. Gokbulut et al. / The Veterinary Journal 172 (2006) 166–172 0.55
Dry-faecal concentration (µg/g)
200
0.50 0.45
FBZSO FBZSO2
0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0
10
20
30
40
50
150
FBZSO FBZ
100
50
0 0
Time (h)
20
40
60
80
100
120
Time (h)
Fig. 2. Mean (±SEM) plasma concentrations (lg/mL) of oxfendazole (FBZSO) and its sulphone metabolite (FBZSO2) following oral administration of oxfendazole (FBZSO) at 10 mg/kg bodyweight in donkeys (n = 7).
Plasma concentration (µg/mL)
0.08
0.06
ABZSO ABZSO 2 0.04
0.02
0.00 0
5
10
15
20
25
30
35
40
45
50
Time (h)
Fig. 3. Mean (±SEM) plasma concentrations (lg/mL) of albendazole sulphoxide (ABZSO) and albendazole sulphone (ABZSO2) following oral administration of albendazole (ABZ) at 10 mg/kg bodyweight in donkeys (n = 7).
that of the sulphone metabolite (0.50 lg h/mL). Although residence times for sulphoxide forms of both drugs were similar, MRT of FBZSO2 was significantly longer than that of sulphone metabolite of ABZ.
Fig. 4. Mean (±SEM) dry faecal concentrations (lg/g) of oxfendazole (FBZSO) and its sulphide metabolite, fenbendazole (FBZ) following oral administration of oxfendazole at 10 mg/kg bodyweight in donkeys (n = 7).
The dry-faecal concentration time curves for FBZ, FBZSO, ABZ and ABZSO following oral administration of FBZSO, FBZ and ABZ are shown in Figs. 4– 6, respectively. The faecal kinetic data associated with each of these drug administrations and respective metabolites are given in Table 2. Following each administration no drug could be detected in faeces for at least 8 h and the maximum mean concentration occurred between 28.0 and 34.7 h. Concentrations had declined to below the limit of analytical detection in most samples by 96 h after oral administration. In faeces, the sulphide metabolite or parent was present at significantly higher concentrations than the more oxidised moieties. After ABZ administration, the parent molecule was significantly metabolised to sulphoxide metabolite (ABZSO) and it showed a similar excretion profile to parent molecule in the faecal samples. The Cmax and AUC of parent FBZ (722.2 lg/g, 20 487 lg h/g) molecule were significantly higher and larger, respectively, than those of FBZSO (59.2 lg/g, 1830 lg h/g) and ABZ (221.7 lg/ g, 5876 lg h/g) in faecal samples.
Table 1 Mean ± SEM pharmacokinetic parameters of oxfendazole (FBZSO), fenbendazole sulphone (FBZSO2), albendazole sulphoxide (ABZSO) and albendazole sulphone (ABZSO2) following oral administration of oxfendazole (10 mg/kg) and albendazole (10 mg/kg) to donkeys Pharmacokinetic parameters
tmax (h) Cmax (lg/mL) AUC (lg h/mL) t1=2kz a (h) AUMC (lg h2/mL) MRT (h)
FBZSO administered
ABZ administered
FBZSO
FBZSO2
ABZSO
ABZSO2
5.67 ± 2.89 0.49 ± 0.11 5.17 ± 0.82 4.49 ± 0.74 58.12 ± 14.18 10.95 ± 1.92
8.00 ± 2.53 0.60 ± 0.09b 10.33 ± 0.87b 7.53 ± 0.35 161.00 ± 22.99b 15.38 ± 1.50b
5.71 ± 0.87 0.08 ± 0.01 0.84 ± 0.14 6.65 ± 3.22 8.27 ± 1.86 9.15 ± 1.20
8.00 ± 2.31 0.04 ± 0.00 0.50 ± 0.11 7.44 ± 1.19 5.68 ± 1.93 9.98 ± 1.58
Cmax: peak plasma concentration; tmax: time to reach peak plasma concentration; AUC: area under the (zero moment) curve from time 0 to the last detectable concentration, AUMC: area under the moment curve from time 0 to t last detectable concentration; MRT: mean residence time; t1=2kz : terminal half-life. a Values represent the harmonic mean for t1=2kz . b Mean parameters of FBZSO2 are significantly different from those obtained for ABZSO2.
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Dry-faecal concentration (µ g/g)
1000 900 800 700 600
FBZ FBZSO
500 400 300 200 100 0 0
20
40
60
80
100
120
Time (h)
Fig. 5. Mean (±SEM) dry faecal concentrations (lg/g) of fenbendazole (FBZ) and its sulphoxide metabolites (FBZSO) following oral administration of fenbendazole at 10 mg/kg bodyweight in donkeys (n = 7).
Dry-faecal concentration (µg/g)
350 300
ABZ ABZSO
250 200 150 100 50 0 0
20
40
60
80
100
120
Time (h)
Fig. 6. Mean (±SEM) dry faecal concentrations (lg/g) of albendazole (ABZ) and its sulphoxide metabolites (ABZSO) following oral administration of albendazole at 10 mg/kg bodyweight in donkeys (n = 7).
4. Discussion The pharmacokinetics and activity of benzimidazoles are particularly influenced by the physicochemical properties and metabolic pathways of the active molecules.
171
The benzimidazole anthelmintics are only sparingly soluble in water and their absorption and pharmacokinetics are affected by their aqueous solubility (Ngomuo et al., 1984). The results reported here indicate that FBZ and its metabolites (FBZSO and FBZSO2) were not detected in plasma at any time, and the plasma concentration of FBZSO was significantly greater than that of ABZ following oral administration at same dose rate in the donkey. This probably reflects better water solubility and subsequent absorption of FBZSO than FBZ and ABZ since, at least for FBZ, the same metabolites are produced by each compound and would be expected to have similar metabolic and excretory rates. Although the plasma dispositions of FBZSO and its sulphone metabolite showed a similar profile in donkeys to those found in horses (McKellar et al., 2002; Marriner and Bogan, 1985), FBZ and its sulphoxide and sulphone metabolites were not detectable in plasma any time and this differs from horses. It is likely that FBZ absorption from the gastrointestinal track is substantially lower than absorptions of FBZSO and ABZ in donkeys and the larger faecal excretion of FBZ than FBZSO and ABZ supports this. The much higher solubility of FBZSO (3.01 mg/L) than FBZ (0.07 mg/L) (Marriner and Bogan, 1985) may explain its greater absorption in donkeys. It is also apparent that the sulphone and sulphoxide metabolites were predominant in plasma following administration of FBZSO and ABZ administration, respectively. It was demonstrated that horses metabolise FBZ and FBZSO to their sulphone metabolites more quickly than ruminants (McKellar et al., 2002; Marriner and Bogan, 1985). Although bioavailability of ABZ was lower than ruminants, its metabolic pathways displayed a similar profile to that found in ruminants. Plasma disposition of ABZ was different compared to FBZSO and FBZ in donkeys. Although the parent ABZ was not detected in plasma, its sulphoxide and sulphone metabolites appeared in plasma following oral ABZ administration. In addition Cmax and AUC of the
Table 2 Mean ± SEM faecal kinetic parameters of oxfendazole (FBZSO), fenbendazole (FBZ), albendazole (ABZ) and albendazole sulphoxide (ABZSO) following oral administration of oxfendazole, fenbendazole and albendazole to donkeys FBZSO administered
tmax (h) Cmax(lg/g) tlast (h) AUC (lg h/g) MRT(h)
FBZ administered
ABZ administered
FBZSO
FBZ
FBZ
FBZSO
ABZ
ABZSO
32.00 ± 3.32 59.2 ± 19.4 56.00 ± 4.69 1830 ± 579 38.7 ± 3.25
31.71 ± 8.82 133.3 ± 27.4 68.00 ± 3.44 4595 ± 932 40.2 ± 2.87
34.29 ± 3.80 722.2 ± 155.5a 88.00 ± 10.31a 20487 ± 4098a 35.9 ± 3.42
34.67 ± 4.63 20.8 ± 5.0 50.67 ± 7.63 594 ± 15 0 46.2 ± 4.97
29.71 ± 1.48 221.7 ± 92.6b 64.00 ± 4.86 5876 ± 2382b 37.6 ± 3.76
28.00 ± 1.96 176.9 ± 41.6 72.00 ± 5.25 6925 ± 1604 39.5 ± 3.43
Cmax: peak faecal concentration; tmax: time to reach peak faecal concentration; AUClast: area under the (zero moment) curve from time 0 to the last detectable concentration, MRTlast: mean residence time. a Mean parameters of FBZ are significantly different from those obtained for parent FBZSO and ABZ. b Mean parameters of ABZ are significantly different from those obtained for parent FBZSO.
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sulphoxide metabolite (0.08 lg and 0.84 lg h/mL, respectively) which is thought to be anthelmintically active were significantly greater than those of the sulphone metabolite (0.04 lg/mL and 0.5 lg h/mL, respectively) which is thought to be anthelmintically inactive. In the present study, the times to maximum faecal concentrations (32 h for FBZSO, 34 h for FBZ and 30 h for ABZ) reflect gut transit time following oral administration in donkeys and these values were longer than those of horses (24 h for FBZ and FBZSO – McKellar et al., 2002). It is likely that hay-based diet and different anatomical characterisations caused this delay in donkeys compared with horses which were at grass pasture. In sheep and cattle, it has been reported that the reduction of FBZSO to sulphide (FBZ) occurred in ruminal fluid (Beretta et al., 1987). The reductive environment of the gastrointestinal tract of the donkey could be responsible for metabolic reduction. Following FBZ administration, the parent drug was predominant and its sulphoxide metabolite was present in extremely low concentrations in faecal samples. After ABZ administration, its sulphoxide metabolite was present at higher concentrations in faeces compared with the sulphoxide metabolite produced following FBZ administration. In addition, the ratios of faecal AUC of the parent molecule:sulphoxide metabolite were 1:1 and 34:1 after ABZ and FBZ administration, respectively. In addition, following FBZSO administration, the parent molecule was extensively metabolised to its sulphide metabolite (FBZ), which displayed a greater faecal excretion profile than the parent FBZSO. Despite this, the sulphide metabolite was not detected in any plasma samples. It is possible that high first-pass metabolism decreased the bioavailability of the sulphide metabolite by converting it once again into the parent molecule again. This may explain why the plasma concentration of FBZSO was significantly greater than that of FBZ and ABZ following oral administration at same dose rate in donkeys. In conclusion, this study has demonstrated that the plasma concentration of FBZSO was significantly higher than that of ABZ, whereas the plasma concentration of ABZ was very low and that of FBZ did not reach detectable concentrations following oral administration of each compound at the same dose rate (10 mg/kg) in donkeys. High intestinal concentrations could be effective against gastrointestinal nematodes that inhabit the gut lumen, but very low plasma concentrations of ABZ may not be effective against migrating fourth-larval stages of large strongyles or lungworms. Repeated dosage regimes of ABZ or co-administration with metabolic inhibitors could be used to treat migrating larval or tissue stages of strongyles and lungworms in donkeys, although such strategies should be confirmed before they can be recommended.
Acknowledgements The technical assistance of Mr. U. Karademir and Mr. M. Boyacioglu is gratefully acknowledged.
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