Transplacental transport of netobimin metabolites in ewes

EUROPEAN JOURNAL OF DRUG METABOLISM AND PHARMACOKINETICS, 1995, Vol. 20, No.3, pp. 167-171

Transplacental transport of netobimin metabolites • In ewes C. CRISTOFOL', A. CARRETER0 , M, FERNANDEZ , M. NAVARR02 , J. SAUTET3, 2

2

J. RUBERTE and M. ARBOIX

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I

I Unitat de Farmacologia and 2 Unitat d'Anatomia i Embriologia, Facultat de Yeterinaria. UAR, Bellaterra, Spain 3Labornto;re d'Anatomie et Teratologie, Ecole Nationale Veterillaire de Toulouse, Toulouse, France

Receivedfor publication : November 8, 1993

Keywords: Pharmacokinetics, transplacental-transport, albendazole-metabolites, netobimin

SUMMARY Neither netobunin (NTB) nor its metabolite albendazole (ABZ) were found in plasma after an oral administration of 20 mglkg of NTB to pregnant ewes during the last third of gestation. ABZ metabolites, albendazole sulphoxide (ABZSO) and albendazole sulphone (ABZS02) were found in plasma 30 min and 2 h. respectively, after administration. The maximal plasma concentration (C max) of ABZSO was detected at I 1.6 ± 1.0 h and for ABZSOz at 16.5 ± 2.3 h. The plasma levels of the latter remained constant for 36 h, and decreased as ABZSO was removed from the blood. Jugular plasma levels of both metabolites did not differ significantly from those observed in the ovarian vein, suggesting that there were no exchanges between foetal and placental tissues. Both metabolite concentrations were similar in the umbilical vein and artery and in the amniotic and allantoic fluids, their values were half the maternal plasma concentration. leading to the conclusion that there was transplacental movement of metabolites. Both metabolites reached the foetus and could be responsible for the teratogenicity of NTB in sheep.

INTRODUCTION The benzimidazole derivatives are mainly used as antihelmintics. These drugs have been identified as potential teratogens (1-3). One of the benzimidazoles frequently used as an antihelmintic in animals is albendazole. This drug is converted by hepatic metabolism to albendazole sulphoxide (ABZSO), which subsequently is oxidised to the sulphone metabolite (ABZS02) (4.5). ABZ is rapidly metabolized; significant levels of ABZSO can be found during the first 30 min period

Please send reprint requests to : Dr M. Arboix. Unitat de Farrnacologia, Facultat de Veterinaria, UAB. 08193 Bellaterra. Spain.

after i.v. and oral administration (6,7). Studies performed on different species showed that the maximum levels of ABZSO and its metabolite ABZS02 occurred between 7-18 hand 20-30 h, respectively, after ABZ administration (7,8). Netobimin (NTB) is an antihelmintic compound. Recent data showed that NTB is transformed into ABZ by gastrointestinal bacteria (9). ABZSO and ABZS02 have been detected after NTB administration in rats (9), sheep (10) and cattle (11,12). ABZ and ABZSO have been shown to be active metabolites, and thus it seems likely that the antihelmintic efficacy of NTB depends on the level and disposition kinetics of these metabolites in the host. In studies on pregnant rats treated with ABZ and

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ABZSO. Delatour et al. (2) observed that the embryotoxic effects were largely due to the metabolite ABZSO. Similar data have been found for febendazole sulphoxide and febendazole sulphone (3). Studies with ABZ and ABZSO made in cultures of rodent emhryonic cells, revealed that both metabolites were significantly cytotoxic; ABZ was 50-fold more potent than ABZSO (13). Sheep are sensitive to the teratogenic effects of benzimidazoles ( 1,14.15). Following treatment with an oral dose of NTB (20 rug/kg) to pregnant ewes, up to 7Ylr of the lambs were found to have congenital skeletal malformations and there was embryonic mortality (14). These authors suggested that the main metaholites of the antihelminthic drug could be responsible for the toxicity as ABZ or its metabolites must reach the embryo through the placental barrier. The present study analyzes the plasma concentrations of NTB and its metabolites in pregnant ewes and the permeability of the placental tissue to these compounds after an oral administration of 20 mg/kg of the drug; the aim was to determine which compounds reached the foetus and the rate at which they were retained or transformed in foetal tissues.

MATERIALS AND METHODS Fig. I : Resin cast of ovaric vein.

QV, ovaric vein; UC,

uterine caruncle.

Animals Five pregnant ewes (last third of gestation), with an average weight of 35 kg were used. The animals were treated orally with 20 rng/kg of NTB. After administration, maternal blood samples were collected from the jugular vein (0.5, I, 2, 4, 6, 8, 10. 12. 14. 15. 16, 17. 18,21. 30. 36,50,72 h) and ovarian vein (11,12,13,14, IS, 16, 17, 18 h). The methodology employed to catheterize the ovarian vein was similar to that used by Comline and Silver (16). For the catheterisation of the ovarian vein, the animals were anaesthetized with an i.v. dose of 6 mg/kg of propofol. After anaesthetizing, the animal was cannulated for administration of epidural anaesthesia with 1.8 mg/kg of bupivacaine, given through a cannula inserted in the epidural space. The anaesthesia lasted between 2.5-3 h: after this time the dose was repeated. The right paralumbar fossa was shaved and disinfected. An incision was made in the abdominal wall. allowing access to the peritoneal cavity at the level of the free end of the caecum and descending colon. On forward separation of the caecum. the mesovarium which is transversed by the ovarian artery

and vein appears deep down. As can be seen, this vein carries blood from the uterine placentomes of the ewe. Once located, the ovarian vein was cannulated with a heparinized polyethylene tube. The tube was brought out at the level of the abdominal wall and the incision sutured. The ovarian vein was catheterized 10 h after NTB administration and caesarian section was performed at 20 h. At that time, the metabolite ABZSO reached peak concentrations and the ABZS0:2 was detected in significant quantities, reaching peak concentrations after 16 h. Finally a caesarian section was performed to collect umbilical and arterial blood, as amniotic and allantoic fluid. Figure I shows a vascular cast of the ovaric vein, where the cannula was placed, and the uterine caruncle.

Analytical methods NTB was provided by Essex Lab. (Spain). ABZ, ABZSO and ABZS02 were obtained from Shering

C. Cristofol et al.• Disposition of netobimin metabolites

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ABZSO

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Fig. 2 : Mean plasmalevels of ABZSO and ABZS02 after the administration of NTB (20 mglkg)to pregnantewes.

Plough (USA) and the internal standard was oxibendazole from Labiana Analitica Lab. (Spain). NTB. ABZ. ABZSO and ABZSOz concentrations of plasma. amniotic and allantoic fluids were measured by HPLC (10). The column used was a 7 11m CI8 Nucleosil. The mobile phase for product separation was acetonitrile and ammonium acetate 0.025M (40:60).

RESULTS Mean plasma concentrations from 5 animals versus time for ABZSO and ABZSOz. are shown in Figure 2. Neither NTB nor ABZ were detected in plasma. Significant plasma concentrations (0.05 ug/ml) were found at 30 min for ABZSO. and ABZSOz detection (0.02

ug/rnl) began at 2 h. These graphics show how ABZSO (first metabolite) increased at a high rate reaching a maximum value 11 h after NTB administration. In contrast, the values for ABZSOz were much lower (16% of the ABZSO values) and reached a maximum from 14-36 h. It must be emphasized that the elimination rate of both metabolites was slow; i.e. significant levels were maintained for more than 36 h. Figure 3 shows the corresponding curves for ABZSO and ABZSOz during the time interval (11-18 h) that the jugular and ovarian veins were cannulated. The two metabolites maintained similar concentrations in both veins. Table I shows the average values of plasma concentrations of ABZSO and ABZSQz in the umbilical vein and artery, the ovaric vein, the jugular vein, allantoic fluid and amniotic fluid 20 h after NTB admin-

Table I : Comparative levels of ABZSO and ABZSOz 20 h after the administration of NTB (20 mglkg).

Allantoic fluid Amniotic fluid Fetal venous plasma Fetal arterial plasma Ovaric vein plasma Jugular vein plasma

ABZSO concentration (~g/ml)

ABZS02 concentration (~g/ml)

mean ± SD

mean ± SD

1.415 ± 0.669

0.263 ± 0.019

1.476 ± 0.517

0.217 ±0.065

1.568 ± 0.610

0.388 ± 0.079

1.473 ± 0.675

0.388 ± 0.097

2.433 ± 0.642

0.635 ± 0.130

2.900 ± 1.291

0.797 ± 0.170

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time (h) Fig. 3 : Comparative levels of ABZSO and ABZS02 between plasmas of jugular vein and ovaric vein after the administration of NTB (20 mglkgl to pregnant ewes.

istration. The metabolite levels in the ovarian and jugular veins did not show significant differences. The levels in the other blood vessels and fluids showed similar values and were approximately half those observed in the jugular vein. Neither ABZ nor NTB were detected in any of the fluids sampled.

DISCUSSION The study of the disposition of NTB and its metabolites in pregnant ewes treated orally with 20 mglkg NTB. suggests that NTB is rapidly transformed by the ruminal microflora into ABZ which, in turn, is highly metabolized by the liver at first pass, since neither was

found in plasma samples collected at the different post-administration sampling times. A rapid appearance in plasma of ABZSO (30 min) was observed, showing a high rate of hepatic metabolism. It should also be noted that in some cases during the second hour ABZS02 was detected in plasma samples. These data are in agreement with those reported by other authors for sheep (6,7,17) and in other species such as calves (11,12) and camels (18). In ewes, peak plasma concentrations of ABZSO and ABZSOz were found at 11.6 ± 0.96 and 16.5 ± 2.3 h, respectively, and ABZS02 levels did not decrease until 30 h after NTB administration; these values are close to those found in sheep by other authors (6-8) after albendazole administration; they

C. Cristofol et al.. Disposition of netobimin metabolites

observed a C max at 7.5 h. 3 hours earlier than in the present study. which could be due to direct administration of ABZ rather than its precursor NTB. Lanusse and Prichard (10). after administering 20 mg/kg NTB in suspension to sheep. found a C max value for ABZSO at 17 h and for ABZS02 at 25 h maintained until 35 h. The concentrations of ABZS02 remained at similar levels in the organism for long periods (12-36 h), subsequently they declined coinciding with the elimination of ABZSO. These results would confirm the sequential oxidation paths followed by ABZ metabolism in the microsomal hepatic system after cycling of NTB (6.9). The plasma concentrations of ABZSO and ABZS02 found in the samples obtained from the jugular and ovarian veins in the 11-18 h period after NTB administration (in which the highest levels of the metabolites were found in the body), were similar in both veins; this suggests that they are not significantly transformed on passing through placental and fetal tissues (Fig. 2). The concentrations of the two metabolites observed in the umbilical vein and artery and in the allantoic and amniotic fluids were similar; they were half the concentrations found in the jugular and ovarian veins (Table I). These results indicate that both metabolites pass through the placental barrier and reach significant levels in the foetus. By analysing malformations observed in rats treated with ABZSO, Delatour et al. (2) suggest that the metabolites, particularly ABZSO, were responsible for the toxic effects. In in vitro studies on cultures of rodent embryonic cells (13), the authors observed toxicity in the cell growth. differentiation and cytotoxicity produced by ABZ and ABZSO. The fact that NTB and ABZ are rapidly converted to ABZSO and ABZS02 which maintain significant levels in the body for hours, and neither NTB nor ABZ was ever detected in ewe and foetal plasma. suggests that in vivo the metabolites would he responsible for embryotoxicity and would cause malformations in ewes as Fabre et al. observed (14). This indicates the necessity for a more exhaustive study of the emhryotoxic activity of ABZSO and ABZS02.

ACKNOWLEDGEMENTS This study was conducted with the support of a CICYT (R&D) grant from the Spanish Government.

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