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Vol. 27, Issue 6, 736-740, June 1999
Laboratory of Animal Physiology, Faculty of Veterinary, University of León, León, Spain
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Albendazole (ABZ) presystemic clearance was studied in rat by perfusion of a 25 µM ABZ solution in isolated intestinal loops. Significant secretion of the active metabolite, ABZSO, into the lumen was observed. The metabolite was also present in mesenteric blood. After 30 min of intestinal perfusion, 64% of the ABZ dose had disappeared from lumen. The total amount of ABZSO measured was 0.341 ± 0.04 nmol/cm with 0.176 ± 0.03 nmol/cm in mesenteric blood. The metabolite secretion to intestinal lumen was 0.165 ± 0.05 nmol/cm. Intestinal sulfoxidation was induced by repeated administration of ABZ and ABZ coadministered with surfactants, especially polysorbate 80. The enantioselectivity of the in vitro intestinal sulfoxidation of ABZ showed that the relative contribution of P-450 and flavin-containing monooxygenase was quite similar, but after the induction by ABZ coadministered with polysorbate 80, the cytochrome P-450 system contribution was significantly increased. The appearance of ABZSO in mesenteric blood clearance was also increased under these conditions.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Albendazole
[ABZ;1
methyl(5-proylthio)1H-benzimidazole-2yl] carbamate is
one of the most important benzimidazole derivatives used against liver
flukes, tapeworms, and lung and gastrointestinal roundworms. It is
effective in the treatment of human hydatidosis (Gil-Grande et al.,
1993
) and microsporidial species, emerging agents as opportunistic
pathogens in persons infected with the human immunodeficiency virus
(HIV) (Kelly et al., 1996; Didier, 1997).
After oral administration, ABZ is oxidized to a sulfoxide
(ABZSO), which is in part further oxidized to a
sulfone (ABZSO2); albendazole sulfoxide is
the main metabolite in vivo. The formation of ABZSO is
directly associated with two different microsomal enzymatic systems,
cytochrome P-450 and flavin-containing monooxygenase (FMO). Previous
studies into liver and intestine microsomes of rat (Moroni et al.,
1995; Villaverde et al., 1995) have shown that both systems are
similarly involved. ABZSO has two antipodes, enantiomers
(
) and (+), produced from the chiral thioether albendazole, which can
be separated by HPLC; (+)ABZSO is associated with the
activity of FMO, whereas the P-450 system participates in the
production of ()ABZSO (Delatour et al., 1991).
The low aqueous solubility of albendazole influences its absorption.
Recently, we obtained results on the coadministration of ABZ with
surfactants, which modify the pharmacokinetic profile of
ABZSO, increasing the area under curve (AUC). When
ABZ was administered to rats in a formulation that included surfactants sodium taurocholate at 5 and 10 mM and polysorbate 80 (P80) at 0.0022%
(0.016 mM), ABZSO mean residence time values showed longer-lasting levels, particularly for the 0.016 mM P80 treatment (Redondo et al., 1998).
The liver has been considered a predominant organ of drug
bioconversion. However, recent research has suggested a significant role of intestinal metabolism that contributes to the presystemic clearance for orally administered drugs such as cyclosporine (Kolars et
al., 1992; Watkins, 1992).
To determine the contribution of the gut to the first-pass metabolism
of ABZ, we decided to use the rat jejunum by means of the in situ
physiological model of intestinal absorption. The method described by
Doluisio et al. (1969) was partially modified by a surgical technique
allowing us to collect the mesenteric blood drained by that segment.
This experimental approach maintained the perfused intestinal segment
under proper physiological conditions, with the possibility of
isolating the enterocyte metabolism from the systemic (liver)
metabolism. The simultaneous disappearance of ABZ and formation of its
metabolite are aspects not covered in a previous study on the
biotransformation of ABZ in isolated rat intestine, where the perfusion
procedure was performed in vitro (Lawrenz et al., 1992). The isolated
loop model has been previously used to characterize the absorption
and/or intestinal metabolism of different xenobiotics (Goon and
Klaassen, 1989; Albers and Rosenberg, 1991; Choi et al., 1995).
Nevertheless, one disadvantage of this method is the need to determine
the water reabsorption rate when the loss is greater than 10%
(Martín-Villodré et al., 1986).
The aim of the present study was to investigate the role of the intestine in the presystemic biotransformation of albendazole in rat. The experimental preparation was an isolated intestinal loop with complete venous collection. Because the biotransformation process can be induced by long-term ABZ administration, the study was carried out after having administered different formulations of ABZ for 21 days. To elucidate the enzyme system involved under each condition, the enantioselectivity of the microsomal sulfoxidation by rat intestinal microsomes was studied.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Chemicals. Albendazole (mw 265), albendazole sulfoxide (mw 281), and albendazole sulfone (mw 297) used to carry out the study and to prepare the standard solutions were kindly supplied by Smith-Kline & Beecham S.A. (Madrid, Spain). Mebendazole used as internal standard was supplied by the laboratory of Dr. Esteve (Barcelona, Spain). Sodium taurocholate (mw 537.7) and P80 (polyoxyethylene sorbitol mono-oleate; mw 1309.68) were purchased from Sigma Chemical Co. (St. Louis, MO). Other analytical-grade chemicals were obtained from the following sources: methanol and acetic acid from Merck (Darmstadt, Germany), acetonitrile from BDH (Poole, UK), and ethyl acetate from Sigma-Aldrich (Dorset, UK).
Animals and Experimental Protocol. Male Wistar rats (250 ± 10 g), purchased from IFFA CREDO (Barcelona, Spain), were housed, three per cage, in a temperature-controlled room (21-23°C), with 40-60% humidity and 12:12 h light/dark cycle. Rats were accustomed to these conditions for at least 1 week before any experimentation. Standard rodent diet (Panlab SA, Barcelona, Spain) and water were available ad libitum.
The study of intestinal metabolism of ABZ was carried out in four experimental groups. A control group included animals not receiving ABZ pretreatment (group 1). For induction studies, rats were treated by oral administration with ABZ (5 mg/kg) for 21 consecutive days with the following formulations: ABZ administered in saline (1% dimethyl sulfoxide; group 2), ABZ plus 10 mM sodium taurocholate (STC; group 3), and ABZ plus 0.016 mM P80 (P80; group 4).Intestinal Biotransformation In Situ.
The preparation of isolated intestinal loops in situ and implantation
of the mesenteric venous cannula was performed as described by Doluisio
et al. (1969) and Goon and Klaassen (1989), respectively.
. Afterwards, warm isotonic saline buffered to pH 6.4 was infused to wash the internal surface of the gut. The mesenteric vein was cannulated by inserting a 23-gauge needle attached to Silastic
tubing filled with heparinized saline. The cannula was anchored by
applying a drop of 
-cyanoacrylate adhesive at the entry point.
An ABZ solution (1% dimethyl sulfoxide), was introduced into the lumen
of isolated loop (length, 10.5 ± 2.8 cm) in a volume of 1.5 ml.
The range of measured dose concentrations was 10.07 ± 4.10 µM.
All mesenteric venous blood from the loop was continuously collected
for 30 min at 5-min intervals. A constant blood volume of the rat was
maintained by infusing an heparinized blood-saline mixture (4:1
v/v) into the jugular vein at a rate approximately equal to
the rate of blood drained from the mesenteric cannula (12 ml/h).
Replacement blood used to perform the infusion was collected from
ether-anesthetized donor rats by retroorbicular puncture.
Blood samples were collected in preweighed tubes and the volume of each
sample was gravimetrically determined (specific gravity of 1.054).
After 30 min, the intestinal loop was excised and measured and the
luminal contents collected. It was necessary to determine the
reabsorption of water when the loss was greater than 10%
(Martín-Villodré et al., 1986) to avoid the
excessive distortion of the final concentration of the remaining
perfused solution. In our study, the method used to correct the final
concentrations of solutes was the determination of phenol red
concentration (a nonabsorbable dye) in relation to its initial perfused
concentration (Miller and Schedl, 1972). Five animals were used for
each of the experimental groups studied.
Intestinal Biotransformation In Vitro. The intestinal bioconversion of ABZ was calculated by measuring the sulfoxidase activity through the amount of ABZSO produced by intestinal microsomes.
Cells from the upper and midvillus regions were used as a source of intestinal material. The method used to isolate the enterocytes include evertion of the gut and incubation in chelating medium with EDTA and dithiothreitol, substances that removed the enterocytes from the gut (Weiser 1973). Microsomes from enterocytes were prepared essentially by
methods developed for liver microsomes.
The sulfoxidase activity corresponding to the different experimental
groups was obtained in incubations with 25 µM ABZ in a volume of 1 ml
containing the microsomal fraction (1 mg of microsomal protein). ABZ
was dissolved in methanol (10 µl). The incubations (15 min) were
performed in an oscillating bath at 37°C, according to the technique
of Galtier et al. (1986). After incubation, ABZ and its metabolites
were extracted with dichloromethane. Six animals were used for each of
the experimental groups studied.
Analytical Method.
Plasma and lumenal samples were analyzed for the concentration of ABZ
and the two known metabolites (ABZSO and
ABZSO2) using reversed-phase HPLC. The
extraction method has been previously described by Alvinerie and
Galtier (1984). The HPLC parameters and the quantitation method used
for the analysis is described by Redondo et al. (1998).
Enantiomeric Analysis.
During the chromatographic analysis of in vitro samples, the
ABZSO fraction was collected, extracted, and evaporated under a nitrogen stream. The subsequent residue was then
chromatographed on a chiral column in which stationary phase was an
1-glycoprotein immobilized on silica (Chiral AGP; ChromTech AB,
Hägersten, Sweden) using the conditions previously described
by Delatour et al. (1991). This step provided the relative proportions
of both ()ABZSO and
(+)ABZSO enantiomers. Results were expressed as enantiomeric excess (e.e.), which is defined as:
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Statistical Analysis. Data are presented as mean ± S.D. Mean values for each experimental group were compared by ANOVA. When a significant F value was found, a Newman-Keuls multiple range test was performed to test the difference between means.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Intestinal Biotransformation In Situ. Table 1 shows the percentage of disappeared ABZ from lumen intestinal after an experimental period of 30 min. The range of measured ABZ dose concentration initially perfused was 10.07 ± 4.10 µM.
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). ABZSO2 was not
detected in any sample. ABZSO was quantified in mesenteric
blood and lumenal fluid. The total amount of ABZSO
produced was obtained as the addition of ABZSO in mesenteric
blood and lumenal fluid.
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1 cm1)
significantly increased with respect to control rats when ABZ was
administered with surfactants, whereas the administration of ABZ
without surfactants did not alter the formation and passage of
ABZSO through the intestinal mucosa to systemic blood.
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Microsomal Metabolism: Sulfoxidase Activity.
Table 4 summarizes the sulfoxidase
activities
(nmol · mg1 · min1)
of rat intestinal microsomes. The sulfoxidation was 0.147 ± 0.043 nmol/min/mg in control rats. However, the activity significantly increased (0.324 ± 0.047 nmol/min/mg) in rats treated with ABZ plus P80.
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)ABZSO,
(+)ABZSO, and e.e. was obtained from the total sulfoxidase
activity. From these data we estimated the percentage of involvement of
the enzymatic systems P-450 and FMO, respectively. The ratio of
()ABZSO/(+)ABZSO in
control rats was 48.07/51.92, which corresponded to an e.e. of 4.13%
(favorable to FMO). When ABZ was administered with P80, the ratio of
both was 71.60/28.40, with an e.e. of 43.20%, favorable to P-450.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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ABZSO Intestinal Formation.
The intestine is considered to be an absorptive organ but some drugs
are excreted from the systemic circulation to the intestinal lumen
through an active transport. This same efflux process can be considered
as causing presystemic elimination of an orally dosed drug, influencing
drug bioavailability (Yasuhara et al., 1984; Lennernäs and
Regårdh, 1993). The process becomes more evident when the enterocyte
takes part in the metabolism of a drug orally administered.
) and cyclosporine (Kolars et al., 1992).
The excretion of ABZSO from the enterocyte to the lumen, at
least in a proportion similar to the one transferred to the blood,
means an important modification in the theory stating that the low
bioavailability of the ABZ in blood is due to their low absorption. The
presence of ABZSO in the lumen is highly beneficial to the
treatment of the intestinal parasitoses because the metabolite is more
active than the parent compound.
The results expressed in Table 2 show the importance of intestinal
metabolism of albendazole. The total amount of ABZSO in
control rats was 0.341 ± 0.04 nmol/cm of intestine, and when the
percentage of ABZ disappeared in the lumen at the same time it was
63.71 ± 15.82%, as shown in Table 1.
In our experimental conditions, the lack of the sulfone metabolite in
the analyzed samples suggests that the biotransformation of the ABZ by
the intestine may be limited to its first step, the sulfoxidation.
Owing to the fact that ABZSO was collected continuously,
drained by mesenteric vein, it is not possible to completely discount
sulfonation. However, the absence of sulfonation by intestine was also
one of the conclusions drawn from the work by Lawrenz et al. (1992),
after recirculating 1000 ng/mL through isolated intestinal segments.
The observed increase in the total production of ABZSO
(Table 2) in the experimental groups treated with ABZ for 21 days, with
statistically significant differences when coadministered with
surfactants (10 mM STC, 1.042 ± 0.18; 0.016 mM P80, 1.214 ± 0.13) in relation to the control value (0.341 ± 0.04 nmol/cm), seems to be related to a process of induction of the P-450 system (Souhaili-El Amri et al., 1988; Aix et al., 1994). Intestinal induction
was also observed in studies on the role of the cytochrome P-450 in the
metabolism of cyclosporine, where the pretreatment of animals with
dexamethasone, an inducer of cytochrome P-4503A produces a
significant increase in the rate of metabolite formation (Kolars et
al., 1992). On the other hand, the induction by surfactants observed in
our experiment does not seem to be related to the one described
in vivo by Souhaili-El Amri et al. (1988), which points out an
induction of the hepatic P-450 system in rat that increases the
sulfonation of the ABZSO.
Benzimidazoles (in their sulfoxidated form) are considered as active
inducers of the P-450 system, as studies using fenbendazole show that
the parental compound possesses less inducing power of the isoform
P-4501A than their corresponding sulfoxide metabolite oxfendazol
(Gleizes et al., 1991). The presence of the sulfur atom in the molecule
of benzimidazole is not considered a prerequisite for the potential
induction of P-450 (Rey-Grobellet et al., 1996).
The long-term administration of ABZ suspension (5 mg/kg) did not
increase the sulfoxidation process significantly (Table 2). This fact
does not contradict the important increases observed with the use of
surfactants because they facilitate the ABZ absorption and the passage
into enterocyte. To explain these increases, some aspects must be
considered. First, the long-term administration of the drug could
modify the composition of the enterocyte membrane (Miyamoto et al.,
1983; Jones, 1992), which favors the absorption of ABZ. Even if such
effect is reversible, it may influence the absorption process (Kakemi
et al., 1970) and the apparent rate of sulfoxidation. Secondly, the
surfactant itself could exert an inductive effect on the
biotransformation system. This point will be discussed below.
Another interesting observation reflected in Table 2 and Fig. 1 is that
ABZSO was quantified in the mesenteric blood (0.176 ± 0.03 nmol/cm) and in the intestinal lumen in a similar proportion
(0.165 ± 0.05 nmol/cm).
The reasons of the luminal excretion from the enterocyte could be due
to the hydrophilic nature of the formed metabolites and to the
existence of carriers. In the specific case of the metabolism of the
7-ethoxycoumarin, the free and sulfated metabolites are equally
distributed between blood and lumen, without the participation of
active transport, but the glucuronide conjugate metabolites are
liberated into mesenteric blood by a carrier in the basolateral membrane of the enterocyte (Albers and Rosenberg, 1991).
A slight modification in the ratio of ABZSO in blood and
lumenal perfusate takes place when ABZ is administered for 21 days
(Table 2 and Fig. 1), producing a greater transference of the sulfoxide
to the mesenteric blood. This was particularly evident when ABZ was
administered with polysorbate vehicle. This excretion of
ABZSO toward the enterocyte basal membrane could be related
to the intracellular distribution of the biotransformation enzymes
involved in the ABZ sulfoxidation.
Microsomal Intestinal FMO and P-450 Contribution.
Differential enantioselectivity of ABZO was used in our study to
determine the P-450 and FMO contribution to ABZ sulfoxidation. Specific
inhibitors such as clotrimazole (P-450) and methimazole (FMO) have been
used by some authors with the same purpose (Galtier et al., 1986,
Villaverde et al.,1995; Moroni et al., 1995).
)ABZSO/(+)ABZSO
obtained is 48.07/51.92 being 4.13% the e.e. of the
S-(+) enantiomer (Table 4). These data agree with the
results of Villaverde et al. (1995). In liver the contribution of the
enzymatic systems is different because the P-450 system is more
relevant than FMO. The mean ratio ()ABZSO/(+)ABZSO
55.75/43.36, corresponds to an e.e. of 13.33% for
()ABZSO (Moroni et al., 1995).
In the rat and in the human the major enterocyte P-450 appears to be a
member of the P-4503A subfamily and it could be involved in the
sulfoxidation process (Watkins et al., 1987; Fasco et al., 1993). FMO1
is present in the intestinal system but the enzymatic activity measured
by the oxidation of thiourea is lower in intestine than in liver
(Larsen-Su and Willians, 1996).
Effects of Administration of Surfactants. Surfactants, especially P80, operated at the intestinal level by increasing the metabolic conversion rate of ABZ into ABZSO and also caused marked changes in the kinetic disposition of the latter.
The increase in the sulfoxidase activity when ABZ was administered with 0.016 mM P80 was due predominantly to P-450, because it correlates well with the stereoselective production of ()ABZSO (enantiomeric
ratios: 71.6/28.4 versus 48.07/51.9 in the control). The surfactants
increased the intestinal absorption of ABZ (del Estal et al., 1991),
which could enhance the cytochrome transcriptional mechanisms (Aix et
al., 1994). Because in our work the gastrointestinal tract is
repeatedly exposed to ABZ, long-term oral administration could modulate
the metabolizing enzyme at the site of a primary entry point of the
systemic circulation.
Intestinal microsomal activity could be modified by increased ABZ
absorption, but considering that the increase in ABZ absorption from
64% under the control condition to 85% of the administered dose in
the presence of surfactants seems very slight in comparison with the
increase in ABZ sulfoxidation in vitro and in situ, the possibility
that the surfactants have a direct P-450 inductive effect must be
considered. Also, the membrane perturbation produced by detergents
increased microsomal hexose-6-phosphate dehydrogenase (Kulkarni and
Hodgson, 1982) and glucuronyltransferase activities (Berry et al.,
1975), possibly due to an increased cofactor or substrate
accessibility to the enzyme. In fact, hexose-6-phosphate dehydrogenase
is related to monooxygenation and therefore, also to the P-450
catalytic system. Yun et al. (1997) showed that the addition of
detergents to a P-4501A2 solution increased the enzyme activity. The
activity increase of P-4501A2 coincides with the raised helix
content and decreased 
sheet content. The authors concluded that the
interaction with the phospholipid molecules surrounding P-4501A2 its
important for a functional conformation of P-4501A2.
In summary, the present study shows the importance of intestinal
metabolism in relation to the first-pass extraction (via phase I
reactions) of ABZ. Unfortunately the experimental design with ABZ
(insoluble drug) did not allow us to determine the first-pass extraction ratio. Furthermore, the lumenal efflux of ABZSO has also been proved for the first time. Finally, the presystemic biotransformation increased when ABZ was coadministered with surfactants.
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Acknowledgments |
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We thank Smith-Kline & Beecham S.A. (Madrid, Spain) for the free supply of ABZ, ABZSO, and ABZSO2 and to the Dr. Esteve Laboratory (Barcelona, Spain) for supplying the mebendazole. We are also grateful to María Luisa Fernández Rojo for translating the manuscript.
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Footnotes |
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Received April 2, 1998; accepted March 1, 1999.
This work was supported by Comision Interministerial de Ciencia y Tecnologia (CICYT) (Spain) (FAR 91/0748 project).
Send reprint requests to: Julio G. Prieto, Laboratory of Animal Physiology, Faculty of Veterinary, University of León, E24071, León, Spain. E-mail: dfijpf{at}unileon.es
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Abbreviations |
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Abbreviations used are: ABZ, albendazole; ABZSO, albendazole sulfoxide; ABZSO2, albendazole sulfone; e.e., enantiomeric excess.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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, Mesenteric blood; 
,
intestinal lumen.
