Moredun Research Institute, Penicuik, Scotland, United Kingdom
(Q.A.M); Adnan Menderes University, Faculty of Veterinary Medicine,
Department of Pharmacology and Toxicology, Isikli-Aydin, Turkey (C.G.);
Graduate School of Veterinary Medicine, Laboratory of Toxicology,
Department of Environmental Science, Hokkaido University, Sapporo,
Japan (K.M.); and Pfizer Central Research Ltd, Animal Health Clinical
Affairs, Sandwich, Kent, United Kingdom (H.B.)
The present study was designed to describe the pharmacokinetics and
fecal excretion of fenbendazole (FBZ) and fenbendazole sulphoxide (FBZSO) and their metabolites in horses, to investigate the
effects which concurrent feeding has on the absorption and pharmacokinetics of FBZ, and to determine the effect of
coadministration of the metabolic inhibitor piperonyl-butoxide on the
in vivo pharmacokinetics and in vitro liver microsomal metabolism of
sulfide and sulfoxide benzimidazoles. The effect of piperonyl-butoxide
on the enantiomeric genesis of the sulfoxide moiety was also
investigated. Following administration of FBZSO and FBZ, the
fenbendazole sulphone metabolite predominated in plasma, and the
Cmax and area under the plasma curve (AUC)
values for each moiety were larger (P < 0.001)
following FBZSO than FBZ. In feces the administered parent molecule
predominated. The combined AUC for active benzimidazole moieties
following oral administration of FBZ (10 mg/kg) in horses was almost 4 times as high in unfed horses (2.19 µg · h/ml) than in fed
horses (0.59 µg · h/ml), and coadministration of
piperonyl-butoxide significantly increased the AUC and
Cmax of active moieties following
intravenous administration of FBZSO and oral administration of FBZ.
When FBZSO was administered i.v. as a racemate, the first enantiomer of
oxfendazole (FBZSO-1) predominated in plasma, however, following
coadministration with piperonyl-butoxide, the second enantiomer of
oxfendazole (FBZSO-2) predominated for 10 h. Piperonyl-butoxide
significantly reduced the oxidative metabolism of FBZSO and FBZ in
equine liver microsomes and altered the ratio of enantiomers
FBZSO-1/FBZSO-2 from >4:1 to 1:1. It is concluded that in horses
efficacy of FBZSO and FBZ could be improved by administration to unfed
animals and coadministration with piperonyl-butoxide.
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Introduction |
Helminth parasites produce pathological changes
in the horse including diarrhea (Mair et al., 1990
), rapid progressive
weight loss (Love, 1992), functional disorders of the intestine
(Ogbourne and Duncan, 1977), and colic and pathological changes in the
mesenteric arteries (Duncan and Dargie, 1975).
Large strongyles (Strongylus vulgaris, Strongylus
edentatus, Strongylus equinus) migrate from the
gastrointestinal tract through viscera and blood vessels where orally
administered anthelmintics with low bioavailability are ineffective.
The small strongyles (Cyathostominae) may become inhibited
within the large intestinal mucosa at the early third and fourth larval
stages, which are largely recalcitrant to available anthelmintics. The
selection of anthelmintic resistant populations has increased since
phenothiazine resistance was first recognized in horses in 1961 (Drudge
and Elam, 1961). Selection of resistance to anthelmintics has developed rapidly in the horse (Love et al., 1989) probably because the epidemiology of equine parasites is less seasonal than that of ruminant
parasites. Horses are consequently treated frequently throughout the
year even in temperate climates thus exerting great selection pressure.
The pharmacokinetics of benzimidazole anthelmintics has been studied in
horses in which the metabolic interconversion of the sulfide and
sulfoxide benzimidazoles [fenbendazole (FBZ1)
and fenbendazole sulphoxide (FBZSO), respectively] seems to differ
substantially from that in ruminants (Marriner and Bogan, 1985).
Sulfide and sulfoxide benzimidazoles are
known to bind nematode tubulin (Lacey et al., 1987) and therefore have
activity against nematodes, although sulfides generally exert
inhibitory activity on tubulin at lower concentrations than sulfoxides.
In most species examined, the sulfoxide 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 sulforeduction.
The absorption and consequent efficacy of benzimidazoles is known to be
influenced by their administration with food. The bioavailability of
sulfide benzimidazoles is markedly reduced in ruminants which have had
unrestricted access to food compared with those given restricted access
prior to treatment. This is thought to be associated with
adsorbence on to, and passage of, particulate associated drug in the
digestive tract (Hennessy et al., 1994). The opposite is true in the
dog (McKellar et al., 1993) in which coadministration with food
increases the bioavailability of the benzimidazole by up to three times.
The coadministration of metabolic inhibitors has been used to reduce
the rate of metabolic oxidation of benzimidazole sulfides and
sulfoxides to sulfones, which are inactive and thus improve their
efficacy (Hennessy et al., 1985, Lanusse and Prichard, 1991). Piperonyl-butoxide is a potent inhibitor of cytochrome P450 oxidation, which has been shown to alter the pharmacokinetic profile of FBZ and to
potentiate its nematocidal activity in sheep (Benchaoui and McKellar,
1996). The potential for improved efficacy of benzimidazoles coadministered with inhibitors of oxidative metabolism is greater in
the horse than in ruminants since the rate of oxidative metabolism and
thus inactivation of sulfoxide moieties in the horse seems to be
extremely rapid (Marriner and Bogan, 1985).
The excretion of anthelmintics in the feces of livestock has given rise
to concern since it was observed that the avermectins have adverse
effects in dipteran flies and coleopteran beetles, which inhabit and
feed in dung (Wall and Strong, 1987). Whereas it is recognized that
benzimidazoles are unlikely to affect dung-dwelling arthropods
(McKellar, 1997), environmental impact is not limited to specific
effect on scavenger insects, and their excretion in the feces of horses
has not been characterized, and consequently the associated
environmental impact is not known.
The present studies were designed to describe the pharmacokinetics and
fecal excretion of FBZ and FBZSO and their metabolites in horses, to
investigate the effects which concurrent feeding has on the absorption
and pharmacokinetics of FBZ, and to determine the effect of
coadministration of the metabolic inhibitor piperonyl-butoxide on the
in vivo pharmacokinetics and in vitro equine liver microsomal metabolism of sulfide and sulfoxide benzimidazoles. The effect of
piperonyl-butoxide on the enantiomeric genesis of the sulfoxide moiety
was also investigated.
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Materials and Methods |
Animals and Experimental Design.
Oral pharmacokinetic and
fecal excretion study
Sixteen horses of mixed breed weighing 390 to 720 kg were allocated in
a restricted random fashion to two groups of eight, which were balanced
for weight. Animals were kept under field conditions with unrestricted
access to herbage and water. They were only corralled for the period of
drug administration, and all other procedures were carried out with
minimum restraint in the field. Commercially available formulations of
FBZ (Panacur, 18.75% w/w; Hoechst Pharmaceutical Research Labs, Milton
Keynes, Bedfordshire, UK) and oxfendazole [(FBZSO) (Systamex 906, 9.06% w/v; Mallinckrodt Veterinary Ltd., Uxbridge, UK)] were
administered orally as a single bolus dose on the back of the tongue at
10 mg/kg bodyweight. Heparinized blood samples (approximately 10 ml)
were collected by jugular venepuncture and fecal samples
(>10g) were collected per rectum at predetermined times
until 120 h after drug administration.
Intravenous enantioselective pharmacokinetics of oxfendazole
alone and following coadministration with piperonyl-butoxide.
Seven ponies weighing 164 to 250 kg were randomly allocated to two
groups comprising four and three animals. Ponies were kept indoors and
had hay and water available ad libitum throughout the course of the
study. Oxfendazole [(FBZSO) 99.9%; Schering-Plough, Uxbridge, UK]
was prepared in dimethyl sulfoxide (DMSO, 500 mg/ml; Sigma-Aldrich,
Gillingham, UK) and administered at a dose rate of 10 mg/kg
bodyweight by right jugular venepuncture to one group of ponies. The
other group was administered piperonyl-butoxide [90%; Sigma-Aldrich]
by nasogastric intubation at a dose rate of 31 mg/kg bodyweight and 30 min later was administered FBZSO as described above. Heparinized blood
samples were collected by jugular venepuncture from the left jugular
vein at predetermined times until 96 h after drug administration.
After a 4 week washout period, the experiment was repeated with the
groups reversed according to a two-phase crossover design.
Oral pharmacokinetics of fenbendazole administered with and
without food and following coadministration with piperonyl-butoxide.
Six ponies weighing 94 to 216 kg were randomly allocated to two groups
of three. They were kept indoors and had hay and water available ad
libitum except for the period immediately (for 12 h) prior to each
drug administration when the hay and any straw bedding were removed.
Three animals were administered FBZ (Panacur 2.5% w/w; Hoechst
Pharmaceutical Research Labs) orally as a single dose by nasogastric
intubation at 10 mg/kg bodyweight. Three animals received FBZ as above
30 min after receiving a 63 mg/kg dose of piperonyl-butoxide also by
nasogastric intubation. This study was repeated after a 4-week washout
period with the groups reversed. Animals were fed four h after drug
administration. Three months after the above study was completed, it
was repeated identically except that the horses were given
approximately 0.5 kg of cereal concentrates 1 h before drug
administration and a further 0.5 kg just before drug administration.
Heparinized blood samples were collected by jugular venepuncture at
predetermined times until 96 h after drug administration. All
animals were given access to hay ad libitum 4 h after FBZ administration.
Oral administration of fenbendazole and oxfendazole to a horse
with a chronic cecal fistula.
A pony weighing 260 kg with a chronic cecal fistula, which had been
inserted by the method of Boyd (1985) was used. Fenbendazole and FBZSO
were administered orally and intra cecally at a dose of 10 mg/kg with a
week washout period between drug administrations. Blood was collected
by jugular venepuncture at predetermined times (identical to those used
for the oral administration studies). All analytical methods were the
same as those used in the other oral studies.
The effect of piperonyl-butoxide on the metabolism and chirality
of benzimidazoles in microsomes from equine liver.
The livers were collected from seven horses being euthanized for
clinical reasons unrelated to hepatic disease. After the horse was
killed, its liver was removed and perfused with ice-cold saline (0.9%
NaCl solution) through the hepatic veins. A portion of liver was
dissected free, drained of excess moisture, and weighed to make
100 g from each animal.
Drug Analysis.
Blood samples were centrifuged at 1825g for 30 min, and
plasma was transferred to plastic tubes. All the plasma and fecal samples were stored at 
20°C until estimation of drug concentration. Plasma and wet-fecal concentration of FBZ, FBZSO,
FBZSO2, and hydroxy-fenbendazole (OH-FBZ) were
estimated by high performance liquid chromatography (HPLC) with a
liquid phase extraction procedure adapted from that of Marriner and
Bogan (1980). Standard compounds of FBZ, FBZSO,
FBZSO2, and OH-FBZ were obtained from Hoechst
(Frankfurt, Germany).
Drug-free plasma samples (1 ml) were spiked with standards of
FBZ, and its metabolites (FBZSO, FBZSO2, and
OH-FBZ) for the FBZ study, FBZSO and its metabolites (FBZ,
FBZSO2 and OH-FBZ) for the FBZSO study, and
oxibendazole as an internal standard to reach the following final
concentrations: 0.01, 0.05, 0.1, 0.5, and 1 µg/ml. Ammonium hydroxide
(200 µg/ml, 0.1 N, pH 10) was added to 10 ml-ground glass tubes
containing 1 ml of spiked or experimental plasma samples. After
vortexing for 15 s, 6 ml of chloroform (Rathburn Chemical Ltd.,
Walkerburn, Scotland, UK) was added. The tubes were shaken on a slow
rotary mixer for 10 min. After centrifugation at 1825g for
15 min, the supernatant was removed with a Pasteur pipette. The organic
phase (4 ml) was transferred to a thin-walled 10 ml-concial glass tube
and evaporated to dryness at 43°C in a sample
concentrator (model SC10A; Savant Instrument Inc., Holbrook, NY). The
dry residue was resuspended with 50 µl of DMSO and 200 µl of 25%
acetonitrile. The tubes were placed in an ultrasonic bath and 50 µl
of this solution was injected into the chromatographic system.
Wet fecal samples were mixed finely with a spatula to obtain
homogeneous concentrations. Drug-free wet feces samples (0.5 g) were
spiked with benzimidazole standards, described above, to reach the
following final concentrations: 1, 5, 50, 100, 200 µg/g. Sodium
hydroxide buffer (200 µl, 0.4M, pH 10) and 2 ml of acetonitrile were
added to 10 ml-ground glass tubes containing 0.5 g of spiked or
experimental wet-fecal samples. After vortexing for 15 s, 8 ml of
chloroform was added. The tubes were shaken on a slow rotary mixer for
15 min. After centrifugation at 1825g for 15 min, the
supernatant was removed with a Pasteur pipette. The organic phase (5 ml) was transferred to a thin-walled 10 ml-conical glass tube and
evaporated to dryness at 43°C in the sample
concentrator. The dry residue was resuspended with 50 µl of DMSO and
diluted appropriately with 35% acetonitrile. After ultrasonication,
the samples were filtered with glass microfibre filter (Whatman
International Ltd, Maidstone, Kent, UK) Finally, 50 µl of this
solution was injected into the chromatographic system.
The HPLC system (PC1000; Spectra Physics Analytical Inc., Manchester,
UK) comprised a gradient pump (model SP4000), a UV-detector (SP Focus)
set at 292 nm, an autosampler (model AS 3000), and a controller (model
SN 4000). 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 (Genesis nukleosil C18 4 µm, 150 mm × 4.6 mm; Crawford Scientific, Strathaven, Lanarkshire, Scotland, UK)
at a flow rate of 1 ml/min in a linear gradient from 35:65
(acetonitrile-water) to 60:40 for 9 min, 60:40 to 35:65 for 1 min and
35:65 for 2 min. The retention times were 5.5 (FBZSO), 6.8 (OH-FBZ),
7.4 (FBZSO2), and 10.2 (FBZ) min (Fig.
1). Recovery of parent molecules was
determined by comparison of peak areas on chromatograms from spiked
plasma and fecal samples with areas from direct injection of standards.
The interassay precision was evaluated by processing replicate aliquots
of spiked horse plasma or feces on different days.
For chiral analysis of FBZSO, plasma samples were extracted as
described above and the residue resuspended with 50 µl of DMSO and
150 µl of H2O, 50 µl of which was injected
into the chromatographic system. A mobile phase of acetonitrile/water
(7:93) was pumped at a flow rate of 0.9 ml/min through a chiral-AGP
column (5µ, 100 × 40 mm; BAS Instruments Ltd., Congleton,
Cheshire, UK) with ultraviolet detection at 296 nm. Retention
times of the enantiomers were 7.87 min for FBZSO-1 and 10.43 min for
FBZSO-2.
For analysis of microsome incubation mixture, the whole sample was
recovered and the tube washed with 1 ml of acetonitrile. Chloroform (6 ml) was added and the tube shaken on a slow rotary mixer for 10 min.
The tube was then centrifuged at 1825g for 15 min and two 3 ml of aliquots collected for achiral and chiral analysis. The HPLC
systems were as described above, but for achiral analysis the gradient
profile of the mobile phase changed from 25:75 (acetonitrile:water) to
45:55 in 6 min, to 75:25 in 11 min and then to 25:75 for 13 min. The
flow rate was 1.5 ml/min, and the retention times were 3.48 min
(FBZSO), 4.68min (OH-FBZ), 5.58 min (FBZSO2), and
8.41 (FBZ).
Analysis of fecal samples was carried out on a wet weight basis, and a
10 g sample of feces taken and dried in an oven at 70°C for
10 h. All fecal drug concentrations have been converted and are
described as concentrations per gram fecal dry matter.
Liver Microsome Preparations.
One hundred grams of finely chopped liver tissue was placed in 300 ml
of 1.15% KCl solution and homogenized using a Potter-Elvehjem homogenizer (Cambridge, UK). The homogenate was centrifuged for 20 min
at 9000g to remove debris, nuclei, and mitochondria. The surface fat layer was removed and the supernatant decanted and further
centrifuged at 105,000g for 75 min in a Beckman L8-70 refrigerated ultracentrifuge (Beckman Coulter Ltd., High Wycombe, Buckinghamshire, UK). The cytosolic supernatant was removed and the microsomal pellet resuspended in 60 ml of 0.1 M tris-phosphate buffer (pH 7.4) containing 20% (v/v) glycerol. The protein content of
the microsomal suspensions were determined using the Coomassie blue
protein assay reagent.
Drug incubations were carried out in triplicate in a shaking-water bath
of 37°C for 1 h. Glass test tubes (10 ml) were used. One assay
of an incubation mixture contained 4 mg of microsomal protein, 5 µl
of test drugs (0.5, 1, and 2.5 µM of FBZSO, FBZ, and OBZ standards
were each dissolved in DMSO) alone or with 5 µl of piperonyl-butoxide
and 1 ml of cofactor solution. Piperonyl-butoxide was used at a
constant concentration of 5 µM. Tubes without microsome were used as
controls for possible nonenzymatic drug conversion. After incubation
the tubes containing reaction mixture were placed in boiling water for
2 min to terminate the reaction then immediately stored at 20°C
until analysis.
Pharmacokinetic and Statistical Analysis.
The plasma concentration time curves obtained after each treatment in
individual animals were fitted with the WinNonLin software program
(Statistical Consulting Inc. Pharsight Corporation, Cary, NC). For all
oral drug administration studies, noncompartmental model analysis with
extravascular input was used. The Cmax
and time to reach Cmax
(Tmax) were obtained from the plotted
concentration time curve for each moiety in each animal. The linear
trapezoidal rule was used to calculate the area under the plasma
concentration time curve (AUC) and area under the first moment curve
(AUMC). The mean residence time (MRT) was determined as AUMC 
AUC.
The pharmacokinetic data are reported as mean ± S.E.M., and
pharmacokinetic parameters for each drug moiety obtained following intravenous and following oral administration were statistically compared using the Mann-Whitney U test. The Wilcoxon sign rank test was
used to determine the statistical differences associated with feeding.
In the microsomal incubation studies, the extent of conversion and
amount of unchanged drug with and without metabolic inhibition were
compared by one-way analysis of variance. Results were considered
significant when P < 0.05.
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Results |
Analytical Methods.
The percentage recoveries from plasma and interassay coefficients of
variation were determined for FBZ, FBZSO, and
FBZSO2 over the concentrations 0.05, 0.10, 0.25, 0.50, and 1.00 µg/ml. The mean (n = 40 for each)
recovery (r) and CV were FBZ r = 84.16%, CV = 9.05; FBZSO r = 94.61%, CV = 9.44; and
FBZSO2 r = 93.51%, CV = 6.23. The limit of detection of the assays was 0.005 µg/ml for plasma
and 0.2 mg/g for fecal samples. During chiral analysis the limit of
detection for both FBZSO enantiomers was 0.02 µg/ml, and recoveries
were 85.28% (CV = 7.8) and 86.69% (CV = 7.4%) for FBZSO-1
and FBZSO-2, respectively. OH-FBZ was not detected in plasma following
either FBZ or FBZSO administration.
Oral Pharmacokinetic and Fecal Excretion Study.
The plasma concentration time curves for FBZ, FBZSO, and
FBZSO2 following oral administration of FBZSO and
FBZ are shown in Figs. 2 and
3, respectively. The pharmacokinetic
data associated with each of these drug administrations are given in
Table 1.
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Fig. 2.
Mean (±S.E.M.) plasma concentrations
(µg/ml) of FBZSO and its metabolites, FBZ and FBZSO2
following oral administration of FBZSO at 10 mg/kg bodyweight in
horses.
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Fig. 3.
Mean (±S.E.M.) plasma concentrations
(µg/ml) of FBZ and its metabolites, FBZSO and FBZSO2
following oral administration of FBZ at 10 mg/kg bodyweight in
horses.
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TABLE 1
Mean ± S.E.M. pharmacokinetic parameters of FBZSO, FBZ, and
FBZSO2 following oral administration of oxfendazole (10 mg/kg)
and fenbendazole (10 mg/kg) to horses (n = 8)
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Following administration of FBZSO and FBZ as parent drugs, the
FBZSO2 metabolic moiety predominated in plasma.
The AUC ratio of sulfoxide/sulfide/sulfone was approximately 3:1:9
following FBZSO administration and 1:4:7 following FBZ administration.
The Cmax and AUC values for each
moiety were significantly (P < 0.001) larger following
administration of FBZSO than FBZ, and the two known active moieties
achieved approximately 25.6 times (FBZSO) and 2.3 times (FBZ) the AUC
values following administration of FBZSO than FBZ as the parent
compound. The residence times of the two active moieties were longer
(but not significantly longer) following administration of FBZSO than
FBZ but that of the sulfone metabolite was very similar following each
administration (15.45 ± 2.10 h versus 16.50 ± 1.00 h, respectively).
The fecal concentration time curves for FBZ, FBZSO, and
FBZSO2 following oral administration of FBZSO and
FBZ are shown in Figs. 4 and
5, respectively. Following each
administration, no drug could be detected in feces for at least 12 h, and the maximal mean concentration occurred at 24h. Concentrations
had declined to below the limit of analytical detection in most samples
by 72 h after administration. In feces the parent molecule
predominated and wheras the sulfide was present at a concentration of
0.21 mg/g following administration of the sulfoxide, the more oxidized metabolites never exceeded 0.017 mg/g in feces following FBZ
administration.
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Fig. 4.
Mean (±S.E.M.) dry fecal concentrations
(mg/g) of FBZSO and its metabolites, FBZ and FBZSO2
following oral administration of FBZSO at 10 mg/kg bodyweight in
horses.
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Fig. 5.
Mean (±S.E.M.) dry fecal concentrations
(mg/g) of FBZ and its metabolites, FBZSO and FBZSO2
following oral administration of fenbendazole at 10 mg/kg in horses
(n = 8).
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Intravenous Enantioselective Pharmacokinetics of FBZSO Alone and
following Coadministration with Piperonyl-butoxide.
The plasma concentration time curves of FBZSO, FBZ, and
FBZSO2, respectively, following administration of
FBZSO alone or in combination with piperonyl-butoxide are given in
Figs. 6, 7,
and 8, and pharmacokinetic data are
presented in Table 2. When FBZSO was
administered alone, the FBZSO moiety did not display a typical exponential decline over time. After an initial decline phase lasting
approximately 45 min, the concentrations of FBZSO plateaued at between
0.94 and 1.5 µg/ml until approximately 12 h from which time
concentrations declined to the limit of detection by 96 h. The
concentrations of FBZSO following administration of FBZSO plus
piperonyl-butoxide declined from administration for 30 min then
increased until 7 h by which time the plasma concentration was
3.69 ± 0.50 µg/ml. Concentrations decreased from this peak until by 30 h, they were lower than concentrations following
administration of FBZSO alone. The AUC and
Cmax of FBZSO were significantly
larger (P < 0.01) when FBZSO was administered with
piperonyl-butoxide than on its own.
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Fig. 6.
Mean (±S.E.M.) plasma concentrations
(µg/ml) of FBZSO following intravenous administration of oxfendazole
(10 mg/kg) alone or with piperonyl-butoxide coadministered orally (31 mg/kg) in ponies (n = 6).
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Fig. 7.
Mean (±S.E.M.) plasma concentrations
(µg/ml) of FBZ (mean ± S.E.M.) following intravenous
administration of oxfendazole (10 mg/kg) alone or with
piperonyl-butoxide coadministered orally (31 mg/kg) in ponies
(n = 6).
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Fig. 8.
Mean (±S.E.M.) plasma concentrations
(µg/ml) of FBZ.SO2 following intravenous administration
of oxfendazole (10 mg/kg) alone or with piperonyl-butoxide
coadministered orally (31 mg/kg) in ponies (n = 6).
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TABLE 2
Mean ± S.E.M. pharmacokinetic parameters of FBZSO, FBZ, and
FBZSO2 following intravenous administration of oxfendazole (10 mg/kg) alone or with piperonyl-butoxide administered p.o. at 31 mg/kg
in ponies (n = 6)
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The concentration of FBZ following administration of FBZSO alone or in
combination with piperonyl-butoxide displayed a similar pattern to
those of the parent molecule except that the maximal concentrations
were achieved later (at 16 h) compared with those of FBZSO (7 or
8 h). Concentrations of FBZ were lower than those of FBZSO, and
the AUC and Cmax of FBZ were
significantly (P < 0.01) lower following
administration of FBZSO alone compared with administration of FBZSO
with piperonyl-butoxide.
The concentration of FBZSO2 increased to
1.49 ± 0.12 µg/ml at 10 h following administration of
FBZSO alone. When FBZSO was given in combination with
piperonyl-butoxide, a similar maximal mean concentration was achieved
(1.46 ± 0.13 µg/ml), but it did not occur until 16 h after
administration. The AUC ratios for sulfide/sulfoxide/sulfone were
33:1:46 following FBZSO alone and 21:1:19 following FBZSO in
combination with piperonyl-butoxide.
The plasma concentration time curves of the enantiomers of FBZSO
following administration of FBZSO (as a racemate) either alone or in
combination with piperonyl-butoxide are shown in Fig. 9, and the ratios (as percentages) of
each enantiomer are given in Fig. 10.
When FBZSO (racemate) was administered alone, the FBZSO-1 enantiomer
predominated in plasma from 45 min after drug administration. From
approximately 5 h after administration until 24 h after
administration, the ratio was approximately 60:40 in favor of FBZSO-1.
Between 24 h and 48 h the ratio changed toward that of a
racemate.
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Fig. 9.
Mean (±S.E.M.) plasma concentrations
(µg/ml) of FBZSO-1 and FBZSO-2 in ponies following i.v.
administration of oxfendazole (10 mg/kg) either alone (A) or in
combination (B) with PB (31 mg/kg).
PB, piperonyl-butoxide.
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Fig. 10.
Ratio of the percentage of FBZSO-1 and
FBZSO-2 (µg/ml) in ponies following i.v. administration of
oxfendazole (10 mg/kg) either alone (A) or in combination with
piperonyl-butoxide (31 mg/kg) (B).
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The coadministration of piperonyl-butoxide had a dramatic effect on the
absolute and relative concentrations of each enantiomer. The FBZSO-2
enantiomer predominated from the time of administration until 10 h
approaching a 60:40 ratio at 5 h after administration. From
10 h the FBZSO-1 enantiomer predominated, and the ratio
FBZSO-1:FBZSO-2 increased until 30 h after administration and then
decreased until 48 h at which time it approached a racemate.
Oral Pharmacokinetics of Fenbendazole Administered with and without
Food and following Coadministration with Piperonyl-butoxide.
There were no statistically significant differences in the
pharmacokinetic parameters of the benzimidazole moieties when FBZ was
administered with or without food. Nevertheless the total-combined AUC
for the active benzimidazole moieties (FBZSO and FBZ) was almost 4 times as high in unfed horses (2.19 µg · h/ml) than in fed
horses (0.59 µg · h/ml). The coadministration of
piperonyl-butoxide significantly (P < 0.05) increased
the AUC and Cmax of FBZ in fed and
unfed horses. In fed horses piperonyl-butoxide coadministration increased the AUC of the active moieties FBZSO and FBZ by 13.9 times
and 13.2 times, respectively. In unfed horses the piperonyl-butoxide increased the AUC of the parent FBZ by 11 times but decreased the AUC
of the FBZSO moiety by 2.3 times. The effect on combined active
moieties was an increase in AUC of 2.0 times when FBZ was given with
piperonyl-butoxide.
The Absorption of Fenbendazole and Oxfendazole following Intracecal
Administration in a Horse.
Fenbendazole was absorbed following intracecal administration in the
horse indicating absorption processes in the hind gut. Concentrations
of the parent molecule (expressed as AUC and
Cmax) were as high following
intracecal administration as oral administration, although neither of
the more oxidized metabolites achieved
such good apparent bioavailability following intracecal administration (Table 3). Oxfendazole was also
absorbed from the cecum, but its bioavailability even as the parent
molecule was substantially lower than when administered orally.
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TABLE 3
The AUC and Cmax in a single horse following oral and
intracecal administration of fenbendazole and oxfendazole at a dose
rate of 10 mg/kg.
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The Effect of Piperonyl-butoxide on the Metabolism and Chirality of
Benzimidazoles in Microsomes from Equine Liver.
Oxfendazole was incubated in liver microsome preparations alone or with
piperonyl-butoxide. Following a 1 h incubation period, significantly (P < 0.001) more FBZSO remained in the
incubation mixture when the incubation was carried out in the presence
of piperonyl-butoxide (data not shown). This was associated with a
concurrent significant (P < 0.001) reduction in the
generation of the sulfone (FBZSO2) metabolite
when the substrate was incubated with piperonyl-butoxide (Fig.
11).
Piperonyl-butoxide coincubation also significantly reduced the
metabolism of FBZ in microsome preparations and significantly (P < 0.001) reduced the generation of FBZSO in the 2.5 µM incubation only (data not shown). The generation of the sulfone
metabolite was reduced at all incubation concentrations (Fig.
12); however, the generation of OH-FBZ
was not affected by piperonyl-butoxide (data not shown).
The enantioselective metabolism of FBZSO was determined following
incubations of FBZSO substrate as a racemate with and without piperonyl-butoxide. Microsomal metabolism was apparently
enantioselective since the FBZSO-1 and FBZSO-2 enantiomers were present
in the incubation medium after the incubation period in a ratio of
>4:1 when FBZSO was incubated alone (Fig.
13). There was a marked change in the
ratio when racemic FBZSO was incubated with piperonyl-butoxide such
that the ratio of FBZSO-1/FBZSO-2 approached 1:1 (Fig.
14). Fenbendazole metabolism to the
sulfoxide (FBZSO) was also shown to be enantioselective since FBZSO-1
predominated in reaction mixtures following incubation of FBZ.
Piperonyl-butoxide affected the enantioselective character of the
metabolism since the ratio of FBZSO-1/FBZSO-2 changed from 11 to 15:1
when FBZ was incubated alone to 3 to 6:1 when FBZ was incubated with
piperonyl-butoxide (data not shown).
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Fig. 14.
Total FBZ.SO-1 and FBZ.SO-2 remaining in
microsome reaction mixture following oxfendazole (0.5, 1, and 2.5 µM)
incubation with (5 µM) piperonyl-butoxide (PB).
PB, piperonyl-butoxide.
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Discussion |
The oral pharmacokinetic and excretion study confirms and extends
the work of Marriner and Bogan (1985) who demonstrated that in the
horse the bioavailability and residence time of the tested benzimidazoles (and metabolites) were lower and shorter, respectively than in ruminants (Marriner and Bogan, 1981a,b). The relatively low
plasma concentrations of active moieties (FBZ, FBZSO) following administration of fenbendazole probably accounts for the increased dosage required to treat migrating larval and tissue stages of large
strongyles and encysted mucosal stages of the cyathostomes (MAFF,
1983). It is also apparent that the sulfone metabolite predominates in
plasma following administration of either the sulfoxide (FBZSO) or the
sulfide (FBZ), and since this is known to confer relatively little
anthelmintic activity, this could contribute to its relatively poorer
efficacy. It is likely that the horse metabolizes sulfide and sulfoxide
benzimidazoles to their sulfone metabolites more quickly than ruminants
since the large AUC of the sulfone in the horse is not associated with
a substantially increased MRT. In goats FBZSO2
had a MRT of 34.4 ± 1.52 h following administration of
fenbendazole at 7.5 mg/kg (Benchaoui and McKellar, 1996), whereas in
the present study, the MRT of the sulfone was 16.50 ± 1.00 h
following fenbendazole administered at 10 mg/kg in the horse. The
plasma bioavailability of oxfendazole was much greater than that of
fenbendazole when each were administered orally at 10 mg/kg in the
present study. This probably reflects better absorption of the parent
molecule since the same metabolites are produced by each compound, and these would be expected to have similar metabolic and excretory rates.
It is of interest that the sulfide moiety achieved 2.3 times greater
concentrations (expressed as AUC) in horses administered FBZSO than FBZ
as the parent molecule. It seems likely that the greater solubility of
FBZSO than FBZ (3.01 and 0.07 mg/l, respectively, in buffer at pH 6.0 and 37°; Marriner and Bogan, 1985) permits its absorption at a rate
exceeding its oxidative clearance and thus provide sufficient substrate
for reductive metabolism to the sulfide. There is a paucity of
information on the reductive mechanisms for benzimidazoles (Galtier,
1991), although it is clear from the present study that FBZSO is
reduced to FBZ in the horse following oral and intravenous
administration of FBZSO. This is most likely by hepatic processes,
although secretion to and reduction in the gut with subsequent
reabsorbtion of reduced moieties is also possible. Following
administration of the sulfide as parent, its limited rate of absorption
may be matched by rapid oxidative metabolism with consequent low
concentrations achieved in plasma. The 12 h delay in appearance of
benzimidazoles in feces following administration and the times to
maximal concentration (24 h) and until no drug could be detected (72h)
reflect the gut transit time of the horse, which has been shown to vary
from 25.9 ± 4.5 h (Wolter et al., 1974) to 37.9 ± 5.3 h (Vander Noot et al., 1967).
The intravenous administration of FBZSO generated an atypical plasma
concentration time curve. In the present study, FBZSO was dissolved in
DMSO for administration, and for practical administration purposes, a
very concentrated solution was prepared (500 mg/ml FBZSO) such that the
volumes for delivery were 2 ml/100kg (10 mg/kg). It is possible that
upon delivery FBZSO came out of solution and deposited as a reservoir
which, released over time, could have accounted for the increases in
plasma concentration. This hypothesis is supported by recent work
carried out in lambs, in which administration of FBZSO in DMSO as 8 and
16% solutions generated atypical monoexponential plasma decline curves
whereas a 4% solution generated a typical biexponential curve, and
parallel in vitro dissolution experiments, in which recovery from
plasma spiked at the same overall w/v rate with 8 and 16% FBZSO
preparations was proportionately less than with a 4% solution (Sanchez
et al., 2000). It is possible that extravasation during delivery could have affected the absorption pattern, although great care was taken to
avoid this, and there was no evidence of it at the time of delivery.
When FBZSO was coadministered with piperonyl-butoxide, the parent FBZSO
and the FBZ metabolite achieved significantly (P < 0.01) greater concentrations (AUC and
Cmax) in plasma, and this was
apparently associated with inhibited metabolism of the FBZSO to
FBZSO2 since the elimination rates of each moiety
appeared to be the same or faster (Figs. 6-8) when FBZSO was
administered with piperonyl-butoxide, and because the drug was
administered intravenously, absorption factors were precluded. The use
of DMSO as the solvent for delivery of FBZSO could also have affected its metabolism. The reduction product of DMSO, dimethylsulphide is a known substrate for flavin-containing monooxygenase (FMO), and
since FMO is responsible for the sulfoxidation of FBZ to FBZSO and for
the sulforeduction of FBZSO to FBZ, the presence of an alternate
substrate could affect these processes. Dimethyl sulfoxide is also
known to inhibit several cytochrome P450 subtypes (2C9, 2C19, 2E1, and
3A4) in a concentration dependent manner (Hickman et al., 1998;
Easterbrook et al., 2001). While these confounding factors could not
explain the early increase in concentration of FBZSO following
intravenous administration, they could have contributed to reduced
metabolism of benzimidazole moieties in both intravenous treatment
groups (FBZSO alone and FBZSO with piperonyl-butoxide). The
ratio of AUC-FBZSO/FBZSO2 was 1: 2.98 in the
horses given FBZSO orally in a preparation that did not contain DMSO
and 1:1.38 following intravenous administration in DMSO, and although
other factors such as concentration-dependent metabolism could have had
an effect, this observation supports the hypothesis that DMSO affected
the metabolism of FBZSO. Since DMSO was administered intravenously at
the same dosage in horses given FBZSO alone and in those given FBZSO
with piperonyl-butoxide, the differences in kinetics between the groups
are not associated with DMSO. It is likely that the major effect of
piperonyl-butoxide on FBZSO pharmacokinetics was associated with
inhibited metabolism, and this is supported by liver microsomal studies
described below. Nevertheless it is also possible that
piperonyl-butoxide could have effects on blood flow and tissue
perfusion and that the pharmacokinetic observations are the result of
several pharmacological and physiological interactions.
Oxfendazole displayed enantioselective pharmacokinetics since the
FBZSO-1 enantiomer predominated following administration of the
racemate, and the FBZSO-1/FBZSO-2 ratio was 60:40 throughout most of
the disposition period. The coadministration of piperonyl-butoxide dramatically altered the enantioselective pharmacokinetics of FBZSO
since the FBZSO-2 enantiomer predominated for the first 12 h
following administration after which the ratio changed in favor of
FBZSO-1. The administration of DMSO could have affected the absolute
proportions of enantiomers in the present study since inhibition of FMO
oxidation/reduction by DMSO/dimethylsulphide could have reduced
oxidation of FBZ or caused an accumulation of FBZSO thus enhancing its
stereoselective reduction. Since DMSO was given to both groups of
animals (with and without piperonyl-butoxide), the enantioselective
changes in pharmacokinetics can be attributed to the
piperonyl-butoxide.
The metabolism of sulfide to sulfoxide benzimidazoles is thought to be
principally catalyzed by the FMO system (Galtier et al., 1986) whereas
metabolism of sulfoxide to sulfone is thought to be catalyzed by
hepatic cytochrome P450 (Souhaili-El Amri et al., 1988). It has also
been demonstrated that the FMO system is stereo-selective in favor of
the (+) sulfoxide of the related benzimidazole albendazole whereas
cytochrome P450 systems specifically use () albendazole sulfoxide as
substrate (Morani et al., 1995).
In the present study piperonyl-butoxide coadministration dramatically
altered the generation of FBZSO enantiomers in favor of FBZSO-2,
suggesting that the cytochrome P450 systems on which it acts may be
responsible for FBZSO-2 metabolism. The eudismic (potency) ratio of the
benzimidazole sulfoxides is unknown, however, the alterations in
enantiomer generation together with the alteration in achiral
metabolism of benzimidazoles by piperonyl-butoxide could have a major
impact on the efficacy of benzimidazole sulfides and sulfoxides in the horse.
The small number of animals (n = 6) and large
interanimal variation in results meant that no significant differences
could be detected for pharmacokinetic parameters of the separate drug moieties following oral administration of fenbendazole with or without
food. Nevertheless when the AUC values of active moieties (FBZSO and
FBZ) were summed, they achieved almost four times the concentration in
unfed (2.19 µg · h/ml) than fed (0.59 µg · h/ml) horses, and it would seem appropriate that for systemic parasitic infections, fenbendazole be given following food restriction.
It is known that solutes move through the gut more rapidly than
particles and that small particles move more rapidly than large ones
(Warner, 1981). Benzimidazoles are administered orally as suspensions,
and their particulate size relative to gut transport is more likely to
be associated with the size of concurrent food particles to which they
become adsorbed. Consequently, it would be expected that transit would
be faster on an empty stomach and thus systemic absorption reduced.
However, it is known that in the hind gut of the rabbit and some other
animals, solutes and small particles are selectively retained (Warner,
1981) and both FBZ and FBZSO were extensively absorbed when
administered directly into the cecum of the horse (see Table 3). The
anatomy of the equine ileo-cecal valve makes retrograde delivery of the
drug into the distal ileum from the cecum unlikely, although not impossible.
The coadministration of piperonyl-butoxide p.o. at 63 mg/kg
increased the AUC of the active moieties (FBZSO + FBZ) of FBZ administered at 10 mg/kg in fed ponies by 13 times, and this may represent a strategy for improving the efficacy of benzimidazoles in
the horse. Benchaoui and McKellar (1996) have shown that
piperonyl-butoxide coadministration greatly improves the activity of
FBZ against nematodes resistant to benzimidazoles in sheep when
administered at normal therapeutic doses. They have also demonstrated
in a dose titration study that a dose of 31 mg/kg piperonyl-butoxide significantly improved the bioavailability of benzimidazoles in sheep,
and given the very significant increases in bioavailability demonstrated in the present study when piperonyl-butoxide was administered at 31 mg/kg (intravenous experiment) and 63 mg/kg (oral
experiment), it is likely that it will prove effective at lower
dosages. It is of interest that in the present oral administration study, piperonyl-butoxide produced a greater effect on benzimidazole bioavailability when it was given with food (Table
4), and since this effect is the opposite
of that described above when the benzimidazole was given with food in
the absence of piperonyl-butoxide, it is possible that the food
increased the absorption of the piperonyl-butoxide, thus improving its
dynamic effects on liver metabolism. Piperonyl-butoxide concentrations
were not measured in the present study.
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TABLE 4
Pharmacokinetic parameters of FBZSO, FBZ, and FBZSO2 following
oral administration of fenbendazole (10 mg/kg) with or without food,
and with or without piperonyl-butoxide (PB) (63 mg/kg).
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The equine liver microsomal studies support much of the in vivo
work described above. Piperonyl-butoxide significantly inhibited the
sulfonation of FBZSO and the sulfoxidation and sulfonation of FBZ. It
was also apparent that FBZSO-2 was metabolized more rapidly than
FBZSO-1 in equine liver microsomes and that piperonyl-butoxide altered
the metabolism such that the ratio of FBZSO-1/FBZSO-2 remaining after
incubation was much closer to unity. The eudismic ratio of FBZSO is
unknown, but it is apparent that the effects of piperonyl-butoxide on
efficacy of benzimidazoles could be due to changes in enantiomer ratio
as well as absolute increases in active moiety in plasma. In the horse
the relationship between gastrointestinal concentrations, plasma
concentrations, and efficacy of benzimidazoles has not been clearly
defined. In ruminants it is thought that redistribution from plasma
into gut is responsible for much of the activity of the drug and that
bulk flow through the gastrointestinal tract is less important (Baggot
and McKellar, 1994).
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Abstract
Introduction
