Georg Tzimas

Identification of 9-cis-retinoic acid, 9,13-di-cis-retinoic acid, and 14-hydroxy-4,14-retro-retinol in human plasma after liver consumption

Vitamin A is a well-established teratogen in several animal species. Case reports as well as a recent epidemiological study suggest that vitamin A intake in excess of 25,000 or 10,000 IU respectively, can result in retinoid-specific defects in the offspring. A single meal of liver contains, on the average, a 10- to 20-fold higher amount of vitamin A than what is already suspected to be teratogenic. To evaluate the risk of liver consumption during pregnancy, we have studied levels of vitamin A and a number of potentially active retinoid metabolites in plasma of ten healthy male volunteers following consumption of fried turkey liver (2 g raw weight/kg body weight). HPLC, UV spectroscopy and mass spectrometry were used for identification and quantitation of retinoids in plasma. As shown previously, vitamin A intake via liver consumption resulted in greatly increased plasma levels of 13-cis-retinoic acid (13-cis-RA) and 13-cis-4-oxo-RA, and low levels of all-trans-RA and all-trans-4-oxo-RA. In our present investigation 9-cis-RA, 9,13-di-cis-RA, and 14-hydroxy-4,14-retro-retinol (14-HRR) were identified for the first time in humans as physiological metabolites of vitamin A. 9-cis-RA is a potent teratogen as well as a high affinity ligand of retinoid receptors, and 14-HRR was previously shown to promote lymphocyte activation in vitro. The present study bears on the issue of a possible teratogenic risk of liver consumption, as active retinoids were identified in human plasma, and their levels could be related to previous human studies as well as to experimental studies in sensitive animal species.

The role of metabolism and toxicokinetics in retinoid teratogenesis.

Retinoids (vitamin A and its analogs) exert profound effects on a wide variety of life processes, including morphogenesis and embryonic development. Several retinoids are also effective drugs for therapy of skin diseases and some types of cancer. However, the applicability of this class of compounds is limited by their teratogenic activity. A major question in retinoid toxicology has been the marked interspecies differences in the lowest teratogenic doses of 13-cis-retinoic acid and retinol. In addition, great attention has been drawn to the risk assessment of embryotoxicity resulting from excessive intake of vitamin A by pregnant women. The present review first gives an overview of the biochemistry, metabolism and mode of action of retinoids as well as their role in embryonic development. It then summarizes the results of recent studies on retinoid metabolism, toxicokinetics, and embryonic exposure and discusses how the available information provides explanation of the aforementioned interspecies variations. Finally, it presents some approaches for risk assessment of high vitamin A intake by humans based on various animal models and epidemiological studies.

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

Previous studies suggested that the rabbit is much more susceptible to the teratogenic action of 13-cis-retinoic acid (13-cis-RA) than the mouse or the rat, while the teratogenicity of all-trans-RA was comparable in these species. In the present study we investigated if pharmacokinetics can explain these species- and structure-related differences. The embryotoxic and teratogenic potential of all-trans-retinoid acid (all-trans-RA) and 13-cis-RA were evaluated in the Swiss hare rabbit after oral administration of daily doses of the two drugs throughout organogenesis, from gestation day (GD) 6 to 18 (plug day=GD 0). All-trans-RA was given at dose levels of 0.7, 2 or 6 mg/kg body weight per day and 13-cis-RA at 3, 7.5 or 10 mg/kg per day. The doses needed to elicit a minimum teratogenic response were found to be 6 mg/kg per day for all-trans-RA and 10 mg/kg per day for 13-cis-RA. Using these doses, transplacental pharmacokinetics of all-trans- and 13-cis-RA were performed. Pregnant rabbits were treated once daily from GD 7 to 12 and plasma and embryo samples were collected for HPLC analysis at various time intervals after the final dose. The main plasma metabolites of all-trans-and 13-cis-RA were all-trans-β-glucuronide (all-trans-RAG) and 13-cis-4-oxo-RA, respectively. The elimination of 13-cis-RA and its metabolites from maternal plasma were much slower than of all-trans-RA resulting in accumulation of the 13-cis-isomers in plasma. Marked differences in the placental transfer of the two drugs and their metabolites were observed. All-trans-RA and all-trans-4-oxo-RA were efficiently transferred to the rabbit embryo, reaching concentrations similar to the plasma levels. On the contrary, the 13-cis-isomers reached the embryo to a lesser extent. Despite its limited placental transfer, a considerable embryonic exposure to 13-cis-RA and 13-cis-4-oxo-RA was noticed after treatment with isotretinoin, as indicated by their area-under-the-concentration-time-curve (AUC) values in the embryo, which were in the same range as the corresponding AUC value of all-trans-RA after treatment with the all-trans-isomer. Our results suggest that the high sensitivity of the rabbit to the teratogenic effects of 13-cis-RA can be attributed mainly to the 13-cis-isomers and not to isomerization to all-trans-RA. The significant exposure of the rabbit embryo to 13-cis-RA and its 4-oxo metabolite is a result of their very slow excretion rates from the maternal organism. Furthermore, this study supports the view that embryonic AUC values should be considered as the most suitable pharmacokinetic correlate to retinoid induced teratogenesis.

ANTIEPILEPTIC DRUGS ALTER ENDOGENOUS RETINOID CONCENTRATIONS : A POSSIBLE MECHANISM OF TERATOGENESIS OF ANTICONVULSANT THERAPY

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 therefore studied if the teratogenic action of antiepileptic agents could be mediated via alteration of the endogenous vitamin A--retinoid metabolism. Retinol and its oxidative metabolites all-trans-, 13-cis- and 13-cis-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. 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. The plasma levels of the oxidative metabolites 13-cis- and 13-cis-4-oxo-retinoic acids were strongly decreased in all patient groups treated with phenytoin, phenobarbital, carbamazepine and ethosuximide, in combination with valproic acid, to levels which were below 1/3rd and 1/10th of corresponding control values, respectively. Little changes were 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 profound effect on the endogenous retinoid metabolism. Because of the importance of retinoids for the signaling of crucial biological events during embryonic development, such altered retinoid metabolism may be highly significant in regard to antiepileptic drug teratogenesis.

Identification of 14-hydroxy-4,14-retro-retinol as an in vivo metabolite of vitamin A

Retinol (vitamin A alcohol) undergoes extensive metabolism in vertebrates. We report here (i) the identification of a yet undescribed in vivo metabolite of retinol as 14-hydroxy-4,14-retro-retinol in pregnant mice, rats and rabbits following dosing with vitamin A, and (ii) the preferential accumulation of 14-hydroxy-4,14-retro-retinol in maternal and embryonic tissues, rather than in material plasma.

9-Cis-retinoyl-β-glucuronide is a major metabolite of 9-Cis-retinoic acid

Abstract The in vivo metabolism of 9-cis-retinoic acid (9-c-RA), an endogenous ligand of retinoid X receptors (RXRs), which can also bind to retinoic acid receptors (RARs), was examined in pregnant mice and rats following a single oral dose of 100 mg 9-cis- retinaldehyde (9-c-RAL) / kg body weight. 9-Cis-retinoyl-β-glucuronide (9-c-RAG), a metabolite not found in vivo before, was a major metabolite of 9-c-RA in mouse plasma and was also present in all mouse tissues examined as well as in rat plasma. In both species putative oxidation products of retinoic acids and high levels of retinyl esters were found. Concentrations of retinoic acid isomers and retinoyl-β-D-glucuronides in the mouse plasma greatly exceeded those of the rat plasma. The finding of high levels of 9-c-RAG underlines the importance of glucuronidation in the metabolism of retinoids.

Retinoid teratogenicity in the macaque: Verification of dosing regimen

Abstract: To further define teratogenicity associated with 13‐cis‐retinoic acid (13‐cis‐RA) in the cynomolgus monkey, the drug was orally administered on three different treatment regimens. Experiment (Exp.) 1 (2.5 mg/kg/day, gestational day [GD] 12–27, n = 11) investigated the teratogenicity of a single daily dose of 13‐cis‐RA administered shortly after embryo implantation. Pharmacokinetic sampling was done to determine retinoid profiles on the first (GD12) and last (GD27) days of treatment. Exposure to 13‐cis‐RA during early organogenesis in Exp. 2 (2.5 mg/kg/day, GD20–27, and 2 × 2.5 mg/kg/day, GD28–30, n = 5) investigated the potential adverse effects of 13‐cis‐RA on the developing limb. The use of multiple doses of 13‐cis‐RA in Exp. 3 (2 × 2.5 mg/kg/day, GD26–27, n = 5) investigated the necessity of double dosing on the induction of retinoid embryopathy in the macaque. Malformations of retinoid target organs as well as embryolethality were most prevalent when single daily doses of 13‐cis‐RA were administered during pre‐ and early organogenesis in Exp. 1. Moreover, multiple doses on GD26–27 failed to induce any manifestation of abnormal development in Exp. 3. These results confirm that the lowest observed adverse effect level (LOAEL) in macaques is 2.5 rather than 5.0 times greater than that observed in human pregnancies. Exposure during forelimb development (GD20–30) in Exp. 2 was unsuccessful in inducing defects of this skeletal region, although defects in several retinoid target organs (i.e., cerebellum and internal ear) were present, indicating that a teratogenic threshold was achieved. Pharmacokinetic analysis of 13‐cis‐RA and its metabolites on GD12 and 27 in Exp. 1 showed considerable exposure to the administered drug and its 4‐oxo‐metabolite. In contrast, the exposure to all‐trans‐RA was negligible. The results support the use of a specific treatment schedule in early gestation in the macaque as the most appropriate model for characterizing the teratogenic potential of retinoids in humans.

Retinoid metabolism and transplacental pharmacokinetics in the cynomolgus monkey following a nonteratogenic dosing regimen with all-trans-retinoic acid

Retinoids often exhibit a complex metabolic pattern and differential transplacental kinetics, which make it difficult to pinpoint the proximate compound responsible for the observed teratogenic effect. We have therefore studied the pharmacokinetics and metabolism of all-trans-retinoic acid (all-trans-RA) in cynomolgus monkeys following application of a nonteratogenic dosing regimen and compared the results with corresponding data from a previous study with a teratogenic dosing regimen with 13-cis-RA [Hummler et al. (1994) Teratology 50:184-193]. All-trans-RA was administered to pregnant cynomolgus monkeys (Macaca fascicularis) by nasogastric intubation at a dose of 5 mg/kg body wt once daily from gestational day (GD) 16 to 26 and twice daily at 8-h intervals from GD 27 to 31. Examination of the fetuses of four dams on GD 100 +/- 2 showed no embryotoxic or teratogenic effects of the applied dosing regimen (Experiment 1). Maternal plasma retinoid pharmacokinetics on GD 16, 26, and 31 as well as embryonic retinoid profiles after the last drug administration on GD 31 were determined in thirteen further dams (Experiment 2). All-trans-RA reached much lower plasma concentrations after the last two treatments on GD 31 than after the first one on GD 16 and the eleventh one on GD 26 (0-24-h area-under-the-concentration-time-curve (AUC) values: 104 +/- 59 ng x h/ml (after the last treatment on GD 31), 189 +/- 110 (GD 16) and 393 +/- 305 ng x h/ml (GD 26). The predominant plasma metabolites of all-trans-RA were its beta-glucuronide and the beta-glucuronide of all-trans-4-oxo-RA. Both of these retinoids accumulated in the plasma during the period of treatment and displayed AUC values 5- to 30-fold higher than those of all-trans-RA. Embryonic concentrations of all-trans-RA were not increased over endogenous levels after the last administration on GD 31 when plasma concentrations were low. To evaluate the placental transport of all-trans-RA in the presence of high plasma concentrations, a further experiment was performed, in which a single dose of all-trans-RA (10 mg/kg body wt) was given to four pregnant monkeys on GD 31, and plasma pharmacokinetics as well as embryonic concentrations of retinoids at 4 h post-treatment were determined (Experiment 3). This dosing schedule yielded high plasma concentrations of all-trans-RA, while embryonic concentrations were about 40% of plasma levels. Based on the plasma AUC values on GDs 16 and 26 obtained in Experiment 2 and the degree of placental transfer, as determined on GD 31 in the presence of high plasma levels in Experiment 3, we estimated embryonic AUC values for the 24-h period following the nonteratogenic doses on GDs 16 and 26 in Experiment 2. These AUC values were similarly high to the embryonic AUC value of all-trans-RA obtained after application of the teratogenic dosing regimen with 13-cis-RA [Hummler et al. (1994) Teratology 50:184-193]. In addition, plasma AUC values of all-trans-RA were 2- to 7-fold higher after all-trans-RA administration (present study) than after dosing with the teratogenic dose of 13-cis-RA. These results strengthen our recent suggestion that the teratogenic effects induced in cynomolgus monkeys by 13-cis-RA treatment cannot solely result from the action of all-trans-RA, but may involve 13-cis-RA and 13-cis-4-oxo-RA, which could act directly or function as transport vehicle.