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Chemical structure—teratogenicity relationships, toxicokinetics and metabolism in risk assessment of retinoids
Toxicology Letters ELSEVIER
Toxicology Letters 82/83 (1995) 975-979
Chemical structure - teratogenicity relationships, toxicokinetics and metabolism in risk assessment of retinoids Heinz Nau* Imtmte of
To.~~cology and Embr_vophartnacology,
Free Universgv Be&n,
Garystrape .f D- lJl9_f Berlln. Germanv
Abstract Retinoic acid, an oxidative metabolite of vitamin A, is involved in the control of many biological processes including embryonic development and excess as well as deficiency of retinoids has been found to be teratogenic. The effects of retinoids in normal as well as abnormal development may be mediated by two members of retinoid receptors. the RARs and RXRs, which exhibit specific temporal and spatial expression during development.
Evidence accumulates that any alteration of this complex retinoid system may be related to teratogenic effects. Here we investigate the influence of toxicokinetic parameters, including aspects of metabolism and placental transfer. on the teratogenic potency of retinoids. It is demonstrated that activation (oxidation of retinoic acids: hydrolysis of glycoconjugates) and deactivation reactions (isomerization from trans- into c&configuration; /?glucuronidation) relate to teratogenesis. The P-glucuronides of retinoic acids show poor placental transfer and prolonged presence in the maternal organism. Non-retinoid compounds such as antiepileptic agents may exert some of their teratogenicity via alteration of endogenous retinoid levels. Keywords: Vitamin A; Retinoic acid; Teratogenesis; transfer
Retinoyl-P-glucuronides;
1. Introduction Retinoic acid, an oxidative metabolite of vitamin A, plays a key role in many biological processes including growth and differentiation of epithelial tissues, spermatogenesis, vision and embryonic development [1,2]. This extremely
* Correspondmg +49-30-831-6139.
author. Tel: +49-30-838-5053/6312; fax:
Abbrevlatlons: AUC. area under the concentration-time carve; c‘,,,,,, maxlmal concentration: CRBP, CRABP, cellular retmol and retmolc acid binding protein, respectively; RAR. retmoic acid receptor; RXR, retinoid X receptor. 037%4274/95/$09..50 0 1995 Elsevler SSDI 0378-4274(9.5)03614-T
Science
Ireland
Retinoid receptor ligands; Placental
broad range of effects is thought to be mediated by nuclear retinoid receptors as well as cytosolic retinoid binding proteins [3]. There are two , groups of retinoid receptors: RARq RARP and RARy as well as RXRa, RXRP and RXRy [4]. These receptors belong to the steroid/thyroid hormone receptor superfamily and function as ligand-activated transcription factors controlling the expression of numerous responsive genes. All-truns-retinoic acid acts as ligand for the RAR-receptors, while 9-cis-retinoic acid can bind to both the RAR- and RXR-receptors. The specific expression of these receptors (and their isoforms), as well as the concentrations of the
Ltd. All rights reserved
976
H. Nau I Toxicology Letters 82183 (1995) 975-979
retinoic acid ligands may be crucial factors in the control of the transcription of responsive genes. Vitamin A excess and deficiency, as well as the administration of low doses of retinoic acid have been shown to result in teratogenic effects [5-81. All-truns-retinoic acid is present endogenously in the embryo and shows a specific distribution within the embryo. The RAR and RXR families of retinoic acid receptors also show a very distinct spatial as well as temporal distribution within the developing embryo. In addition, the cytosolic retinol binding proteins (CRBPs) as well as the retinoic acid binding proteins (CRABPs) also exhibit a very specific expression pattern in the embryo, which could also be crucial for the action for endogenous and exogenous retinoids [9]. These proteins may be involved in the metabolism of retinol and retinoic acid as well as in the control of the ‘free’ concentration of all-trans-retinoic acid, which is available for nuclear receptor binding. Thus, the multiplicity of the receptors (RARs and RXRs and their isoforms), the formation of homo- and hetero-receptor dimers, the two natural ligands (all-truns- and 9-cis-retinoic acids), the multiplicity of cytosolic retinoid binding proteins (CRABPs and CRBPs) and the very specific temporal and spatial expression of retinoid receptors, binding proteins and ligands within the embryo could enable an extremely wide range of signaling pathways that control normal development. We have therefore investigated toxicokinetic and metabolic parameters in maternal plasma and embryo and related these patterns to the teratogenic activity of the administered retinoid compounds [6-81. We have also demonstrated that antiepileptic agents can alter plasma concentrations of retinol and several retinoic acids, and these effects may be involved in the mechanism of anticonvulsant teratogenesis [lo].
2. Results and discussion 2.1. Placental transfer of retinoids The chemical structure of retinoids has a major influence on placental transfer. All-truns-retinol
and all-truns-retinoic acid show extensive placental transfer, while the c&-isomers of retinoic acid (9-cis, 13-cis, 9,13-di-cis-retinoic acid) show a much more limited placental transfer in the mouse, rat and rabbit (Table 1). The reasons behind these structure-specific differences are not clear, and may reside in the differential binding affinities of these retinoic acids to protein(s) which may possibly exist for their specific transport into the embryonic compartment. Interestingly, the placental transfer of 13-cis-retinoic acid was much greater in the monkey than in the rodent species, and the differing placental structures between the rodents (yolk sac placenta) and the monkey (chorioallantoic placenta) were suggested to be one reason for these interesting and potentially important differences. 2.2. Plasma clearance and metabolism The 13-cis-retinoic acid is a potent teratogen in monkeys, and even more potent in the human, and extensive transfer to the monkey embryo may be important in this aspect. Also the primary metabolic pathway proceeds to the 13-cis4-oxo-retinoic acid in the monkey, which may serve as an activation pathway of teratogenesis. In contrast, in rats and mice the main plasma metabolite is the 13-cis-retinoyl-&glucuronide which shows very poor placental transfer and may have little intrinsic teratogenic activity. Furthermore, the plasma clearance of 13-cis-retinoic acid is much greater in rats and mice than in monkeys. The low teratogenic potency of 13-cisTable 1 Placental transfer of retinoids (mouse, rat, rabbit) Retinoid
Embryo/maternal
RAG all-trans 13-cis 9x1s RA 9,13-di-czs 134s 9-k all-truns Data
from
0.01-0.03 0.01-0.03 0.01-0.03 0.01-0.03 0.05-0.1 0.15 0.5-2 Refs. [ll-161.
plasma
ratio
H. Nau I Toxicology Letters &‘I83 (1995) 975-979
977
Table 2 Species variation of 13-cis-retinoic acid teratogenesis Parameter
Mouse/rat
Rabbit
Monkey/human
Clearance AUC Placental transfer Maternal metabolism
Fast Low
Slow High co.1 Activation to 4-oxo-met.
Slow High 0.4 (monkey)
Data from Refs. [11,12,14-17,19,20].
retinoic acid in the rat and mouse may therefore be explained by three factors (Table 2): limited placental transfer, rapid plasma clearance and extensive metabolic detoxification; on the other hand, the high teratogenic activity of this retinoid in the monkey (and possibly the human as well) is the result of more extensive placental transfer, slower plasma clearance and extensive metabolism to the active 4-oxo-metabolite (Table 2). 2.3. Alteration of endogenous retinoid levels as possible mechanism of teratogenesis of antiepileptic agents The major antiepileptic drugs used for the control of seizures can induce developmental toxicity when administered during pregnancy. Vitamin A and retinoids are thought to control many processes of embryonic development including growth, differentiation and morphogenesis. We have tested the hypothesis that the teratogenic action of antiepileptic agents is mediated via alteration of the endogenous vitamin A - retinoid metabolism. Retinol and its Table 3 Plasma concentrations Group
Controls VPA VPA + PT VPA + CM VPA + PB VPA + ES VPA + combination Abbreviations:
oxidative metabolites, all-trans-, 13-k+ and 13cis-4-oxo-retinoic acid were measured in the plasma of 75 infants and children treated with various antiepileptic drugs for the control of seizures, and in 29 untreated controls of comparable age [lo]. Retinol levels increased with age, while the concentrations of retinoic acid compounds did not exhibit age-dependency. Valproic acid monotherapy increased retinol levels in the young age group and a trend toward increased retinol concentrations was also observed in all other patient groups (Table 3). The levels of the oxidative metabolites 13-cis- and 13-cis-4-0x0retinoic acids were strongly decreased in all patient groups treated with phenytoin, phenobarbital, carbamazepine and ethosuximide, in combination with valproic acid, to levels which were 27% and 6% of corresponding control values, respectively (Table 4). Little change was observed with all-trans-retinoic acid except in one patient group treated with valproic acid ethosuximide cotherapy where increased levels of this retinoid were found. Our study indicates that therapy with antiepileptic agents can have a
of retinol in infants and children treated with antiepileptic drugs Age group: retinol plasma cont. (ng/ml; means 5S.D.) O-6 years
>6 years
All subjects
232 ? 36 (n = 15) 312 f 73 (n = 10)”
351? 62 (n = 12) 379 t 102 (n = 27) 393 -c 116 (n = 4) 436/.544 (n = 2)b 414 ? 39 (n = 4) 431 t 137 (n = 6) 420 ? 77.2 (n = 6)
285 -c 77 (n = 27) 363 -c 99 (n = 39)b 393 ? 116 (n = 4)” 392 t 123 (n = 4)b 389 t 47 (n = 7)” 405 ? 143 (n = 7)” 399 ? 69 (n = 9)d
272/317 (n = 2)b 357+40 (n=3)d c 36O-c20 (n=3)d
CM, carbamazepine; ES, ethosuximide; PB, phenobarbital; PT, phenytoin; VPA, valproic acid. Data from Ref. [lo] Significantly higher than in controls (Student’s t-test): ’ p < 0.005; b p < 0.05 ’ p < 0.01; d p < 0.0005; ’ insufficient number of patients.
H. Nau I Toxicology Letters 82/83 (1995) 975-979
978 Table 4 Plasma concentrations Group
Controls VPA VPA + PT VPA + CM VPA + PB VPA + ES VPA in comb.d
of polar retinoids in infants and children treated with antiepileptic
drugs
Plasma cont. of retinoic acids (RA) (nglml: means -CS.D.) n
all-trans-RA
13-cis-RA
13-cis-4-oxo-RA
29 40 5 4 7 8 11
1.07 5 0.26 1.13 -c 0.39 1.07 ir 0.36 1.17 2 0.12 0.92 k 0.22 1.35 k 0.23’ 0.91 ? 0.31
0.97 t 1.19 5 0.30 k 0.34 + 0.35 t 0.58 t 0.23 2
2.26 2 0.93 2.22 2 1.a2 0.172 0.38” 0.22 ? O&tb 0.28 k 0.48” 1.08 2 0.71’ 0.14 -c 0.48”
0.31 0.89 0.19” O.llb 0.26” 0.27’ 0.31 a
n, number of subjects. For abbreviations of antiepileptic drugs see Table 3. Data from Ref. [lo] Significantly different from corresponding controls (Student’s c-test): a p < 0.0001; ’ p < 0.001; ’ p < 0.01; d Patients treated with VPA and two or three additional-antiepileptic drugs.
significant effect on endogenous retinoid metabolism. Because of the importance of retinoids for signaling crucial biological events during embryonic development, altered retinoid metabolism may be an important factor in antiepileptic drug teratogenesis. It will be important to study how such altered plasma retinoid concentrations will affect tissue retinoid levels, particularly in those areas most susceptible to retinoid excess and deficiency. The biological consequences of altered tissue retinoid concentrations could be wide-spread. Although all-[runs-retinoic acid, considered to be the most active endogenous retinoid, was not greatly altered in our present study, the effects of anticonvulsants on retinol, 13-cis-retinoic acid and its 4-oxo-metabolite are likely to be of great importance. The coadministration of phenobarbital also drastically decreased plasma levels of 13-cis-retinoic acid and its 4-oxo-metabolite [lo]. Phenytoin and carbamazepine may have acted similarly to phenobarbital in the reduction of endogenous retinoids. It is interesting to speculate why the levels of all-truns-retinoic acid were not significantly altered in these patients by administration of the inducing antiepileptic agents. It may be that the coadministered valproic acid, previously shown to be an inhibitor of several enzymatic reactions, may have offset the inducing activity of the other anticonvulsants; this hypothesis is presently under investigation.
2.4. Pharmacokinetic
parameters:
AUC
vs. C,,,
We have demonstrated that the AUC-values (area under the concentration-time curve values) of active retinoids attained in the embryonic compartment during sensitive stages of gestation are appropriately correlated with observed teratogenic activity [6,7,20]. Using AUC-values, a rational species comparison is possible and experimental results may be extrapolated to human exposures. It has been shown that the teratogenic effects of other drugs such as caffeine [21] and valproic acid [22] can be related to the maximal concentrations (C,,,)-values rather than the AUCs. It is even possible that one particular effect (digit malformation) induced by a drug such as methoxyacetic acid can be correlated with the AUC-values, while another effect (exencephaly) induced by the same drug can be correlated with C,,, -values [23]. It may be inferred from that study that the crucial toxicokinetic parameter will depend not only on a particular drug, but also on a particular organ system and developmental period.
Acknowledgements This work was supported by a grant from the European Commission (BIOTECH program BIO-CI93-0471), and the Deutsche Forschungsgemeinschaft (Sfb 174; C6).
H. Nau I Toxicology Letters 82183 (1995) 975-979
References L.J. (1994) Retinoids and vertebrate development. J. Biol. Chem. 269, 15399-15402. PI Maden, M. (1994) Vitamin A in embryonic development. Nub. Rev. 52, S3-S12. [31 Chambon. P. (1993) The molecular and genetic dissection of the retinoid signalling pathway. Gene 135, 223228. D.J., Umesono, K. and Evans, R.M. (1994) 141 Mangelsdorf, The retinoid receptors. In: M.B. Sporn, A.B. Roberts and D.S. Goodman (Eds.), The Retinoids: Biology, Chemistry, and Medicine, Raven Press, Ltd., New York, pp. 319-349. F.O. (1986) [51 Rosa, F.W.. Wilk, A.L. and Kelsey, Teratogen update: Vitamin A congeners. Teratology 33, 355-364. of transplacental and materWI Nau, H. (1990) Correlation nal pharmacokinetics of retinoids during organogenesis with teratogenicity. Methods Enzymol. 190, 437-448. teratogenesis: toxicokinettcs [71 Nau. H. (1993) Retinoid and structure-specificity. Arch. Toxic01 (Suppl. 16). 18% 127. I., Dencker, L., Lammer, E.J. and PI Nau, H , Chahoud. Scott. W.J. (1994) Teratogenicity of vitamin A and retinoids. In: R. Blomhoff (Ed.), Vitamin A m Health and Disease, Marcel Dekker, New York, pp. 615-664. 191 Scott Jr., W.J., Walter, R.. Tzimas, G., Sass. J-O.. Nau, H. and Collins, M.D. (1994) Endogenous status of retmoids and their cytosolic binding proteins in limb buds of chick vs. mouse embryos. Dev. Biol. 165, 397-409. [lOI Nau. H.. Tzimas, G., Mondry, M., Plum, C. and Spohr. H.L. (1995) Antiepileptic drugs alter endogenous retinotd concentrattons: A possible mechanism of teratogenesis of anticonvulsant therapy. Life Sci. 57. 53-60 (111 Creech Kraft. J., Eckhoff, C., Kochhar, D.M., Bochert. G.. Chahoud, I. and Nau. H. (1991a) Isotretinom (13ca-retinoic acid) metabolism, &-tram isomerization, glucuronidation and transfer to the mouse embryo: Consequences for teratogemcity. Teratogen. Carcinogen. Mutagen. 11, 21-30. [I21 Creech Kraft, J., Sliker Jr., W., Bailey, J.R.. Roberts, L.G., Fischer, B.. Wittfoht, W. and Nau. H. (1991b) Plasma pharmacokinetics and metabolism of 13-cis- and all-rrans-retinoic acid in the Cynomolgus monkey and the identification of 13.crs- and all-trans-retinoyl-P-glue uromdes. A comparison to one human case study with isotretinom. Drug Metab. Dispos. 19. 317-324. [13] Sass, J.O.. Tzimas, G. and Nau, H. (1994) 9-ca-retinoylP-o-glucuromde is a major metabolite of 9-cis-retinotc acid. Life Sci. 54. PL 69-74.
P41 Tzimas, G., Biirgin, H., Collins, M.D., Hummler,
Ul Gudas,
[I51
iI61
u71
WI
[I91
PO1
PI
WI
[23]
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H. and Nau, H. (1994) The high sensitivity of the rabbit to the teratogenic effects of 13-cis-retinoic acid (isotretinoin) is a consequence of prolonged exposure of the embryo to 13-cis-retinoic acid and 13-cis-4-oxo-retinoic acid, and not of isomerization to all-trans-retinoic acid. Arch. Toxicol. 68, 119-128. Tzimas, G., Sass, J.O., Wittfoht, W., Elmazar, M.M.A., Ehlers, K. and Nau, H. (1994) Identification of 9,13dicis-retinoic acid as a major plasma metabolite of 9-c&retinoic acid and limited transfer of 9-cis-retinoic acid and 9,13-dicis-retinoic acid to the mouse and rat embryos. Drug Metab. Dispos. 22. 928-936. Collins, M.D., Tzimas, G., Hummler, H.. Burgin, H. and Nau, H. (1994) Comparative teratology and transplacental pharmacokinetics of all-trans-retinoic acid, 13.crsretinoic acid, and retinyl palmitate following daily administrations in rats. Toxicol. Appl. Pharmacol. 127, 132-144. Tzimas. G., Sass, J.O., Rtihl, R., Klug, S., Collins, M.D. and Nau. H. (1994) Proximate retinoid teratogens. In: M.A Livrea and G. Vidali (Eds.), From Basic Science to Clinical Applications, Birkhduser Verlag, Basel. pp. 179195. Colhns, M.D., Tzimas, G., Biirgin, H., Hummler, H. and Nau, H. (1995) Single versus multiple dose admmistration of all-frans-retinotc acid during organogenests: differential metabolism and transplacental kinetics m rat and rabbit. Toxicol. Appl. Pharmacol. 130, 9-18. Hummler, H., Hendrickx, A.G. and Nau. H. (1994) Maternal toxicokinetics. metabolism and embryo exposure following a teratogenic dosing regimen with 13-cisretinoic acid (isotretmoin) m the Cynomolgus monkey. Teratology 50, 184-193. Nau. H. (1994) Toxicokinetics and structure-activity relationships in retinoid teratogenesis. Ann. Oncol. 5 (Suppl. 9) s39-s43. Sullivan, F.M., Smith, S.E. and McElhatton. P.R. (1987) Interpretation of animal experiments as illustrated by studies on caffeine. In: H. Nau and W.J. Scott (Eds.). Pharmacokinetics m Teratogenesis, CRC Press, Inc.. Boca Raton, FL,Vol. I, pp. 124-127. Nau, H. (1985) Teratogenic valproic acid concentrations: Infusion by implanted minipumps vs. conventional injection regimen m the mouse. Toxicol. Appl. Pharmacol. 80, 243-250. Terry. K.K., Elswick, B.A., Stedman, D.B. and Welsch. F. (1994) Developmental phase alters dosimetryteratogenicity relationship for 2-methoxyethanol in CD1 mice. Teratology 49. 218-227.
Toxicology Letters 82/83 (1995) 975-979
Chemical structure - teratogenicity relationships, toxicokinetics and metabolism in risk assessment of retinoids Heinz Nau* Imtmte of
To.~~cology and Embr_vophartnacology,
Free Universgv Be&n,
Garystrape .f D- lJl9_f Berlln. Germanv
Abstract Retinoic acid, an oxidative metabolite of vitamin A, is involved in the control of many biological processes including embryonic development and excess as well as deficiency of retinoids has been found to be teratogenic. The effects of retinoids in normal as well as abnormal development may be mediated by two members of retinoid receptors. the RARs and RXRs, which exhibit specific temporal and spatial expression during development.
Evidence accumulates that any alteration of this complex retinoid system may be related to teratogenic effects. Here we investigate the influence of toxicokinetic parameters, including aspects of metabolism and placental transfer. on the teratogenic potency of retinoids. It is demonstrated that activation (oxidation of retinoic acids: hydrolysis of glycoconjugates) and deactivation reactions (isomerization from trans- into c&configuration; /?glucuronidation) relate to teratogenesis. The P-glucuronides of retinoic acids show poor placental transfer and prolonged presence in the maternal organism. Non-retinoid compounds such as antiepileptic agents may exert some of their teratogenicity via alteration of endogenous retinoid levels. Keywords: Vitamin A; Retinoic acid; Teratogenesis; transfer
Retinoyl-P-glucuronides;
1. Introduction Retinoic acid, an oxidative metabolite of vitamin A, plays a key role in many biological processes including growth and differentiation of epithelial tissues, spermatogenesis, vision and embryonic development [1,2]. This extremely
* Correspondmg +49-30-831-6139.
author. Tel: +49-30-838-5053/6312; fax:
Abbrevlatlons: AUC. area under the concentration-time carve; c‘,,,,,, maxlmal concentration: CRBP, CRABP, cellular retmol and retmolc acid binding protein, respectively; RAR. retmoic acid receptor; RXR, retinoid X receptor. 037%4274/95/$09..50 0 1995 Elsevler SSDI 0378-4274(9.5)03614-T
Science
Ireland
Retinoid receptor ligands; Placental
broad range of effects is thought to be mediated by nuclear retinoid receptors as well as cytosolic retinoid binding proteins [3]. There are two , groups of retinoid receptors: RARq RARP and RARy as well as RXRa, RXRP and RXRy [4]. These receptors belong to the steroid/thyroid hormone receptor superfamily and function as ligand-activated transcription factors controlling the expression of numerous responsive genes. All-truns-retinoic acid acts as ligand for the RAR-receptors, while 9-cis-retinoic acid can bind to both the RAR- and RXR-receptors. The specific expression of these receptors (and their isoforms), as well as the concentrations of the
Ltd. All rights reserved
976
H. Nau I Toxicology Letters 82183 (1995) 975-979
retinoic acid ligands may be crucial factors in the control of the transcription of responsive genes. Vitamin A excess and deficiency, as well as the administration of low doses of retinoic acid have been shown to result in teratogenic effects [5-81. All-truns-retinoic acid is present endogenously in the embryo and shows a specific distribution within the embryo. The RAR and RXR families of retinoic acid receptors also show a very distinct spatial as well as temporal distribution within the developing embryo. In addition, the cytosolic retinol binding proteins (CRBPs) as well as the retinoic acid binding proteins (CRABPs) also exhibit a very specific expression pattern in the embryo, which could also be crucial for the action for endogenous and exogenous retinoids [9]. These proteins may be involved in the metabolism of retinol and retinoic acid as well as in the control of the ‘free’ concentration of all-trans-retinoic acid, which is available for nuclear receptor binding. Thus, the multiplicity of the receptors (RARs and RXRs and their isoforms), the formation of homo- and hetero-receptor dimers, the two natural ligands (all-truns- and 9-cis-retinoic acids), the multiplicity of cytosolic retinoid binding proteins (CRABPs and CRBPs) and the very specific temporal and spatial expression of retinoid receptors, binding proteins and ligands within the embryo could enable an extremely wide range of signaling pathways that control normal development. We have therefore investigated toxicokinetic and metabolic parameters in maternal plasma and embryo and related these patterns to the teratogenic activity of the administered retinoid compounds [6-81. We have also demonstrated that antiepileptic agents can alter plasma concentrations of retinol and several retinoic acids, and these effects may be involved in the mechanism of anticonvulsant teratogenesis [lo].
2. Results and discussion 2.1. Placental transfer of retinoids The chemical structure of retinoids has a major influence on placental transfer. All-truns-retinol
and all-truns-retinoic acid show extensive placental transfer, while the c&-isomers of retinoic acid (9-cis, 13-cis, 9,13-di-cis-retinoic acid) show a much more limited placental transfer in the mouse, rat and rabbit (Table 1). The reasons behind these structure-specific differences are not clear, and may reside in the differential binding affinities of these retinoic acids to protein(s) which may possibly exist for their specific transport into the embryonic compartment. Interestingly, the placental transfer of 13-cis-retinoic acid was much greater in the monkey than in the rodent species, and the differing placental structures between the rodents (yolk sac placenta) and the monkey (chorioallantoic placenta) were suggested to be one reason for these interesting and potentially important differences. 2.2. Plasma clearance and metabolism The 13-cis-retinoic acid is a potent teratogen in monkeys, and even more potent in the human, and extensive transfer to the monkey embryo may be important in this aspect. Also the primary metabolic pathway proceeds to the 13-cis4-oxo-retinoic acid in the monkey, which may serve as an activation pathway of teratogenesis. In contrast, in rats and mice the main plasma metabolite is the 13-cis-retinoyl-&glucuronide which shows very poor placental transfer and may have little intrinsic teratogenic activity. Furthermore, the plasma clearance of 13-cis-retinoic acid is much greater in rats and mice than in monkeys. The low teratogenic potency of 13-cisTable 1 Placental transfer of retinoids (mouse, rat, rabbit) Retinoid
Embryo/maternal
RAG all-trans 13-cis 9x1s RA 9,13-di-czs 134s 9-k all-truns Data
from
0.01-0.03 0.01-0.03 0.01-0.03 0.01-0.03 0.05-0.1 0.15 0.5-2 Refs. [ll-161.
plasma
ratio
H. Nau I Toxicology Letters &‘I83 (1995) 975-979
977
Table 2 Species variation of 13-cis-retinoic acid teratogenesis Parameter
Mouse/rat
Rabbit
Monkey/human
Clearance AUC Placental transfer Maternal metabolism
Fast Low
Slow High co.1 Activation to 4-oxo-met.
Slow High 0.4 (monkey)
Data from Refs. [11,12,14-17,19,20].
retinoic acid in the rat and mouse may therefore be explained by three factors (Table 2): limited placental transfer, rapid plasma clearance and extensive metabolic detoxification; on the other hand, the high teratogenic activity of this retinoid in the monkey (and possibly the human as well) is the result of more extensive placental transfer, slower plasma clearance and extensive metabolism to the active 4-oxo-metabolite (Table 2). 2.3. Alteration of endogenous retinoid levels as possible mechanism of teratogenesis of antiepileptic agents The major antiepileptic drugs used for the control of seizures can induce developmental toxicity when administered during pregnancy. Vitamin A and retinoids are thought to control many processes of embryonic development including growth, differentiation and morphogenesis. We have tested the hypothesis that the teratogenic action of antiepileptic agents is mediated via alteration of the endogenous vitamin A - retinoid metabolism. Retinol and its Table 3 Plasma concentrations Group
Controls VPA VPA + PT VPA + CM VPA + PB VPA + ES VPA + combination Abbreviations:
oxidative metabolites, all-trans-, 13-k+ and 13cis-4-oxo-retinoic acid were measured in the plasma of 75 infants and children treated with various antiepileptic drugs for the control of seizures, and in 29 untreated controls of comparable age [lo]. Retinol levels increased with age, while the concentrations of retinoic acid compounds did not exhibit age-dependency. Valproic acid monotherapy increased retinol levels in the young age group and a trend toward increased retinol concentrations was also observed in all other patient groups (Table 3). The levels of the oxidative metabolites 13-cis- and 13-cis-4-0x0retinoic acids were strongly decreased in all patient groups treated with phenytoin, phenobarbital, carbamazepine and ethosuximide, in combination with valproic acid, to levels which were 27% and 6% of corresponding control values, respectively (Table 4). Little change was observed with all-trans-retinoic acid except in one patient group treated with valproic acid ethosuximide cotherapy where increased levels of this retinoid were found. Our study indicates that therapy with antiepileptic agents can have a
of retinol in infants and children treated with antiepileptic drugs Age group: retinol plasma cont. (ng/ml; means 5S.D.) O-6 years
>6 years
All subjects
232 ? 36 (n = 15) 312 f 73 (n = 10)”
351? 62 (n = 12) 379 t 102 (n = 27) 393 -c 116 (n = 4) 436/.544 (n = 2)b 414 ? 39 (n = 4) 431 t 137 (n = 6) 420 ? 77.2 (n = 6)
285 -c 77 (n = 27) 363 -c 99 (n = 39)b 393 ? 116 (n = 4)” 392 t 123 (n = 4)b 389 t 47 (n = 7)” 405 ? 143 (n = 7)” 399 ? 69 (n = 9)d
272/317 (n = 2)b 357+40 (n=3)d c 36O-c20 (n=3)d
CM, carbamazepine; ES, ethosuximide; PB, phenobarbital; PT, phenytoin; VPA, valproic acid. Data from Ref. [lo] Significantly higher than in controls (Student’s t-test): ’ p < 0.005; b p < 0.05 ’ p < 0.01; d p < 0.0005; ’ insufficient number of patients.
H. Nau I Toxicology Letters 82/83 (1995) 975-979
978 Table 4 Plasma concentrations Group
Controls VPA VPA + PT VPA + CM VPA + PB VPA + ES VPA in comb.d
of polar retinoids in infants and children treated with antiepileptic
drugs
Plasma cont. of retinoic acids (RA) (nglml: means -CS.D.) n
all-trans-RA
13-cis-RA
13-cis-4-oxo-RA
29 40 5 4 7 8 11
1.07 5 0.26 1.13 -c 0.39 1.07 ir 0.36 1.17 2 0.12 0.92 k 0.22 1.35 k 0.23’ 0.91 ? 0.31
0.97 t 1.19 5 0.30 k 0.34 + 0.35 t 0.58 t 0.23 2
2.26 2 0.93 2.22 2 1.a2 0.172 0.38” 0.22 ? O&tb 0.28 k 0.48” 1.08 2 0.71’ 0.14 -c 0.48”
0.31 0.89 0.19” O.llb 0.26” 0.27’ 0.31 a
n, number of subjects. For abbreviations of antiepileptic drugs see Table 3. Data from Ref. [lo] Significantly different from corresponding controls (Student’s c-test): a p < 0.0001; ’ p < 0.001; ’ p < 0.01; d Patients treated with VPA and two or three additional-antiepileptic drugs.
significant effect on endogenous retinoid metabolism. Because of the importance of retinoids for signaling crucial biological events during embryonic development, altered retinoid metabolism may be an important factor in antiepileptic drug teratogenesis. It will be important to study how such altered plasma retinoid concentrations will affect tissue retinoid levels, particularly in those areas most susceptible to retinoid excess and deficiency. The biological consequences of altered tissue retinoid concentrations could be wide-spread. Although all-[runs-retinoic acid, considered to be the most active endogenous retinoid, was not greatly altered in our present study, the effects of anticonvulsants on retinol, 13-cis-retinoic acid and its 4-oxo-metabolite are likely to be of great importance. The coadministration of phenobarbital also drastically decreased plasma levels of 13-cis-retinoic acid and its 4-oxo-metabolite [lo]. Phenytoin and carbamazepine may have acted similarly to phenobarbital in the reduction of endogenous retinoids. It is interesting to speculate why the levels of all-truns-retinoic acid were not significantly altered in these patients by administration of the inducing antiepileptic agents. It may be that the coadministered valproic acid, previously shown to be an inhibitor of several enzymatic reactions, may have offset the inducing activity of the other anticonvulsants; this hypothesis is presently under investigation.
2.4. Pharmacokinetic
parameters:
AUC
vs. C,,,
We have demonstrated that the AUC-values (area under the concentration-time curve values) of active retinoids attained in the embryonic compartment during sensitive stages of gestation are appropriately correlated with observed teratogenic activity [6,7,20]. Using AUC-values, a rational species comparison is possible and experimental results may be extrapolated to human exposures. It has been shown that the teratogenic effects of other drugs such as caffeine [21] and valproic acid [22] can be related to the maximal concentrations (C,,,)-values rather than the AUCs. It is even possible that one particular effect (digit malformation) induced by a drug such as methoxyacetic acid can be correlated with the AUC-values, while another effect (exencephaly) induced by the same drug can be correlated with C,,, -values [23]. It may be inferred from that study that the crucial toxicokinetic parameter will depend not only on a particular drug, but also on a particular organ system and developmental period.
Acknowledgements This work was supported by a grant from the European Commission (BIOTECH program BIO-CI93-0471), and the Deutsche Forschungsgemeinschaft (Sfb 174; C6).
H. Nau I Toxicology Letters 82183 (1995) 975-979
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