Transplacental pharmacokinetics of teratogenic doses of etretinate and other aromatic retinoids in mice

Reprodnctlve 7'oxico(,'gy , Vol. 2. pp. 19- 29. 1988 Printed in tbe U.S.A .

Cop yrighl

0890·52381B8 B .OO + .00 © 1988 Pergamon Press pIc

TRANSPLACENTAL PHARMACOKINETICS OF TERATOGENIC DOSES OF ETRETINATE AND OTHER AROMATIC RETINOIDS IN MICE REINERS, B. LOFBERG, J. CREECH KRAFT, D. M. KOCHHAR, * and HEINZ NAU Institute of Toxicology and Embryopharmacology, Free University Berlin, Berlin, F .R.G. and *Department of Anatomy, Jefferson Medical College, Philadelphia, Pennsylvania, USA

J.

Abstract - The transplacental pharmacokinetics of single teratogenic doses of etretinate and motretinide were compared with particular emphasis on distribution and concentrations in the exposed embryos of the free acid metabolite, etretln, The three aromatic retinoids were also tested for their direct inhibitory effect on chondrogenesis in the limb bud mesenchymal cell "micromass" culture assay. After a standard dose of 100 mg/kg administered on day 11 of gestation in NMRI mice, all three compounds were teratogenic, but they differed from each other in potency. Etretinate was most active as a teratogen, equalling the potency of our standard all-transretinoic acid; every exposed fetus was deformed with severe shortening of all limb bones as well as cleft palate. Etretin was less potent than etretinate, and motretinide was considerably less active as a teratogen than the other two. In the in vitro assay, only etretin suppressed chondrogenesis and this activity was equivalent to that of all-tram-retinoic acid (IC50 of 12 ng/IIII). Both etretinate and motretinide (which contain an ethyl ester and ethylamide terminal group, respectively) were essentially inactive in vitro, demonstrating the fact that a free carboxylic group may be a requirement for the in vitro suppression of chondrogenesis. These differences between the results obtained in vivo and in vitro could be resolved by pharmacokinetic investigations using HPLC methods. Both etretinate and motretinide were metabolized in vivo to etretin , their likely common teratogenic metabolite. The high teratogenic potency of etretinate was probably the result of high concentrations as well as AUe values of its metabolite etretin in the embryo. On the other hand, the comparatively low teratogenicity of motretinide could be related to approximately 5 x lower embryonic peak levels as well as AUC values of etretin. A comparison of these results with those previously obtained for all-tralls- and 13-cis-retinoic acids confirms the correlation between embryonic exposure and teratogenic potency in the mouse. Our results indicate that pharmacokinetic studies are essential for the interpretation of relative teratogenic potencies of retinoids as well as apparent differences between in vivo and in vitro teratogenesis. A free carboxyl group at the terminal end of the tetraene chain was necessary for high activity of the retinoids studied. Key Words: Etretinate , Etretin , Motretinide, Retinoid teratogenes is. In vivo/in vitro comparison, Mouse teratogenicity. Transplacental pharm acokinetics, Embryonic concentrations , Limb bud mesenchymal spot culture.

INTRODUCTION

Since several other synthetic retinoids appear to be promising candidates for development into therapeutic drugs, their teratogenic properties will have to be carefully monitored. Correlation of teratogenesis with chemical structure and with other features such as pharmacokinetic parameters, tissue distribution, placental transfer, and metabolism may be essential to consider in the development of safer compounds. In this paper we report correlations between the teratogenic effects of the closely related aromatic retinoids, etre tinate and motret inide, and their common metabolite etretin with certain pharmacokinetic parameters as well as with their direct in vitro effects. Exposure to the aromatic retinoid etretinate has been related up to now to seven human birth defect outcomes (4,5): three cases with meningomyeloceles, three with craniofacial and skeletal abnormalities including limb reduction defects, and

Retinoids (vitamin A and its structural congeners) are widely used for the treatment of dermatologic disorders and are currently being tested as prophylactic and therapeutic agents against cancer (1,2). The fact that retinoids possess teratogenic properties has long been known from animal studies (6,7, see review in Geelen , 31 ) and it is also apparent from recent clinical literature on isotretinoin (13-cis-retinoic acid or Accutane) and etretinate (Tigason) that both are teratogenic in humans (3- 5) .

Addres s correspondence to : Hei nz Nau, Institute of Toxicology and Embryopharmacology, Free University Berlin . Garystrade 5, D-lOOO Berlin 33, F.R.G. Address correspondence in North America to: Dr. D. M. Kochhar, Department of Anatomy, Jefferson Medical College, 1020 Locust Street, Philadelphia, PA 19107. Received 13 January 1988 ; A ccepted 13 March 1988.

19

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Reproductive Toxicology

Volume 2. Number I, 1988

~COOC2H5

ETRETINATE (TIGASON)

CH30

~COOH

ETRETIN

CH30

~CONHC2H5

MOTRETINIDE

CH30

Scheme I. Chemical structure of the aromatic retinoids used in the study.

discontinuation of etretinate therapy (up to two years) has therefore been advocated. Also, the main metabolite of etretinate, etretin, was recently shown to be teratogenic in the mouse, rat and rabbit with lowest teratogenic doses of 0.6, 3, and 15 mg/kg/day, respectively (14). Etretin is the free acid analogue of the ethylester etretinate (Scheme I) and was reported to have a terminal half-life of only two days, but exerted therapeutic effects similar to etretinate (15). Thus, etretin may have some advantages over etretinate for clinical use because concentrations of this free acid apparently decrease rapidly after discontinuation of therapy, which implies a concomitant rapid decrease of the teratogenic risk. Motretinide (Tasmaderm), the ethyl amide analogue of etretinate (Scheme I), is used as topical formulation on the skin (2). It is not known if significant plasma concentrations result from such use.

one with a severe brain defect with anopthalmia. Etretinate was also shown to be teratogenic in the mouse, rat, hamster, rabbit and sub-human primates (7-9) with daily doses (2-5 rug/kg/day) which were approximately 2-10 times higher than the usual therapeutic doses (0.5-1 mg/kg/day); the lowest teratogenic dose in the human (0.2 mg/kg/day) (4) was apparently 10-20 times below the teratogenic doses in the experimental animals. Therapeutic plasma concentrations of etretinate in patients are between 100-500 ng/ml; following cessation of therapy, extremely long terminal elimination half-lives of etretinate (100 days) were observed because of extensive drug storage in deep tissue compartments, particularly in adipose tissue (10-12). Indeed, one pregnancy four months after the last dose of etretinate may have resulted in severe skeletal malformations of the fetus (13). Effective contraception for an extended time period after

Table 1. Comparative teratogenic effects of the three aromatic retinoids and tretinoin in NMRI mice. Single dose (l00 mg/kg) given orally on day 11 (plug day = day 0) of gestation. Fetuses examined on day 18 Vehicle control No. litters treated No. abnormal litters" % abnormal litters No. implantation sites % sites absorbed No. fetuses examined % abnormal fetuses" No. cleft palate No. examIned % cleft palate No. limb defects No. examined % limb defects Average fetal body weight

±SD

9

a a 112 7

104

a

Tretinoin

Etretin

Motretinid

Etretinate

6 6

8 8

8

8 8

100 84

100 102 4

100 88

98 100

78 69

10 76

8

II

100 85 34b 56

49 49

26

39

100 28 28

100 46"

67

100

49 96

39

28

21

100

1.25

100 38 38 100 38 38 100 1.31

1.10

J.l5

1.16

±0.13

±0.15

±O.JO

±0.J1

±O.II

a 52 a a 52 a

8<1

3An abnormal litter contained one or more malformed fetuses. "p < 0.05 compared to vehicle control. COnly 23 out of 46 fetuses had phocomelia; remaining fetuses showed only mild reduction defects. dAII 8 fetuses had mild reduction defects.

28

Aromatic ret inoids • J . REINERS ET AL.

21

Table 2. Relative activities of several retinoids in a mouse limb bud mesenchymal cell assay

Retinoid All-trans-retinoi c acid l3..d s· re t inoic acid 4..oxo-B..c is-retinoic acid Etretinate Etretin Motre tinide

Concentration (M) required for 50% suppression of chondrogenesis" 5 X IO- H (15 ng/ml) 6 x 10- H (18 ng/ml) 6 x 10-H (18 ng/ml)

no suppression at 1.7 x 1O-r. (500 ng/ml) 4 x 10-H (12 ng/ml)

no suppression at 1.7 x 10-· (500 ng/ml)

"Number of cartilage nodules per spot.

Its teratogenic effects have not yet been reported . Published material on the pharmacokinetics of aromatic retinoids in experimental animals is not available up to now. We have therefore studied the maternal plasma and embryonic concentrations of etretinate, etretin, and motretinide as well as their metabolism in the pregnant mouse in order to relate the teratogenic potencies of these retinoids to the relative embryonic exposures. Furthermore, the activities of these compounds were studied in an in vitro system. This in vivo/in vitro comparison allowed us to differentiate if a particular structure exhibits intrinsic activity, or if it must be activated to a teratogenic metabolite. Possible inferences and extrapolations to the human therapeutic situation are also discussed, particularly in regard to differences between etretinate and etretin. MATERIALS AND METHODS Teratological studies in vivo NMRI mice were housed in environmentally controlled rooms under spf-conditions and a 12 h light-dark cycle. They were fed an Altromin diet (AItrogge, FRG) and were given water ad libitum . Female mice were mated from 6-8 a.m. The following 24 h were designated as day 0 if vaginal plugs were detected. Drug solutions were freshly prepared by dissolving 100 mg of the retinoids in 0.4 ml ethanol followed by 4.6 ml soybean oil. Single oral doses (100 mg/kg, 5 rnl/kg) of each of the selected retinoids were administered on the morning of day 11 of gestation, and the reproductive outcome was monitored on the day 18. Upon laparotomy the fetuses were weighed and examined for external malformations. Fifty percent of the fetuses were fixed in 95% ethanol, processed for alizarin staining of the skeleton, and examined under the dissection microscope. The remaining fetuses were fixed in

Bouin's solution and examined by free-hand razor blade sectioning. Control animals either were administered an appropriate volume of the vehicle or were left untreated ; data from the two sets are combined for presentation as no differences between them were encountered. The numbers of animals used and of live fetuses examined are given in Table 1. For the statistical anal ysis of the differences in the incidence of resorptions and malformations between retinoids, the fetus was considered the experimental unit. The groups were compared statistically by a method based on r-tests of arcsin square root transformed percentages (32). Values at the 0.05 probability level were considered significant (7).

Chemicals Etretinate, etretin, and motretinide (these retinoids were in the all-trans configuration) were gifts from Hoffmann-La Roche, Basel, Switzerland. Methanol was HPLC grade; the other chemicals were obtained in the highest available purity from Merck , Darmstadt, FRO. All manipulations involving retinoids such as preparation of solutions , administration to animals , sampling of plasma and embryonic tissue as well as analytical procedures were performed under dim amber light.

Preparation of mouse plasma and embryo samples The blood was drawn from the heart under ether anestheisa, and embryos were removed from the laparotomized animals at selected time intervals after drug application. Between 3-6 animals (depending on the pregnancy rates) were used for each group. The plasma was obtained after centrifugation of heparinized blood for 10 min at 3000 rpm. Plasma and embryos were stored at -80°C.

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Reproductive Toxicology

Volume 2, Number 1, 1988

IJg et reti nate! ml or 9 10

0001 1

after etretinale

4

8

12

24 hours after application

48

72

Fig. 1. Maternal plasma and embryonic concentrations of etretinate following a single oral dose of 100mg/kg on day 11 of gestation in NMRI mice. Values are means :t SD of groups of 3-6 mice.

High-performance liquid chromatography (HPLC) assay Stock solutions of retinoids were prepared by dissolving 10mg of the retinoids in 10 ml methanol. These solutions were kept in the dark at 4°C and were freshly prepared every 3 weeks. Plasma and embryonic tissue homogenates (112, v/v prepared with water and ultrasonication) from control (untreated) animals were spiked with the appropriate amounts of the retinoids and then further diluted with plasma and tissue homogenates to obtain samples containing known amounts of the drugs for the preparation of calibration graphs. Etretinate and etretin were determined by a straight-phase HPLC procedure according to the method of Paravicini and Busslinger (16). The use of a Kratos UV detector made it possible to further increase the sensitivity of the original assay (4 ng/ml detection limit) down to 0.5 ng/ml with a sample size of 0.5 ml. When multiple peaks appeared (cis-trans isomerization), they were added to obtain the total amounts of the substances present. Motretinide and its metabolite etretin were analyzed by a reversed-phase HPLC method with an automated precolumn switching technique developed in our laboratory (Lofbert et a1., to be published). Briefly, an equal volume of isopropanol was added to the plasma samples, and then the mixture was frozen overnight in liquid nitrogen. Embryo samples were treated with two volumes of iso-

propanol, frozen in liquid nitrogen overnight, and then ultrasonified. The supernatant obtained after centrifugation (20 min x 4000 g, 4°C) of the isopropanol mixtures of plasma or embryo were analyzed by a reversed-phase HPLC method using a gradient of methanol from 71% to 100% in 0.04 M ammonium acetate buffer. Limb bud mesenchymal cell culture system In this system, untreated control embryos were aseptically removed from the uteri of ICR mice on the day eleven ofgestation. Forelimbbuds of embryos judged to stage 18 (40 ± 2 somites) (19,22) were processed for preparation of micromass "spot" cultures according to the method of Ahrens et a1. (20), with some modifications. Briefly, whole limb buds were dissociated in trypsin-EDTA solution in calcium-magnesium-free saline G and filtered to remove epithelial fragments and cell clumps. The cells were rinsed in fresh CMRL-1066 medium containing 10 percent fetal calf serum (GIBCO) to which antibiotics and ascorbic acid (150 fLg/ml) had been added. After centrifugation, the cells were resuspended in an appropriate volume of fresh medium to give a cell number of 1 x 107/ml. Twenty ILl aliquots of this cell suspension were plated as spots on 35 mm plastic tissue-culture dishes, five spots per dish, and left for 2 h in a 37°C incubator maintained at 5% CO2 in air. Each dish was then supplied with 2 ml of complete medium. The medium was changed once

Aromatic retinoids • J.

at 48 h. The retinoids were dissolved in absolute ethanol before being added to the medium at 24 h after plating. Three or four concentrations (spaced on a log-scale) of each of the retinoids were investigated (ranging from 3 x 10- 9 to 3 X 10- 5 M or 0.001-10 fLg/ml). The cultures were terminated at 96 h after plating, at which time the medium was removed and the cells were fixed in situ for 1 h in 10% formalin containing 0.5% cetylpyridinium chloride, after which the cultures were rinsed in acidified acetic acid and stained for 1 h in 0.5% alcian blue solution at pH 1.0. After differentiation in 3% acetic acid, they were dehydrated in ethanol and scored for chondrogenesis under the microscope. An absence or reduction in the number of cartilage nodules in stained cultures was taken as a measure of suppression of chondrogenesis by retinoids as reported earlier (21,22). The concentrations of retinoids that produced a 50% reduction of the number of spots were determined (Table 2). RESULTS In vivo teratogenesis These data are summarized in Table 1. The dose 100 mg/kg employed in this study for comparative effects of the three compounds is based on previous experience with our standard all-transretinoic acid (7,23). A single oral dose of the standard compound administered on day 11 of gestation in ICR mice results in a very high incidence of skeletal defects in the offspring without any signs of maternal toxicity or any increase in embryolethality (resorption) over the usual rate. In a preliminary study we found that NMRI mice were also equally sensitive to retinoic acid (tretinoin) where a single oral dose 100 rng/kg given on day 11 resulted in 100% incidence of cleft palate and limb reduction defects in the offspring (Table 1). Using similar parameters of dose and developmental stage, treatment with etretinate resulted in a similarly high incidence of phocomelia and cleft palate in NMRI offspring, with the only difference being that the resorption rate was also increased to 34% compared to only 10% after tretinoin (Table 1). Both etretin and motretinide were also actively teratogenic in NMRI mice, but they differed from etretinate and from each other in the incidence and severity of defects produced in the offspring. Etretin affected 100%of the fetuses with cleft palate, but only 50% with phocomelia, the remaining fetuses escaped with only mild shortening of the limb bones. Motretinide produced cleft palate in two thirds of the fetuses; only 20% had limb defects with

REINERS ET AL.

23

a mild degree of bone shortening. The resorption rate after etretin or motretinide treatment was in the same range as control animals (Table 1). In vitro activities The comparative ability of the three aromatic retinoids to suppress chondrogenesis was assessed in the limb bud mesenchymal cell culture system. The reduction in the number of cartilage nodules in stained cultures was taken as a measure of activity (Table 2). In this system, etretin was the most active substance tested; it was even more active than alltrans- and 13-cis-retinoic acid. Etretin produced a 50% inhibitory effect (Ie-50) at approximately 12 ng/ml. Both etretinate and motretinide were inactive in this system up to concentrations in the very high ng/ml range (Table 2). Transplacental pharmacokinetics Concentrations of the aromatic retinoids and their major metabolites were determined in maternal plasma and embryo at various time intervals after a single oral dose of 100 mg/kg to NMRI mice on day 11. Etretinate was rapidly absorbed and peak plasma levels (about 7.5 fLglml) were reached 1h after application (Figure 1). The initial half-life (during the first day) was about 2.2 h. A much slower elimination phase followed (half-life of about 24 h) and etretinate levels were still detectable three days after drug administration. The embryonic concentrations of etretinate increased more slowly than the plasma levels and reached their maximum at 6 h after administration. The decline in embryonic levels of etretinate (half-life 11 h) was considerably slower than the initial decline of plasma concentrations. Therefore, etretinate concentrations in the embryo exceeded corresponding plasma concentrations several-fold during later time periods. The transplacental kinetics of etretinformed via metabolism of etretinate - are shown in Figure 2. The plasma concentrations of this metabolite increased more slowly than those of the parent drug and reached their peak: between 4 and 8 h after drug application. The metabolite etretin apparently was transferred to the embryo more rapidly than the parent drug, and embryonic concentrations increased in parallel with those of etretinate during the first 4 h after drug application. The decline of both embryonic and maternal plasma concentrations of the metabolite generally mirror the respective patterns of the parent drug. This indicates that the decline of etretin concentrations is dependent on the formation of the metabolite from

24

Volume 2, Number 1, 1988

Reproductive Toxicology

1-'9 etretin after etretlnate

10

Im, erg

3

1 """'"

"

""",-1

.1_ ------------___

embryo

---------------1

0.Q1

plasma

0003 000112468

12

24 hours after application

72

48

Fig. 2. Maternal plasma and embryonic concentrations of etretin following a single oral dose of 100 mg/kg of etretinate on day 11 of gestation in NMRlmice. Values are means ± SD of groups of 3-6 mice.

~g. etretin!ml erg after etretin

10

3

0.3

t

---1--------

0.1

1. ...............

0.03

............

<,

"

...........

............

......................

0.01

"---'1 embryo ..................

0003

or below

plasma 0001 124

8

12

24 hours after application

48

72

Fig, 3. Maternal plasma and embryonic concentrations of etretin following a single oral dose of 100mg/kg of etretin on day J 1 of gestation in NMRI mice. Values are means ± SD of groups of 3-6 mice.

Aromatic retinoids • J.

25

REINERS ET AL.

fJQ motretinide! rnl or 9 after motretinide

, ' .J..-

Q3

1

J._.... L

- ------------~~-----------------------------L

---------------------1 embryo

0.1 plasma

aOO1..L,-,r-r-r-......----r:,-----.-----------,,-----------,..6 8 12 24 72 48 hours after application

Fig. 4. Maternal plasma and embryonic concentrations of motretinide following a single oral dose of 100 mg/kg of motretinide on day 11 of gestation in NMRI mice. Values are means ± SD of groups of 3-6 mice.

the parent drug and not on the elimination of the metabolite . Thus, as with the parent drug, the metabolite could still be detected in maternal plasma and embryo three days after drug application. Also, the embryonic concentrations of both etretinate and etretin were lower than the corresponding maternal plasma levels during the absorption phase, but exceeded the maternal plasma levels during most of the elimination phases. This implies that the embryo constitutes a deep compartment for etretinate. The pharmacokinetics of etretin after its direct administration (Figure 3) is very different from that of etretin as a metabolite of etretinate (Figur e 2). Application of etretin resulted in rapid absorption of this substance, and peak plasma levels were obtained at 1-2 h (about 2 ,uglml). Placental transfer was very rapid and peak embryonic levels were obtained simulta neously with those in maternal plasma. After 6 h, embryonic concentrations declined in parallel with maternal plasma levels . Although embryonic concentrations exceeded maternal plasma levels (Figure 3), this difference was much smaller than after etretinate application (Fig. 3). This implies that the embryo may not constitute a major deep compartment for etretin after direct administration of this substance. The maximal concentrations of motretinide in

plasma were reached 2 h after application (Figure 4) and then decreased with an initial half-life of about 10 h and a terminal half-life of about 30 h. The embryonic concen trations of motretinide reached maximal levels 4 h after drug application and then declined much more slowly than the plasma levels, with initial and terminal half-lives of 15 and 50 h , respectively. This indicates that the embryo constitutes a deep compartment in regard to motretinide pharmacokinetics. Etretin was detected as a metabolite of motretinide (Figure 5) and , just as is the case after application of etretinate or etretin itself, the concentration s of this metabolite in the plasma decreased in parallel with those in the embryo. Most importantl y, however, the levels of etretin after rnotretinide application (Figure 5) were much lower than after application of etre tin itself (Figure 3) and particularly after etretinate appli cation (Figure 2). Apparently, enzymatic hydrolysis of the esteretretinate was much more extensive than ofthe amide motretinide. DISCUSSION

The most important findings of our study are that the relative teratogenic potencies of the retinoids studied in vivo can be related to the transplacental pharmacokinetics and metabolism of

Reproductive Toxicology

26

Volume 2, Number 1, 1988

~g etretin Iml or g

10

after molrellnidEI

)._--------..... ---------------...-----------------------------1---

------------------_~!:'~10 plasma

0001 124

12

24 hours after application

48

7

Fig. 5. Maternal plasma and embryonic concentrations of etretin following a single oral dose of 100 rng/kg of motret inide

on day 11 of gestation in NMRI mice. Values are means ± SD of groups of 4-6 mice.

these substances. during organogenesis. Furthermore, the drastic differences between results obtained in vivo and those obtained in vitro can tentatively be explained by metabolic activation of etretinate and motretinide to a common teratogenic metabolite (etretin) in the intact organism and the apparent absence or low activity of this metabolic reaction in the in vitro system used. Previous studies have shown that aromatic retinoids produced a broad spectrum of congenital defects in mice involving craniofacial, cardiovascular, central nervous system, and skeletal structures (6-8). This wide range was considerably narrowed if single doses were administered during specific gestational stages. A single dose applied orally on day 11 resulted predominantly in cleft palate and limb reduction defects with minimal effects on fetal weight, resorption rates, and maternal toxicity (Table 1). We have selected a single high oral dose of 100 mg/kg to allow a comparison with the results of a previous study involving all-trans retinoic acid and 13-eis-retinoic acid (17). Etretinate was the most potent teratogen, and every fetus developed cleft palate and serious reduction defects of the limbs (Table 1). Etretin produced a similar frequency of defects in NMRI mice, but the limb defects were less severely expressed as compared to the treatment

with etretinate. A previous study indicates that etretin was of considerably lower teratogenicity than etretinate in the less sensitive mouse strain ICR (23). Motretinide was less potent than the other two aromatic retinoids in NMRI mice. Surprisingly, both etretinate and motretinide were essentially inactive in vitro using a limb bud mesenchymal cell assay. Etretin was very active in this system with an IC-50 in the low ng/rnl range which was slightly below the range of Ie-50 values obtained for all-trans-retinoic acid and 13-cisretinoic acid used for comparison (Table 2). These results suggest (1) that both etretinate and motretinide must be activated in vivo to teratogenic metabolites: (2) that the lack of activity of etretinate and motretinide in vitro result from a lack of metabolic activity by the limb bud cells used; and (3) that etretin is teratogenic by itself, presumably because its structure contains a free carboxyl group at the carbon-I5. The other two aromatic retinoids contain an ester or amide function instead. Previous results obtained in vivo also point to the crucial importance of a polar group with an acidic pKa at the terminal end of the tetraene chain in regard to retinoid teratogenesis: Willhite and colleagues (24-26)have shown that the teratogenicity of a wide range of retinoids in the hamster depends

Aromatic ret inoids • J. REINERS ET AL. A UG 40 1-19/ 9

27

(pg!g

e mb ry ox h )

embryo (m aximal concentrations )

4 30

3

/

··•• ·•

n ~i~!i metabolite etretin

20

f

I A

o

........ .......... ·......... ·:::... .. ::

·

"

80

·

"

60 40

~:

;; ::

TI

.. ".. ..

....is :: ..:i:: ·.::it

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C

D

t~ ~: ~

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A

:

·...

"

~

ii

BCD

If

i:

If'~.. E

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: E

(appli ed SU bstance) (b)

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i

10

(appli ed su bstance) (a) 0

!

I

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n : •

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·.:·:: : : :· : : ··:•

~:

iiE

(appli ed su bst ance) (c)

on the presence of a free carboxyl group or a moiety with an equivalent pKa at carbon-IS at the terminal end of the polyene chain . Also, in vitro studies by Kistler (27) showed that the addition of hydrolytic enzymes to the limb bud assay used increased the activity of etretinate ; the nature of the metabolic activation reaction has not been demonstrated. Also, Bechter and Taccard (28) have argued that the teratogenic effects elicited by etretinate in whole embyros in culture may have been the result of metabolism to etretin taking place in this system. It has been suggested that an acidic terminal group on the polyene

Fig. 6. Em bryonic peak concentrations (a), embryonic areas under the concentration-time curves (A Ue) (b) and rates of limb reduction defects in fetuses (c). Single doses of 100 rng/kg of etretin ate (E), etretin (D) and motretinide (C) were administered on day 11 of gestati on in NMRI mice and compared with corresponding data previously obtained for all-zmns-retinoic acid (B) and 13-cis-retinoic acid (A) (17,23) .

chain may be necessary for binding to a putative cytosolic receptor protein as a prerequi site for retinoid activity (24). We now demonstrate that the differences between the in vivo teratogenic activities of the three aromatic retinoids studied are most likely due to differences in metabolism and placental transfer. Since, according to the in vitro results discussed above, etretinate and motretinide do not possess appreciable intrinsic teratogenic activities, it is highly likely that the exposure to the common metabolite etretin (Scheme I) and not to the parent drugs must be considered here . Indee d, th e embryonic exposure to etretin in regard to both embryonic peak concentrations and area under concentration-time curve (AVe) - was highest after etretinate administration, lower after application of etretin itself, and lowest after motretinide application, thus mirroring the pattern of teratogenic potencies of the three aromatic retinoids studied (Figure 6). The relationship between embryonic exposure and teratogenicity becomes particularly apparent if our previous results with all-trans -retinoic acid and 13-cis-retinoic acid are

28

Reproductive Toxicology

included (17). In this comparison, 13-cis-retinoic acid application showed the lowest embryonic peak concentrations, the lower Ave values, and also the lowest teratogenicity of all five retinoids studied up to now; etretinate and all-trans-retinoic acid application yielded high embryonic exposure levels and teratogenicity; motretinide and etretin application yielded medium values. We cannot decide at this time if embryonic peak concentrations or the AVe values or both correspond to the teratogenic effects. Preliminary evidence of ongoing studies suggest that steady-state concentrations of etretinate and etretin maintained via infusion throughout the organogenesis period in mice are highly teratogenic, thus pointing to the crucial importance of AVe values in this regard (29). It is also noteworthy that the elimination of the retinoids after etretinate application was much slower than after etretin application, which is similar to the human situation; Whether these findings point to deep compartments for etretinate in the mouse, which can be demonstrated even after a single drug administration, must be further investigated. It must also be studied whether the relatively low peak concentrations of etretin after the administration of this substance - in comparison to those after etretinate application - are the result of considerable first-pass elimination of etretin in the mouse. ~ This work was supported by the Deutsch Forschungsgemeinschaft by a grant to the Sonderforschungsbereich 174(C-6), and by a National Institute of Health grant to D. M. Kochhar (HD-20925). Expert technical assistance was provided by Th. Sparenberg, John Penner, and Lisa Minutella.The secretarial assistance of Renate Ebbinghaus is gratefully acknowledged.

Acknowledgements

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