The dose-response of the teratogenic potency of the thalidomide (Thd) derivative EM12 was evaluated in the common marmoset (Callithrix jacchus). The smallest daily dose found to be effective was 30 micrograms EM12/kg body wt. This is the lowest dose of a Thd derivative ever reported to induce severe skeletal abnormalities. Ten micrograms EM12/kg body wt may be considered the no-observed-adverse-effect-level (NOAEL) under the experimental conditions chosen. The teratogenic potencies of the two EM12 enantiomers were tested at 100 micrograms/kg body wt, the dose which just induces an almost 100% effect in the case of the racemate. The S(-)-EM12 was found to induce typical severe limb abnormalities such as amelia, phocomelia, and radius aplasia, and none of the exposed fetuses were devoid of skeletal defects. In contrast, only few and minor skeletal defects were observed after application of the R(+) enantiomer. Although a pronounced teratogenic potency of the R(+)-EM12 can now largely be excluded, these low-dose studies are not sufficient to completely rule out any teratogenic potential of this enantiomer, since racemisation to small amounts of the S(-) form may occur in vivo. Further studies with Thd derivatives which are unable to racemise are necessary to prove the assumed complete ineffectiveness of the R(+) enantiomers.
The plasma pharmacokinetics of the enantiomers of 2-(2,6-dioxopiperidine-3-yl)-phthalimidine (EM 12) and the racemic mixture of this substance were investigated in Callithrix jacchus, a thalidomide-sensitive primate. Single doses of 5 mg/kg body wt were administered orally or intraperitoneally. Maximum plasma concentrations were reached 1 h after administration of the enantiomers, and 3 h after application of the racemate. The mean plasma elimination half-life was in the range of 5 h for the enantiomers, as well as for the racemic mixture, although there was a tendency toward slower elimination and higher plasma AUC values of the S-enantiomer: thus, after administration of the (greater than 99%) pure enantiomers, the plasma AUC value of the administered S-enantiomer was found to be more than one-third higher than that of the administered R-enantiomer. Racemisation of the R- and the S-form of EM 12 occurred both in vitro (phosphate buffer, pH 7.4, 37 degrees C) and in vivo. The maximum plasma concentrations of the antipodes produced via chiral inversion were between 13% and 21%; the plasma AUC values of the resulting antipodes were between 24% and 30% of the corresponding values of total EM 12. The plasma pharmacokinetic data, including the extent of the chiral inversion obtained after p.o. and i.p. application of the substances, were in the same range. The results indicate that both enantiomers racemise with appreciable rates; this may be expected to complicate the interpretation of studies designed to evaluate stereoselective differences with respect to teratological activities of EM 12 and related substances such as thalidomide.
Thalidomide administration during early gestation results in specific and dramatic limb defects in primates, but not in laboratory rodents such as the rat and mouse. The thalidomide analogue EM12 [2-(2,6-dioxopiperidine-3-yl)-phthalimidine] was used in the present study because this compound is metabolically more stable and teratogenically more potent than thalidomide in the monkey. We have administered the pure enantiomers, since we have previously shown that S-EM12 proved to be much more teratogenic in the monkey than R-EM12. In maternal plasma, placenta and embryo of the pregnant marmoset monkey (Callithrix jacchus) and Wistar rat, the concentrations were investigated of the enantiomers and their metabolites after administration of R- and S-EM12. With whole body autoradiography the distribution in the embryo, including the target tissue, the embryonic limb bud was examined in the NMRI mouse and marmoset monkey. Our investigations showed that both the R- and the S-enantiomers were transferred to the embryo during organogenesis [monkey, gestation day (GD) 61; rat, GD 12; mouse, GD 10]. The gestation period chosen was toward the end of the thalidomide-sensitive stage, but yielded sufficient gestational material for analysis. Considerable amounts of the enantiomers were produced via racemization of the administered pure enantiomers and were present in maternal plasma as well as in placenta and embryo. In the monkey, the racemization were stereoselective: the S-enantiomer was eliminated more slowly in the monkey than the R-enantiomer, possibly because of stereospecific binding and metabolism. In the plasma and embryo of both rat and monkey, the metabolites were detected in considerably lower concentrations than EM12, emphasizing the importance of the parent drug in regard to the teratogenic effect. The whole-body autoradiography in marmoset and mouse showed high radioactivity in the embryonic CNS, the branchial apparatus and in the limb buds. The S-enantiomer of EM12 was more strongly concentrated than the R-enantiomer in these areas. In the limb buds, the highest concentrations of radioactivity were observed in the periphery, sometimes at the very tip of the buds. Accumulation of radioactivity in limb buds and neural epithelium relative to other areas of the embryo was much more pronounced in the monkey than in the mouse. Future studies must demonstrate if this accumulation has implications for the mechanism of thalidomide teratogenesis in primate species.
The teratogenic potencies of the enantiomers of 2-(2,6-dioxopiperidine-3-yl)-phthalimidine ( = EM 12), a teratogenic thalidomide analogue, were investigated in Callithrix jacchus, a primate very sensitive to the teratogenic action of this thalidomide analogue. The results indicate that the S-(-)-form of EM 12 is clearly more teratogenic than the R-(+)-form. The interpretation of the studies designed to evaluate stereo-selective differences in the teratogenicity of the enantiomers becomes difficult, since both enantiomers racemise in vivo with appreciable rates (Schmahl et al. 1988a, b). Therefore, it cannot be concluded as yet that the R-(+)-form lacks all teratogenic potential.
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