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Disposition of Netobimin, Albendazole, and Its Metabolites in the Pregnant Rat: Developmental Toxicity
TOXICOLOGY AND APPLIED PHARMACOLOGY ARTICLE NO.
144, 56–61 (1997)
TO978114
Disposition of Netobimin, Albendazole, and Its Metabolites in the Pregnant Rat: Developmental Toxicity CARLES CRISTO`FOL,* MARC NAVARRO,† CARME FRANQUELO,* JOSEP ENRIC VALLADARES,* ANA CARRETERO,† JESUS RUBERTE,† AND MARGARITA ARBOIX* *Departament de Farmacologia i de Terape`utica and †Unitat d’Anatomia i Embriologia, Facultat de Veterina`ria, Universidad Auto`noma de Barcelona, 08193 Barcelona, Spain Received August 13, 1996; accepted January 11, 1997
ABZ and ABZSO exhibit significant anthelmintic and toxic effects (Delatour et al., 1981, 1984), the main toxic one being teratogenicity. Several studies suggest that the capacity of benzimidazolic drugs to bind with the tubulin of cellules (Delatour and Parish, 1986) is responsible for the toxic effects on the embryo. Fetuses from ewes treated with NTB in the first third of pregnancy show congenital malformations such as renal agenesis and ectopic kidneys, spina bifida, fused vertebrae and ribs, hypoplasia of the thoracic limb, and vascular anomalies (Fabre et al., 1989; Navarro, 1996), and Cristo`fol et al. (1995) found that ABZSO and ABZSO2 cross the placenta, reaching the fetus in significant amounts. Benzimidazole anthelmintics have also proven to be teratogenic in rats, producing an increased number of resorptions, decreased fetal weight, and external and skeletal malformations (Martin, 1980; Delatour et al., 1984, 1986; Yoshimura et al., 1987). In rats treated with 17.7 mg NTB/ kg, Delatour et al. (1986) found that peak plasma concentrations (Cmax ) were 0.79 mg/ml for ABZSO and 0.27 for ABZSO2 , and that they were achieved 6–9 and 12 hr after dosing, respectively. No studies have investigated the relationship between concentrations of ABZ and its metabolites and toxic effects on the fetus. The aim of this study was to establish the relationship between the NTB dose administered to pregnant rats, the maternal plasma concentration of ABZ and its metabolites, the concentrations of metabolites (ABZSO and ABZSO2 ) reaching the embryo, and the toxic effects observed in the fetuses.
Disposition of Netobimin, Albendazole, and Its Metabolites in the Pregnant Rat: Developmental Toxicity. CRISTO`FOL, C., NAVARRO, M., FRANQUELO, C., VALLADARES, J. E., CARRETERO, A., RUBERTE, J., AND ARBOIX, M. (1997). Toxicol. Appl. Pharmacol. 144, 56–61. Netobimin (NTB), a benzimidazole prodrug with a good anthelmintic spectrum, was administered orally to female rats at a dose of 59.5 mg NTB/kg, to study its pharmacokinetic behavior and the disposition of its most important metabolites, albendazole (ABZ), albendazole sulfoxide (ABZSO), and albendazole sulfone (ABZSO2 ). ABZ was found in plasma after 6 hr. Peak plasma concentrations (Cmax ) and areas under curves (AUC) of ABZSO were eight- and fourfold higher, respectively, than those of ABZSO2 . To study NTB disposition in pregnant rats, three different drug doses (50, 59.5, and 70.7 mg/kg) were given. No significant differences were found between plasma concentrations for each metabolite at the three doses studied. Only ABZ concentrations rose slightly as dose increased. ABZ, ABZSO, and ABZSO2 were found in amniotic sacs and embryos at concentrations that were higher than plasma at the same times. The fetuses obtained after administration of each of the doses of NTB were studied to detect developmental toxicity. A significant correlation was found between rate of developmental toxicity and metabolite concentration. ABZSO embryo concentrations could be the main factor accounting for toxicity. q 1997 Academic Press
Netobimin (NTB) has been shown to exhibit an anthelmintic spectrum against nematodes, cestodes, and trematodes (Williams et al., 1985; Santiago et al., 1985; Richards et al., 1987) and is reported to undergo biotransformation to albendazole (ABZ) when in contact with gastrointestinal bacteria (Delatour et al., 1986). Once ABZ is absorbed, it is metabolized sequentially to ABZSO and ABZSO2 (Gyurick et al., 1981; Souhaili-el Amri et al., 1987, 1988). NTB’s anthelmintic activity may be due in part to bioenergetic disruptions resulting from transmembrane proton discharge. Marked changes in wet weight and glycogen and protein concentrations were observed in tapeworms in animals that received therapeutic doses of benzimidazolic drugs (McCracken and Stillwell, 1991).
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Animals. Female Sprague–Dawley rats (Crl:CD), provided by CIDA Laboratories, were fed on a commercial diet (UAR A04C; Usine d’Alimentation Rationelle, Villemoisson sur Orge, France) and had free access to tap water. The onset of pregnancy was determined by vaginal smears. The day that sperm was found in the smear was called Day 0. The pharmacokinetics of NTB were studied first. Fifty-five nonpregnant rats weighing 300 g were dosed orally with 59.5 mg NTB/kg. Blood samples, obtained by exanguination after halothane overanaesthesia, were collected in heparinized tubes 0.5, 1, 2, 4, 6, 8, 12, 18, 24, 36, and 48 hr after 56
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DEVELOPMENTAL TOXICITY OF NETOBIMIN IN THE RAT drug administration from 5 rats at each collection time. Plasma samples were obtained after centrifugation at 2000 rpm for 20 min and then stored at 0207C until analysis. In the second step, three groups of six rats on the 10th day of pregnancy, weighing a mean of 300 g, were treated orally with 50, 59.5, and 70.7 mg/ kg NTB. Blood samples were taken from the orbital sinus using an heparinized glass capillary tube at 2, 4, and 8 hr after administration. Rats were overanaesthetized with halothane and exsanguinated 12 hr after administration when the maximum plasma concentration of ABZ metabolites had been reached as determined in the pharmacokinetic study in nonpregnant rats. The 10th day of gestation in rats is equivalent to the embryonal stage at which some authors (Fabre et al., 1989; Navarro, 1996) have observed abortion or malformed fetuses in sheep after oral treatment with 20 mg NTB/kg on the 17th day of gestation. Maternal blood, amniotic sacs, and embryos were collected. Embryos, amniotic sacs, and plasma samples were stored at 0207C until analyzed. NTB doses were selected according to Delatour et al. (1982), who observed fetal malformations when pregnant rats were treated orally with NTB doses of 44.2 mg/kg or higher. Finally, three groups of six rats each, on the 10th day of pregnancy, were treated orally with 50, 59.5, or 70.7 mg/kg of NTB. A control group of rats, on the 10th day of gestation, was treated with the same volume of vehicle (0.2% carboximethyl cellulose and 1% Tween 80 in distilled water) and were studied under the same conditions. Rats were overanaesthetized with halothane and exsanguinated 1 day before delivery (20th day of gestation). To determine developmental toxicity, resorptions, fetal weight, and malformations were recorded. Drug analysis. Concentrations of NTB, ABZ, ABZSO, and ABZSO2 in plasma, embryos, and amniotic sacs were determined by high-performance liquid chromatography (Bogan and Marriner, 1980). Malformations. Gross external and internal malformations were examined first. Then, skeletal malformations were determined using a clearing technique described by Staples and Schnell (1964) consisting of maceration in 1% KOH and staining with alizarin red S. Fetuses from each treatment were compared with those from the control group. Kinetic analysis. The pharmacokinetic parameters were determined by noncompartmental modelling using the PKCALC computer program (Shumacker, 1986). The terminal elimination slopes b were calculated by linear least-squares regression analysis. Statistics. Fetal weight and differences in tissue concentrations of ABZSO and ABZSO2 12 hr after administration of the three doses of NTB to pregnant rats were tested for significance by one-way ANOVA testing. Differences in resorptions rate and number of malformations for the three doses of NTB administered were tested for significance using a x2 test. The significance level accepted for differences between doses was p õ 0.05 for all tests. The data were calculated using the fetus as the experimental unit since each fetus possesses its own placenta.
RESULTS
Figure 1 plots plasma levels versus time of ABZ, ABZSO, and ABZSO2 for nonpregnant female rats orally treated with 59.5 mg NTB/kg. No ABZ was found after 6 hr and no NTB was detected at any of the sampling times. Table 1 shows pharmacokinetic parameters for both ABZ metabolites obtained from the analysis of the mean plasma levels of the five animals analyzed in each collection time. ABZSO Cmax and areas under curve (AUC) were eight- and fourfold higher, respectively, than those of ABZSO2 . Table 2 shows the mean AUC and its standard deviation from 0 to 12 hr for ABZSO and ABZSO2 for the three doses
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studied. No statistically significant differences were found for the three doses studied. Table 2 also shows the AUC ratio ABSO2 /ABZSO for the doses studied and, likewise, no significant differences were found. Figure 2 depicts ABZ, ABZSO, and ABZSO2 plasma concentrations in maternal blood from pregnant rats treated orally with NTB at the doses studied (50, 59.5, and 70.7 mg/ kg). No significant differences were found between plasma concentrations of each metabolite among the three doses. Only ABZ concentrations increased slightly at higher doses. Figure 3 shows concentrations of NTB metabolites in plasma, amniotic sacs, and embryos from pregnant rats treated with the three doses. Significant differences were observed for ABZ and ABZSO concentrations in embryos between 50 and 59.5 but not between 59.5 and 70.7 mg NTB/kg doses. Data on developmental toxicity of NTB after oral administration of 50, 59.5, and 70.7 mg/kg to the three groups of pregnant rats are shown in Table 3, and reveal that higher doses of NTB were associated with higher rates of resorptions. The rate for the low-dose group was not statistically different from that of the control group. No internal malformations were found. Decreased fetal weight and external and skeletal malformations were more common in treated animals than in controls but there were no significant differences in these parameters between the 59.5 and 70.7 mg NTB/kg doses. DISCUSSION
NTB administered orally to ruminants or rats is transformed to ABZ by a cyclation process carried out by gastrointestinal bacteria (Delatour et al., 1986; Lanusse and Prichard, 1990). NTB has never been detected in rat blood. However, in our study, ABZ was still detectable in plasma 6 hr after administration, suggesting that ABZ hepatic metabolism in monogastric animals is less efficient than in ruminants, in whom ABZ is immediately converted to ABZSO (Lanusse and Prichard, 1990) and is never measurable in plasma samples. The kinetic profiles of ABZSO and ABZSO2 obtained in the present study agree with the few available data in literature (Delatour et al., 1986; Souhaili-el Amri et al., 1988). Pharmacokinetics and plasma levels of the metabolites studied are similar in nonpregnant and pregnant rats, indicating that the elimination patterns of ABZ and its metabolites are not affected by the early stages of pregnancy. Only ABZ concentration increased significantly with dose while ABZSO and ABZSO2 plasma levels, AUC values, and the ratio of ABZSO2 /ABZSO AUC did not change. As ABZ is metabolized sequentially in the liver to ABZSO and ABZSO2 (Delatour et al., 1986), those metabolic steps are probably saturable. Thus, high doses of NTB may produce a saturation
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FIG. 1. Mean { standard deviation of plasma concentration (n Å 5) of ABZ, ABZSO, and ABZSO2 after administration of 59.5 mg NTB/kg to nonpregnant female rats.
of the enzymes of the hepatic microsomal system, which are responsible for the S-oxidation of ABZ to ABZSO and ABZSO2 . The concentrations of ABZ, ABZSO, and ABZSO2 in placental and embryonal tissues were higher than those observed in maternal blood, suggesting that these metabolites may bind the different molecules in embryonic cells and tissues. On the other hand, Cristo`fol et al. (1995) found that the concentrations of ABZSO and ABZSO2 in fetuses from ewes treated orally during the last third of gestation with 20 mg NTB/kg were half the concentrations in maternal plasma, and that ABZ was never detected; this indicates the existence of interspecies differences that may be related to anatomical differences in their placentas. Rats have two placentas, a discoidal chorioallantoic placenta, and a highly vascularized yolk sac placenta. Until Day 11 or 12, the yolk sac placenta
TABLE 1 Pharmacokinetic Parameters for ABZSO and ABZSO2 after Oral Administration of 59.5 mg NTB/kg to Nonpregnant Female Rats Parameter
ABZSO
ABZSO2
Cmax (mg/ml) Tmax (hr) T1/2b (hr) AUC0 – ` (mgrhr/ml) MRT (hr)
1.6 6.0 0.14 32.7 7.4
0.2 12.0 5.1 7.5 10.1
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is the only route for transport between mother and embryo since the chorioallantoic placenta does not become vascularized before this day. Studies on the placental transfer of material from mother to fetus in the rat did not reveal differences between the two placentas (Baker et al., 1979). However, the structure of rat placenta seems to be more permeable than the thicker syndesmochorial placenta of the ewe. Differences observed in access of the studied drugs to the embryo could also be due, however, to structural dissimilarities in the placenta at the two stages of pregnancy studied, as the first third of gestation was studied in rats and the last third was studied in sheeps. In agreement with results reported by other authors about the toxic effects of NTB and ABZ (Martin, 1980; Delatour et al., 1984, 1986; Mantovani, 1992), we also found correlations between some of the toxic effects examined and the increase in NTB dose administered to pregnant rats. Our data indicate that embryos remained exposed to ABZ for
TABLE 2 Mean AUC0 – 12 and Standard Deviations for ABZSO and ABZSO2 for the Three Doses Studied in Pregnant Rats Dose of NTB (mg/kg)
ABZSO (X { SD)
ABZSO2 (X { SD)
Ratio ABZSO2/ABZSO (X { SD)
50 59.5 70.7
14.6 { 2.5 15.5 { 6.7 13.7 { 5.7
2.7 { 1.0 2.6 { 1.0 1.8 { 1.0
0.18 { 0.04 0.19 { 0.08 0.14 { 0.04
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FIG. 2. Concentration–time curves (mean { SD, n Å 6) of ABZ, ABZSO, and ABZSO2 after administration of 50 (dose 1), 59.5 (dose 2), and 70.7 (dose 3) mg NTB/kg to pregnant rats.
over 12 hr and to ABZSO and ABZSO2 for approximately 36–48 hr. ABZSO2 concentrations, however, were not significantly different between doses that were associated with developmental toxicity. Analysis of the developmental toxicity incidence for the three doses and their correlation with embryo drug concen-
trations for each of the metabolites reveals that the ABZSO concentration could be the main factor accounting for developmental toxicity. The ABZSO concentrations were significantly higher after the 59.5 mg/kg dose of NTB, compared to the lowest dose (50 mg/kg), but we found no significant differences in the concentrations attained after the 59.5 and
FIG. 3. Mean plasma, embryo, and amniotic sacs (a.s.) concentrations { standard deviations (n Å 6) of ABZ, ABZSO, and ABZSO2 after administration of 50, 59.5, and 70.7 mg NTB/kg to pregnant rats.
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TABLE 3 Developmental Toxicity of NTB after Oral Administration of 50, 59.5, and 70.7 mg NTB/kg to Pregnant Rats Dose of NTB (mg/kg)
Number of live fetuses
Resorptions (%)
Control 50 59.5 70.7
66 60 84 38
4.3 4.7 15.5** 51.8***
Fetal weight (g { SE) 5.57 5.06 4.89 4.86
{ { { {
External malformations (% fetuses)
Skeletal malformations (% fetuses)
0 0 13.1** 13.1**
14.2 52.9* 71.4* 68.0*
0.05 0.05* 0.05** 0.08**
* Significantly different from controls. ** Significantly different from controls and from 50 mg/kg dose. *** Significantly different from controls, from 50 mg/kg dose, and from 59.5 mg/kg dose.
70.7 mg NTB/kg doses. On the other hand, statistically significant differences in fetal weight and number of external malformations were observed between NTB doses of 50 and 59.5 mg/kg, whereas there were no such differences between the 59.5 and 70.7 mg/kg doses. No differences in the incidence of resorptions were observed between animals treated with 50 mg/kg NTB and the control group. Increasing the dose to 59.5 mg/kg, however, promoted an increase in the resorption rate. This may be a result of the combined effects of ABZ plus ABZSO. In vitro studies have demonstrated that ABZ appears to be cytotoxic for some embryo cells at a 0.3–0.4 mM concentration, a range that is 50-fold higher than that of ABZSO (Whittaker and Faustman, 1991, 1992). However the affinity of both drugs for tubulin binding is similar, 6.9 mM being the ABZ concentration needed to inhibit mammalian tubulin polymerization (Lacey and Watson, 1985). This could explain why some embryonal cells overcome the lethal effect produced by the presence of ABZ and ABZSO. The surviving cells apparently needed a higher concentrations of these substances for their mitotic microtubular system to adversely affected, producing cell-cycle arrest and cytoskeletal disruption, and thereby chromosome dispersion, and inducing congenital malformations. We conclude that developmental toxicity is related to high concentrations of metabolites after NTB doses of 50, 59.5, and 70.7 mg/kg and that ABZSO may be the main factor responsible for malformations and/or fetal death. ACKNOWLEDGMENTS The authors thank Miquel Pons and Paco Perez for technical assistance.
REFERENCES Baker, H. J., Russell, J., and Weifbroth, S. H. (1979). Embryology and teratology. In The Laboratory Rat. Biology and Diseases, pp. 78–81. American College of Laboratory Animal Medicine Series. Bogan, J. A., and Marriner, S. E. (1980). Analysis of benzimidazoles in body fluids by high performance liquid chromatography. J. Pharm. Sci. 69, 422–423.
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Cristo`fol, C., Carretero, A., Fernandez, M., Navarro, M., Sautet, J., Ruberte, J., and Arboix, M. (1995). Transplacental transport of netobimin metabolites in ewes. Eur. J. Drug Metab. Pharmacokin. 20, 167–171. Delatour, P., Cure, M. C., Benoit, E., and Garnier, F. (1986). Netobimin (Totabin-SCH): Preliminary investigations on metabolism and pharmacology. J. Vet. Pharmacol. Ther. 9, 230–234. Delatour, P., Garnier, F., Benoit, E., and Longin, CH. (1984). A correlation of toxicity of albendazole and oxfendazole with their free metabolites and bound residues. J. Vet. Pharmacol. Ther. 7, 139–145. Delatour, P., and Parish, R. (1986). Benzimidazole anthelmintics and related compounds: Toxicity and evaluation of residues. In Drug Residues in Animals (Rico, A. G., Ed.), pp. 175–203. Academic Press, New York. Delatour, P., Parish, R. C., and Gyurik, R. J. (1981). Albendazole: A comparison of relay embryotoxicity with embryotoxicity of individual metabolites. Ann. Rech. Ve´t. 12, 159–167. Delatour, P., Yoshimura, H., Garnier, F., and Benoit, E. (1982). Embryotoxicite´ compare´e des metabolites de l’oxfendazole. Rec. Me´d. Ve´t. 158, 369–373. Fabre, J. M., Berthelot, X., and Ferney, J. (1989). Embryotoxicite´ des antiparasitaires chez les ovins: Observation clinique. Rev. Me´d. Ve´t. 140, 1089–1095. Gyurik, R. J., Chow, A. W., Zaber, B., Brunner, E. L., Miller, J. A., Villani, A. J., Petka, L. A., and Parish, R. C. (1981). Metabolism of albendazole in cattle, sheep, rats and mice. Drug Metab. Dispos. 9, 503–508. Lacey, E., and Watson, T. R. (1985). Structure-activity relationships of benzimidazole carbamates as inhibitors of mammalian tubulin, in vitro. Biochem. Pharmacol. 34, 1073–1077. Lanusse, C. E., and Prichard, R. K. (1990). Pharmacokinetic behaviour of netobimin and its metabolites in sheep. J. Vet. Pharmacol. Ther. 13, 179–185. Martin, D. (1980). Albendazole: E´tude embryotoxique de dix me´tabolites. Doctoral Thesis, Ecole Nationale Ve´te´rinaire de Lyon, France. Mantovani, A., Macri, C., Stazi, A. V., and Riccciardi, C. (1992). Effects of albendazole on early phases of rat organogenesis ‘‘in vivo’’: Preliminary results. Eur. Teratol. Abst. 46, P22. McCracken, R. O., and Stillwell, W. H. (1991). A possible mode of action for benzimidazole anthelmintics. Int. J. Parasitol. 7, 99–104. Navarro, M. (1996). Efectos embrioto´xicos del netobimin en la oveja, la rata y el pollo. Doctoral Thesis, Publicacions UAB, Barcelona. Richards, L. S., Zimmerman, G. L., Hoberg, E. P., Schons, D. J., and Dawley, S. W. (1987). The anthelmintic efficacy of netobimin against acquired gastrointestinal nematodes in sheep. Vet. Parasitol. 26, 87–94. Santiago, M. A., Da Costa, U. C., and Benevenga, S. F. (1985). Netobimin (Totabin-SCH) efficacy in ruminants in Rio Grande Do Sud Brazil. World Assoc. Adv. Vet. Parasitol. Abst. 130, 30.
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DEVELOPMENTAL TOXICITY OF NETOBIMIN IN THE RAT Schu¨maker, R. C. (1986). Pkcalc: A basic interactive computer program for statistical and pharmacokinetic analysis of data. Drug Metab. Rev. 17, 331–348. Souhaili-el Amri, H., Fargetton, X., Delatour, P., and Bat, A. M. (1987). Sulphoxidation of albendazole by the FAD-containing and cytochrome P-450 dependent monooxigenases from pig liver microsomes. Xenobiotica 17, 1159–1168. Souhaili-el Amri, H., Mothe, O., Totis, M., Batt, A. M., Delatour, P., and Siest, G. (1988). Albendazole sulfonation by rat liver cytochrome P-450c. J. Pharmacol. Exp. Ther. 246(2), 758–764. Staples, R. E., and Schnell, V. L. (1964). Refinements in rapid clearing technic in the KOH-Alizarin red S method for fetal bone. Stain Technol. 39, 61.
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Whittaker, S. G., and Faustman, E. M. (1991). Effects of albendazole sulfoxide on cultures of differentiating rodent embryonic cells. Toxicol. Appl. Pharmacol. 109, 73–84. Whittaker, S. G., and Faustman, E. M. (1992). Effects of benzimidazole analogs on cultures of differentiating rodent embryonic cells. Toxicol. Appl. Pharmacol. 113, 144–151. Williams, J. C., Knox, J. W., Marbury, K. S., Kimball, M. D., Willis, E. R., Sneider, T. G., and Miller, J. E. (1985). Efficacy of SCH 32481 against inhibited larvae of Ostertagia ostertagi. Am. J. Vet. Res. 46, 2188–2192. Yoshimura, H. (1987). Teratogenic evaluation of triclabedazole in rats. Toxicology 43, 283–287.
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144, 56–61 (1997)
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Disposition of Netobimin, Albendazole, and Its Metabolites in the Pregnant Rat: Developmental Toxicity CARLES CRISTO`FOL,* MARC NAVARRO,† CARME FRANQUELO,* JOSEP ENRIC VALLADARES,* ANA CARRETERO,† JESUS RUBERTE,† AND MARGARITA ARBOIX* *Departament de Farmacologia i de Terape`utica and †Unitat d’Anatomia i Embriologia, Facultat de Veterina`ria, Universidad Auto`noma de Barcelona, 08193 Barcelona, Spain Received August 13, 1996; accepted January 11, 1997
ABZ and ABZSO exhibit significant anthelmintic and toxic effects (Delatour et al., 1981, 1984), the main toxic one being teratogenicity. Several studies suggest that the capacity of benzimidazolic drugs to bind with the tubulin of cellules (Delatour and Parish, 1986) is responsible for the toxic effects on the embryo. Fetuses from ewes treated with NTB in the first third of pregnancy show congenital malformations such as renal agenesis and ectopic kidneys, spina bifida, fused vertebrae and ribs, hypoplasia of the thoracic limb, and vascular anomalies (Fabre et al., 1989; Navarro, 1996), and Cristo`fol et al. (1995) found that ABZSO and ABZSO2 cross the placenta, reaching the fetus in significant amounts. Benzimidazole anthelmintics have also proven to be teratogenic in rats, producing an increased number of resorptions, decreased fetal weight, and external and skeletal malformations (Martin, 1980; Delatour et al., 1984, 1986; Yoshimura et al., 1987). In rats treated with 17.7 mg NTB/ kg, Delatour et al. (1986) found that peak plasma concentrations (Cmax ) were 0.79 mg/ml for ABZSO and 0.27 for ABZSO2 , and that they were achieved 6–9 and 12 hr after dosing, respectively. No studies have investigated the relationship between concentrations of ABZ and its metabolites and toxic effects on the fetus. The aim of this study was to establish the relationship between the NTB dose administered to pregnant rats, the maternal plasma concentration of ABZ and its metabolites, the concentrations of metabolites (ABZSO and ABZSO2 ) reaching the embryo, and the toxic effects observed in the fetuses.
Disposition of Netobimin, Albendazole, and Its Metabolites in the Pregnant Rat: Developmental Toxicity. CRISTO`FOL, C., NAVARRO, M., FRANQUELO, C., VALLADARES, J. E., CARRETERO, A., RUBERTE, J., AND ARBOIX, M. (1997). Toxicol. Appl. Pharmacol. 144, 56–61. Netobimin (NTB), a benzimidazole prodrug with a good anthelmintic spectrum, was administered orally to female rats at a dose of 59.5 mg NTB/kg, to study its pharmacokinetic behavior and the disposition of its most important metabolites, albendazole (ABZ), albendazole sulfoxide (ABZSO), and albendazole sulfone (ABZSO2 ). ABZ was found in plasma after 6 hr. Peak plasma concentrations (Cmax ) and areas under curves (AUC) of ABZSO were eight- and fourfold higher, respectively, than those of ABZSO2 . To study NTB disposition in pregnant rats, three different drug doses (50, 59.5, and 70.7 mg/kg) were given. No significant differences were found between plasma concentrations for each metabolite at the three doses studied. Only ABZ concentrations rose slightly as dose increased. ABZ, ABZSO, and ABZSO2 were found in amniotic sacs and embryos at concentrations that were higher than plasma at the same times. The fetuses obtained after administration of each of the doses of NTB were studied to detect developmental toxicity. A significant correlation was found between rate of developmental toxicity and metabolite concentration. ABZSO embryo concentrations could be the main factor accounting for toxicity. q 1997 Academic Press
Netobimin (NTB) has been shown to exhibit an anthelmintic spectrum against nematodes, cestodes, and trematodes (Williams et al., 1985; Santiago et al., 1985; Richards et al., 1987) and is reported to undergo biotransformation to albendazole (ABZ) when in contact with gastrointestinal bacteria (Delatour et al., 1986). Once ABZ is absorbed, it is metabolized sequentially to ABZSO and ABZSO2 (Gyurick et al., 1981; Souhaili-el Amri et al., 1987, 1988). NTB’s anthelmintic activity may be due in part to bioenergetic disruptions resulting from transmembrane proton discharge. Marked changes in wet weight and glycogen and protein concentrations were observed in tapeworms in animals that received therapeutic doses of benzimidazolic drugs (McCracken and Stillwell, 1991).
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Animals. Female Sprague–Dawley rats (Crl:CD), provided by CIDA Laboratories, were fed on a commercial diet (UAR A04C; Usine d’Alimentation Rationelle, Villemoisson sur Orge, France) and had free access to tap water. The onset of pregnancy was determined by vaginal smears. The day that sperm was found in the smear was called Day 0. The pharmacokinetics of NTB were studied first. Fifty-five nonpregnant rats weighing 300 g were dosed orally with 59.5 mg NTB/kg. Blood samples, obtained by exanguination after halothane overanaesthesia, were collected in heparinized tubes 0.5, 1, 2, 4, 6, 8, 12, 18, 24, 36, and 48 hr after 56
0041-008X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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MATERIALS AND METHODS
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DEVELOPMENTAL TOXICITY OF NETOBIMIN IN THE RAT drug administration from 5 rats at each collection time. Plasma samples were obtained after centrifugation at 2000 rpm for 20 min and then stored at 0207C until analysis. In the second step, three groups of six rats on the 10th day of pregnancy, weighing a mean of 300 g, were treated orally with 50, 59.5, and 70.7 mg/ kg NTB. Blood samples were taken from the orbital sinus using an heparinized glass capillary tube at 2, 4, and 8 hr after administration. Rats were overanaesthetized with halothane and exsanguinated 12 hr after administration when the maximum plasma concentration of ABZ metabolites had been reached as determined in the pharmacokinetic study in nonpregnant rats. The 10th day of gestation in rats is equivalent to the embryonal stage at which some authors (Fabre et al., 1989; Navarro, 1996) have observed abortion or malformed fetuses in sheep after oral treatment with 20 mg NTB/kg on the 17th day of gestation. Maternal blood, amniotic sacs, and embryos were collected. Embryos, amniotic sacs, and plasma samples were stored at 0207C until analyzed. NTB doses were selected according to Delatour et al. (1982), who observed fetal malformations when pregnant rats were treated orally with NTB doses of 44.2 mg/kg or higher. Finally, three groups of six rats each, on the 10th day of pregnancy, were treated orally with 50, 59.5, or 70.7 mg/kg of NTB. A control group of rats, on the 10th day of gestation, was treated with the same volume of vehicle (0.2% carboximethyl cellulose and 1% Tween 80 in distilled water) and were studied under the same conditions. Rats were overanaesthetized with halothane and exsanguinated 1 day before delivery (20th day of gestation). To determine developmental toxicity, resorptions, fetal weight, and malformations were recorded. Drug analysis. Concentrations of NTB, ABZ, ABZSO, and ABZSO2 in plasma, embryos, and amniotic sacs were determined by high-performance liquid chromatography (Bogan and Marriner, 1980). Malformations. Gross external and internal malformations were examined first. Then, skeletal malformations were determined using a clearing technique described by Staples and Schnell (1964) consisting of maceration in 1% KOH and staining with alizarin red S. Fetuses from each treatment were compared with those from the control group. Kinetic analysis. The pharmacokinetic parameters were determined by noncompartmental modelling using the PKCALC computer program (Shumacker, 1986). The terminal elimination slopes b were calculated by linear least-squares regression analysis. Statistics. Fetal weight and differences in tissue concentrations of ABZSO and ABZSO2 12 hr after administration of the three doses of NTB to pregnant rats were tested for significance by one-way ANOVA testing. Differences in resorptions rate and number of malformations for the three doses of NTB administered were tested for significance using a x2 test. The significance level accepted for differences between doses was p õ 0.05 for all tests. The data were calculated using the fetus as the experimental unit since each fetus possesses its own placenta.
RESULTS
Figure 1 plots plasma levels versus time of ABZ, ABZSO, and ABZSO2 for nonpregnant female rats orally treated with 59.5 mg NTB/kg. No ABZ was found after 6 hr and no NTB was detected at any of the sampling times. Table 1 shows pharmacokinetic parameters for both ABZ metabolites obtained from the analysis of the mean plasma levels of the five animals analyzed in each collection time. ABZSO Cmax and areas under curve (AUC) were eight- and fourfold higher, respectively, than those of ABZSO2 . Table 2 shows the mean AUC and its standard deviation from 0 to 12 hr for ABZSO and ABZSO2 for the three doses
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studied. No statistically significant differences were found for the three doses studied. Table 2 also shows the AUC ratio ABSO2 /ABZSO for the doses studied and, likewise, no significant differences were found. Figure 2 depicts ABZ, ABZSO, and ABZSO2 plasma concentrations in maternal blood from pregnant rats treated orally with NTB at the doses studied (50, 59.5, and 70.7 mg/ kg). No significant differences were found between plasma concentrations of each metabolite among the three doses. Only ABZ concentrations increased slightly at higher doses. Figure 3 shows concentrations of NTB metabolites in plasma, amniotic sacs, and embryos from pregnant rats treated with the three doses. Significant differences were observed for ABZ and ABZSO concentrations in embryos between 50 and 59.5 but not between 59.5 and 70.7 mg NTB/kg doses. Data on developmental toxicity of NTB after oral administration of 50, 59.5, and 70.7 mg/kg to the three groups of pregnant rats are shown in Table 3, and reveal that higher doses of NTB were associated with higher rates of resorptions. The rate for the low-dose group was not statistically different from that of the control group. No internal malformations were found. Decreased fetal weight and external and skeletal malformations were more common in treated animals than in controls but there were no significant differences in these parameters between the 59.5 and 70.7 mg NTB/kg doses. DISCUSSION
NTB administered orally to ruminants or rats is transformed to ABZ by a cyclation process carried out by gastrointestinal bacteria (Delatour et al., 1986; Lanusse and Prichard, 1990). NTB has never been detected in rat blood. However, in our study, ABZ was still detectable in plasma 6 hr after administration, suggesting that ABZ hepatic metabolism in monogastric animals is less efficient than in ruminants, in whom ABZ is immediately converted to ABZSO (Lanusse and Prichard, 1990) and is never measurable in plasma samples. The kinetic profiles of ABZSO and ABZSO2 obtained in the present study agree with the few available data in literature (Delatour et al., 1986; Souhaili-el Amri et al., 1988). Pharmacokinetics and plasma levels of the metabolites studied are similar in nonpregnant and pregnant rats, indicating that the elimination patterns of ABZ and its metabolites are not affected by the early stages of pregnancy. Only ABZ concentration increased significantly with dose while ABZSO and ABZSO2 plasma levels, AUC values, and the ratio of ABZSO2 /ABZSO AUC did not change. As ABZ is metabolized sequentially in the liver to ABZSO and ABZSO2 (Delatour et al., 1986), those metabolic steps are probably saturable. Thus, high doses of NTB may produce a saturation
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FIG. 1. Mean { standard deviation of plasma concentration (n Å 5) of ABZ, ABZSO, and ABZSO2 after administration of 59.5 mg NTB/kg to nonpregnant female rats.
of the enzymes of the hepatic microsomal system, which are responsible for the S-oxidation of ABZ to ABZSO and ABZSO2 . The concentrations of ABZ, ABZSO, and ABZSO2 in placental and embryonal tissues were higher than those observed in maternal blood, suggesting that these metabolites may bind the different molecules in embryonic cells and tissues. On the other hand, Cristo`fol et al. (1995) found that the concentrations of ABZSO and ABZSO2 in fetuses from ewes treated orally during the last third of gestation with 20 mg NTB/kg were half the concentrations in maternal plasma, and that ABZ was never detected; this indicates the existence of interspecies differences that may be related to anatomical differences in their placentas. Rats have two placentas, a discoidal chorioallantoic placenta, and a highly vascularized yolk sac placenta. Until Day 11 or 12, the yolk sac placenta
TABLE 1 Pharmacokinetic Parameters for ABZSO and ABZSO2 after Oral Administration of 59.5 mg NTB/kg to Nonpregnant Female Rats Parameter
ABZSO
ABZSO2
Cmax (mg/ml) Tmax (hr) T1/2b (hr) AUC0 – ` (mgrhr/ml) MRT (hr)
1.6 6.0 0.14 32.7 7.4
0.2 12.0 5.1 7.5 10.1
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is the only route for transport between mother and embryo since the chorioallantoic placenta does not become vascularized before this day. Studies on the placental transfer of material from mother to fetus in the rat did not reveal differences between the two placentas (Baker et al., 1979). However, the structure of rat placenta seems to be more permeable than the thicker syndesmochorial placenta of the ewe. Differences observed in access of the studied drugs to the embryo could also be due, however, to structural dissimilarities in the placenta at the two stages of pregnancy studied, as the first third of gestation was studied in rats and the last third was studied in sheeps. In agreement with results reported by other authors about the toxic effects of NTB and ABZ (Martin, 1980; Delatour et al., 1984, 1986; Mantovani, 1992), we also found correlations between some of the toxic effects examined and the increase in NTB dose administered to pregnant rats. Our data indicate that embryos remained exposed to ABZ for
TABLE 2 Mean AUC0 – 12 and Standard Deviations for ABZSO and ABZSO2 for the Three Doses Studied in Pregnant Rats Dose of NTB (mg/kg)
ABZSO (X { SD)
ABZSO2 (X { SD)
Ratio ABZSO2/ABZSO (X { SD)
50 59.5 70.7
14.6 { 2.5 15.5 { 6.7 13.7 { 5.7
2.7 { 1.0 2.6 { 1.0 1.8 { 1.0
0.18 { 0.04 0.19 { 0.08 0.14 { 0.04
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FIG. 2. Concentration–time curves (mean { SD, n Å 6) of ABZ, ABZSO, and ABZSO2 after administration of 50 (dose 1), 59.5 (dose 2), and 70.7 (dose 3) mg NTB/kg to pregnant rats.
over 12 hr and to ABZSO and ABZSO2 for approximately 36–48 hr. ABZSO2 concentrations, however, were not significantly different between doses that were associated with developmental toxicity. Analysis of the developmental toxicity incidence for the three doses and their correlation with embryo drug concen-
trations for each of the metabolites reveals that the ABZSO concentration could be the main factor accounting for developmental toxicity. The ABZSO concentrations were significantly higher after the 59.5 mg/kg dose of NTB, compared to the lowest dose (50 mg/kg), but we found no significant differences in the concentrations attained after the 59.5 and
FIG. 3. Mean plasma, embryo, and amniotic sacs (a.s.) concentrations { standard deviations (n Å 6) of ABZ, ABZSO, and ABZSO2 after administration of 50, 59.5, and 70.7 mg NTB/kg to pregnant rats.
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TABLE 3 Developmental Toxicity of NTB after Oral Administration of 50, 59.5, and 70.7 mg NTB/kg to Pregnant Rats Dose of NTB (mg/kg)
Number of live fetuses
Resorptions (%)
Control 50 59.5 70.7
66 60 84 38
4.3 4.7 15.5** 51.8***
Fetal weight (g { SE) 5.57 5.06 4.89 4.86
{ { { {
External malformations (% fetuses)
Skeletal malformations (% fetuses)
0 0 13.1** 13.1**
14.2 52.9* 71.4* 68.0*
0.05 0.05* 0.05** 0.08**
* Significantly different from controls. ** Significantly different from controls and from 50 mg/kg dose. *** Significantly different from controls, from 50 mg/kg dose, and from 59.5 mg/kg dose.
70.7 mg NTB/kg doses. On the other hand, statistically significant differences in fetal weight and number of external malformations were observed between NTB doses of 50 and 59.5 mg/kg, whereas there were no such differences between the 59.5 and 70.7 mg/kg doses. No differences in the incidence of resorptions were observed between animals treated with 50 mg/kg NTB and the control group. Increasing the dose to 59.5 mg/kg, however, promoted an increase in the resorption rate. This may be a result of the combined effects of ABZ plus ABZSO. In vitro studies have demonstrated that ABZ appears to be cytotoxic for some embryo cells at a 0.3–0.4 mM concentration, a range that is 50-fold higher than that of ABZSO (Whittaker and Faustman, 1991, 1992). However the affinity of both drugs for tubulin binding is similar, 6.9 mM being the ABZ concentration needed to inhibit mammalian tubulin polymerization (Lacey and Watson, 1985). This could explain why some embryonal cells overcome the lethal effect produced by the presence of ABZ and ABZSO. The surviving cells apparently needed a higher concentrations of these substances for their mitotic microtubular system to adversely affected, producing cell-cycle arrest and cytoskeletal disruption, and thereby chromosome dispersion, and inducing congenital malformations. We conclude that developmental toxicity is related to high concentrations of metabolites after NTB doses of 50, 59.5, and 70.7 mg/kg and that ABZSO may be the main factor responsible for malformations and/or fetal death. ACKNOWLEDGMENTS The authors thank Miquel Pons and Paco Perez for technical assistance.
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Whittaker, S. G., and Faustman, E. M. (1991). Effects of albendazole sulfoxide on cultures of differentiating rodent embryonic cells. Toxicol. Appl. Pharmacol. 109, 73–84. Whittaker, S. G., and Faustman, E. M. (1992). Effects of benzimidazole analogs on cultures of differentiating rodent embryonic cells. Toxicol. Appl. Pharmacol. 113, 144–151. Williams, J. C., Knox, J. W., Marbury, K. S., Kimball, M. D., Willis, E. R., Sneider, T. G., and Miller, J. E. (1985). Efficacy of SCH 32481 against inhibited larvae of Ostertagia ostertagi. Am. J. Vet. Res. 46, 2188–2192. Yoshimura, H. (1987). Teratogenic evaluation of triclabedazole in rats. Toxicology 43, 283–287.
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