Arch Toxicol (1990) 64 :623-638 Archives of

Toxicology �9 Springer-Verlag 1990

Effects of calcium channel blockers on the development of early rat postimplantation embryos in culture

G a b r i e l e Ste in , Mi th i l e sh K u n m r S r i v a s t a v a * , H a n s - J o a c h i m M e r k e r , and D i e t h e r N e u b e r t

Institut ftir Toxikologie und Embryopharmakologie, Freie Universidk Berlin, Garystrasse 5, 1000 Berlin 33

Received July 17, 1989/Accepted June 19, 1990

A b s t r a c t . Rat embryos (9.5-day-old) were cultured for 48 h in the presence of nifedipine (NIF), nimodipine (NIM), nitrendipine (NIT), gallopamil HCI (GAL), vera- pamil HC1 (VER) and diltiazem HC1 (DIL). The effects on growth and morphogenetic differentiation in vitro were monitored. Dose-response relationships were evaluated, including an assessment of the "no-observed-effect-level" (NOEL) or the "lowest-observed-effect-level" (LOEL), and the lowest concentration tested inducing abnormalities in 100% of the embryos ("100% EL"). The morphological alterations observed at the highest concentrations were very similar for all six drugs. The abnormalities concerned yolk sac circulation and morphology, as well as heartbeat, the morphology of the heart, head, neural tube, or fore- limbs, and the shape of the embryo. The abnormal embryos were also growth retarded (decrease in protein content and crown-rump length). Interference with calcium channel functions seems to represent an interesting model for studying a special kind of abnormal prenatal development, especially the differentiation of certain mesenchymal structures. The concentration ranges between NOELs and 100% ELs were found to be: NIM = 0.1 - 1 pg/ml; NIT and VER = 1-10 ktg/ml; DIL = 1-30 ktg/ml, and LOELs- 100%ELs were: GAL = 1 - 10 p.g/ml; NIF = 10-30 gg/ml.

K e y w o r d s : Rat whole-embryo culture - In vitro - Cal- cium channel blockers - Nifedipine - Nimodipine - Nitrendipine - Gallopamil - Verapamil - Diltiazem

Abbreviations: Nifedipine = NIF; Nimodipine = NIM; Nitrendipine = NIT; Gallopamil HC1 = GAL; Verapamil HC1 = VER; Diltiazem HCI = DIL 100%EL = lowest dose tested inducing abnormalities in 100% of the embryos

* On leave of absence from: Industrial Toxicology Research Centre, Lucknow, India

Offprint requests to: D. Neubert

I n t r o d u c t i o n

Calcium channel blockers are widely prescribed today for therapeutic purposes and some have been recommended for use during pregnancy for defined indications: e. g. VER for treating fetal paroxysmal tachycardia (Wolff et al. 1980; Lilja et al. 1984; Truccone and Mariona 1985), VER and NIF during tocolytic therapy (Strigl et al. 1980; Read and Wellby 1986), and NIF, NIT and VER for treatment of hypertension (Orlandi et al 1986; Constantine et al. 1987; Laragh 1987; Pipkin and Lawrence 1987). These therapeu- tic recommendations all concern treatment late in preg- nancy after organogenesis has already been completed.

For most of the calcium channel blockers data publish- ed on reproductive and developmental toxicity are scarce, and most of the information is in the form of unpublished reports presented to regulatory agencies. At least two of these agents, NIF and DIL, have been shown in animal experiments to be able to induce teratogenic effects and to increase the incidence of embryolethality (e. g. Ariyuki 1975; Fukunishi et al. 1980; Cabov and Palka 1984; Yo- shida et al. 1988).

We report here on results of comparative studies of various calcium channel blockers using the whole-embryo culture system in which adverse effects on early organo- genesis can be evaluated. The aim of our studies was to:

- evaluate whether these agents exhibit typical adverse effects on embryonic development,

- analyse whether the observed effects are basically the same for all the calcium channel blockers, and

- establish quantitative relationships, allowing a com- parative assessment of the potency of these agents to in- duce abnormal development in vitro.

With the results obtained in vitro it may be possible to correlate these data with effects observed in studies on reproductive toxicity in vivo. The relevance of such in vitro data for a risk assessment in pregnant women exposed therapeutically to these agents and the principle problems connected with such extrapolations will be discussed.

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Materia ls and methods

Animal maintenance

Wistar rats (Bor: Wisw/spf, TNO; Fa. Winkelmann, Borchen, FRG) were kept under spf conditions at a constant day/night cycle (light from 9:00 a. m. to 9:00 p. m.). They received Altromin R 1324 pellet feed and tap water ad libitum. One male was caged with three females for mating (period from 6:00 to 8:00 a. m.); 7:00 a.m. was regarded as hour 0 of embryonic age if sperm were detected in the vaginal smears.

Whole-embryo culture

Rat embryos were cultured for 48 h according to the procedure described by Klug et al. (1985). The embryos (1 to 4 somite stage) were explanted aseptically on day 9.5 of gestation (228-232 h after mating). Three to four embryos were cultivated at 38.8•176 in a 50 ml tightly sealed culture bottle containing 6 ml heat-inactivated and sterile-filtered bovine serum and 1 ml Tyrode's buffer enriched with 525 p.g L-methionine/ml buffer, using a roller system. The gas phase consisted of: 10% 02, 5% CO2, 85% N2 during the first 36 h, and 50% 02, 5% CO2, 45% N2 during the final 12 h of the culture period. No antibiotics were added to the culture medium. Embryos were evaluated using the following criteria: number of somites, crown-rump length, yolk sac diameter, protein con- tent, type and frequency of abnormalities, and a score indicating the development of the various organ anlagen. Furthermore, histological examinations were performed on serial sections. To calculate the score, individual morphologically visible organ anlagen of the embryo such as shape, neural tube, head, eye, ear, heart, forelimb, hindlimb, tail and blood were evaluated. The sum of these values gives the score (a high score indicates a more advanced developmental stage). For statistical analysis a Mann-Whitney test was used to assess significant differences in the variables measured as compared to controls. In addition to the evaluation procedure according to Klug et al. (1985), the yolk sac mor- phology was routinely assessed.

Each concentration was tested in at least two different experimental series performed on different days. Embryos from five to seven dams were used for each tested level.

The water-soluble substances diltiazem HCI (Gi3decke, Freiburg, FRG), verapamil HC1 and gallopamil HC1 (Knoll AG, Ludwigshafen, FRG) were added to the culture medium after dissolution in Tyrode's buffer. Nifedipine, nimodipine and nitrendipine (Bayer AG, Wuppertal, FRG) were dissolved in DMSO, and 5 gl of the solution were added to 7 ml culture medium using a 10 gl Hamilton syringe. The solutions were made up freshly immediately before each experiment.

Care was taken not to expose the light-sensitive compounds (NIF, NIM and NIT) to normal light, therefore all work was performed under yellow light (Osram L40W-62).

Histology

Light microscopy. The embryos were fixed in Bouin's solution and embedded in Paraplast R. Subsequently, 5-10 I-tm thick serial sections were cut and stained with hematoxylin/eosin.

Electron microscopy. The embryo-yolk sac unit was fixed in tannic acid (0.5%)-glutaraldehyde (2%) in phosphate buffer (0.1 M, pH 7.2), embedded in Epon R, sectioned using a Reichert microtome and con- trasted with uranyl acetate/lead citrate. The evaluation was performed using a Zeiss EMI0 transmission electron microscope.

Results

Dose-response relationships

A n ove rv iew o f the results ob ta ined with the wate r - so lub le compounds is g iven in Table 1; the results for the water - in- so luble subs tances are l i s ted in Table 2.

To faci l i ta te compar i son , the drug concent ra t ions are d iv ided into three ca tegor ies (Table 3):

1. the N O E L (no-observed-ef fec t - leve l ) ; concerning all tested var iables o f the e m b r y o c o m p a r e d to controls;

2. the L O E L ( lowes t -observed-ef fec t - leve l ) ; growth (often the most sensi t ive var iable) is a l ready defini tely affected, but all morphogene t i c d i f ferent ia t ions are not or only mi ld ly af fec ted and the f requency o f abnormal i t ies only s l ight ly increased. Usua l ly up to 10% abnormal i t ies are accep ted as "normal" under our exper imen ta l condi- t ions.

3. 100%EL, i .e . the lowest concentra t ion tested at which 100% of the e mbryos show abnormal i t i es and the deve lopmen ta l and growth var iables are severe ly affected.

Macroscopic evaluations

To faci l i ta te an ove rv iew of the resul ts the data are dis- cussed according to the effect ca tegor ies observed.

Concentrations: I O0%EL (morphology of embryos and yolk sacs). The types o f abnormal i t i es obse rved when test- ing all six ca lc ium channel b lockers with the in vitro sys- tem were very s imilar : typica l ly , the y o l k sac c i rculat ion was a lmos t abol i shed , and pa tho log ica l changes o f the heart, head, neural tube, and fo re l imbs were seen or the shape o f the e m b r y o was al tered. Some t imes the hear t beat was no longer detectable . Usua l ly the somites were not affected, showing c lear boundar ies and a regula r arrange- ment (Figs. 1, 2 and 3).

In most yo lk sacs the b ranch- l ike pat tern o f the main vesse ls was not es tab l i shed and no c i rcula t ion was visible. The vessels were swol len to di f ferent extents and fo rmed anas tomoses and c i rcular patterns. The morpho log i ca l ap- pearance o f the yo lk sac surface r e sembled a "go l f bal l" (Fig. 3.1). Add i t iona l ly , some yo lk sacs were f lat tened, but not co l lapsed (Fig. 3.2).

Three different pa tho log ica l p h e n o m e n a could be ob- served for the heart: the heart tube showed an addi t ional bulge in the region o f the prospec t ive left ventr ic le (Fig. 3.7); the heart tube i t se l f was swollen (Figs. 3.5 and 3.6), and the pe r i ca rd ium was swol len (Fig. 2.3 b).

Al l neural tubes were c losed (an open caudal neuro- porus was on ly present in three embryos ) , but their wal ls somet imes showed a wavy appearance . This d id not neces- sar i ly include both sides or the whole length o f the tube (Fig. 3.4).

S o m e af fec ted embryos showed a typ ica l abnormal i ty o f the head: the te lencephal ic ves ic les p ro t ruded on both sides in front o f the former p rosencepha l ic region (Fig. 3.5). Addi t iona l ly , smal l and somewha t i r regular ly shaped heads occurred.

Often size d i f ferences be tween the f ight and left fore- l imb were observed (Fig. 3.3). Only obvious size differ- ences were cons ide red abnormal .

S ince the obse rved abnormal i t i es were rather complex , a survey o f the results is g iven in Tab le 4 which shows the type and f requency of the obse rved c lear -cut abnormal i t ies and other observa t ions unusual for day 11.5 control embryos . The lat ter were regarded as re tardat ions .

Table 1. Calcium channel antagonists: verapamil HCI - gallopamil HCI - diltiazem HCI

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Yolk sac Crown- diameter rump Somites Protein (ram) (mm) (n = ) (gg/E)

Score %Abn Abn/E

4.62 3.87 27 211 38 Control 4.44 3.72 27 194 37 0 37 • 0

4.29 3.54 26 167 35 n = 37 n = 32

Verapami 1- 4.62 3.84 28 218 38 HC1 4.44 3.66 27 196 37 5 18 x 0 1 gg/ml 4.32 3.48 26 159 36 1 x 3 n=19 n=18 n=14

Verapamil- 4.92 3.60 27 163 37 19 • 0 HCI 4.68** 3.51"* 26 135"* 36 14 2 x I 3 gg/ml 4.37 3.18 25 115 34 1 x4 n=22 n=21 n= 16

Verapamil- 4.20 2.46 19 76 24 2 x 1 HCI 4.08** 2.40** 18"* 69** 22** 100 3 x 2 10 ~tg/ml 3.99 2.28 17 61 21 12 x 3 n=21 n= 18 4 x 4

Gallopamil 4.68 3.60 27 157 38 -HCI 4.56 3.44** 26 134"* 36 15 17 x 0 1 ~tg/ml 4.28 3.20 25 125 34 3 x 2 n=20 n= 19 n= 16

Gallopamil 4.88 2.46 19 63 22 2 x 2 -HC1 4.62 2.31"* 16"* 57** 20** 100 9 • 3 10/ag/ml 4.38 2.16 15 48 17 10 x4 n=22 I x 5

Diltiazem- 4.68 3.84 28 257 38 HCI 4.47 3.75 27 243** 37 0 20 x 0 1 gg/ml 4.32 3.66 26 206 36 n=20 n=15

Diltiazem- 4.62 3.54 27 172 38 17 x 0 HC1 4.38 3.30** 25* 133"* 35 19 2 x l l0 gg/ml 4.17 2.97 24 121 34 1 x2 n=21 n=20 n= 16 1 •

Diltiazem- 4.37 2.63 19 74 25 1 x 1 HC1 4.08** 2.46** 17"* 70** 23** 100 8 x 2 30 gg/ml 3.92 2.34 14 63 19 3 x3 n=20 n= 18 8x4

For the first 5 columns the boldface numbers represent the median value, the numbers at the top and bottom show the third and the first quartile. * = significantly different from controls at p <0.05 ** = significantly different from controls at p <0.01 %Abn = frequency of abnormalities in % Abn/E= frequency of abnormalities per embryo (i. e. 7 x I = 7 embryos with l abnormality)

Concentrations: LOEL (morphology of embryos and yolk sacs). A funct ioning yolk sac circulat ion is easi ly recognizable in control embryos with thick vessels , trans- parent t issue and with the ref lect ion of light in the f lowing blood cells. In contrast, detect ion o f circulat ion is diff icul t in yolk sacs wi th smaller vessels or with not so transparent tissues. For this reason circulat ion was somet imes diff icul t to detect but the overal l yolk sac appearance could still not be regarded as abnormal . Table 5 summarizes the results on the appearance o f the yolk sacs at concentrat ions corre- sponding to a L O E L .

At the concentrat ions corresponding to a L O E L the variations observed most ly concerned deviat ions in shape ( incomplete f lexions). In addition, single abnormali t ies o f

the heart, head or extremit ies were recorded within the groups o f approximate ly 20 evaluated embryos.

Concentrations: NOEL (morphology of embryos and yolk sacs). At the concentra t ion designated as N O E L the morpho logy of the yolk sac was always normal. The ab- normali t ies found in two embryos were (a) after 1 gg VER/ml : "squi r re l -shaped" (defined as an embryo in which the caudal and cranial parts o f the neural tube are fused and the embryo has failed to rotate; in these embryos several organ anlagen were always affected) and (b) after 0.1/,tg NIM/mI : a head abnormali ty.

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Table 2. Calcium channel antagonists: nifedipine, nitrendipine, nimodipine

Yolk sac- Crown- diameter rump Somites Protein (ram) (mm) (n = ) (~g/E)

Score %Abn Abn/E

4.76 3.86 28 213 38 Control 4.47 3.72 27 175 37 0

4.25 3.48 26 154 36 n = 2 6 n = 2 4

DMSO- 4.62 3.80 28 204 38 Control 4.41 3.54 27 180 37 0

4.20 3.36 25 158 36 n = 26 n = 23

4.50 3.60 28 165 38 Nifedipine 4.32 3.42* 26 135"* 36* 20 10 I.tg/ml 4.11 3.15 26 115 34 n = 25 n = 23 n =20

4.71 2.34 20 63 26 Nifedipine 4.50 2.28** 18"* 57** 21"* 1110 30 p.g/ml 4.29 2.10 17 48 19 n=21 n = 1 9

Nitren- 4.56 3.77 28 212 38 dipine 4.50 3.66 27 194 38* 0 1 lag/ml 4.38 3.60 26 180 36 n = 2 0 n= 15

Nitren- 5.16 2.28 20 62 23 dipine 4.89** 2.19"* 19"* 53** 21"* 100 10 ~tg/ml 4.68 2.04 18 52 19 n = 2 2 n=21

4.74 4.02 28 246 38 Nimodipine 4.56* 3.66 27 191 38 4 0.1 ~tg/ml 4.44 3.48 26 167 36 n=23 n= 18

4.98 2.58 21 60 27 Nimodipine 4.80* 2.28** 20** 51"* 24** 95 1 I.t g/ml 4.38 2.16 17 31 22 n = 1 9 n = 1 8 n = 1 5

2 6 x 0

2 6 x 0

20xO 4 x l l x 4

4 x l 6 x 2 7 x 3 4 x 4

20xO

7 x 2 l l x 3

l x 4 3 x 5

22 x0 l x l

1xO 4 x l

lOx2 4 x 3

For the first 5 columns the boldface numbers represent the median value, the numbers at the top and bottom show the third and the first quartile. * = significantly different from controls at p <0.05 ** = significantly different from controls at p <0.01 %Abn = frequency of abnormalities in % Abn/E = frequency of abnormalities per embryo (i. e. 7 • 1 = 7 embryos with 1 abnormality)

Table 3. Concentrations of the various calcium channel antagonists lead- ing to the corresponding effect categories

NOEL LOEL 100%EL flag/ml) (lag/ml) (lag/ml)

Nifedipine 10 30 Nitrendipine 1 10 Nimodipine 0.1 1 Verapamil HC1 1 3 10 Gallopamil HCI 1 10 Diltiazem HCI 1 10 30

The molecular weights of the six compounds tested are rather similar, ranging from 346 to 521 (i. e. 1 ~g/ml corresponds to 1.9 ~M GAL, 2 ~tM VER, 2.2 p.M DIL, 2.4 ~tM NIM, 2.8 ~tM NIT, 2.9 ~tM NIF)

Micromorphological evaluations

Light microscopic findings. T o e luc ida t e w h e t h e r the e m b r y o s a p p e a r i n g normal u p o n m a c r o s c o p i c in spec t ion m i g h t s h o w s igns o f a b n o r m a l d e v e l o p m e n t at the mic ro - s c o p i c leve l , e m b r y o s e x p o s e d to the va r ious c a l c i u m chan- nel b l o c k e r s at c o n c e n t r a t i o n s c o r r e s p o n d i n g to the N O E L and L O E L w e r e s e c t i o n e d se r ia l ly and i n v e s t i g a t e d his to- log ica l ly .

N e i t h e r con t ro l s no r e m b r y o s t rea ted wi th concen t ra - t ions c o r r e s p o n d i n g to the N O E L or L O E L r e v e a l e d any h i s t o log i ca l a b n o r m a l i t i e s w h e n c o m p a r e d to day 11.5 e m b r y o s in v ivo .

T h e e m b r y o s e x p o s e d to c o n c e n t r a t i o n s c o r r e s p o n d i n g to the 1 0 0 % E L o f the six d rugs s h o w e d s imi l a r h i s to log ica l dev ia t ions . H o w e v e r , the ex t en t to w h i c h the d i f f e ren t re- g ions o f the e m b r y o s w e r e a f f ec t ed was subs t ance depen- dent . G e n e r a l l y , a less d e n s e l y p a c k e d e m b r y o n i c mesen- c h y m e was o b s e r v e d . O n o c c a s i o n this led to unusua l ly

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Fig. 1. Rat embryos after 48 h of culture in the presence of water-soluble compounds: 1 = Con- trol; 2a = 1 lag GAL/ml, 2 b = 10 lag GAL/ml; 3 a = 1 lag VER/ml, 3 b = 3 lag VER/ml, 3 c = 10 lag VER/ml; 4a = 1 lag DIL/ml, 4 b = 10 lag DIL/ml, 4 c = 30 lag DIL/ml. Pic- tures 2 b, 3 c and 4 c show abnormal embryos exposed to the 100%EL category; 2 a, 3 b, and 4 b embryos of the LOEL category and 3 a and 4 a the embryos of the NOEL category

large intra-ernbryonic cavities. Necroses occurred in indi- vidual embryonic parts, sometimes accompanied by the described gross morphological abnormalities. Judged by light microscopy these embryos were not considered "dead", since generalized necroses or nuclear pyknoses as clear-cut signs of cell death were not present (Figs. 4 -9 ) .

During normal development of the embryonic heart the outer one-layered myoepithelium grows in a cone-shaped fashion into the cardiac jelly (Fig. 5 a). At the same time, individual cells that have detached from the endothelium migrate into the cardiac jelly. Invasions from the myo- cardium, as well as from the endothelium, were missing in the swollen cardiac tubes in the ventricle and truncus re- gions of the VER- and GAL-treated embryos. This led to an arrangement of the cardiac tube that consisted of the one-layered myoepithelium, an almost cell-free cardiac jelly, and the endothelium (Figs. 5 b and 5 d). Death of the contractile cells is less likely, since in most cases necroses,

pyknoses, vacuolisations or similar indications of cell le- sion could not be detected by light microscopy. Although morphological investigations did not show any swelling of the cardiac tube in the DIL-, NIF-, NIT- and NIM-treated groups, a varying degree of inhibition of migration was revealed by light microscopic investigation (Fig. 5 c).

Necroses also occurred to a varying degree within the neural tube after exposure to all six substances. The frequency of necroses apparently correlated with the frequency of wavy wails of the neural tube, i.e. single necroses were observed in the presence of VER and DIL, whereas addition of NIF, NIT and NIM to the medium caused many necroses (Figs. 6b, c, d).

Also in the head region, large cavities due to the almost complete absence of mesodermal cells were seen. The visible changes in the shape of the head were possibly due to the lack of a mesoderm which is normally responsible for the shape (Figs. 5b, c, d; 6a, b).

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Fig. 2. Rat embryos after 48 h of cul- ture in the presence of the water-in- soluble compounds: I = DMSO-Con- trol; 2 a = 10 I.tg NIF/ml, 2 b = 30 ~tg NIF/ml; 3a --- 1 ~tg NIT/ml, 3b= lO~tgNIT/ml; 4a=O.1 ~tg NIM/ml, 4 b = 1 ~tg NIM/ml. Pictures 3 a and 4 a show embryos exposed to the NOEL category, 2 a of the LOEL category and 2 b, 3 b and 4 b of the 100%EL category. Arrow indicates heart abnormality (swollen peri- cardium)

Since generally sagittal longitudinal sections were pre- pared for better orientation in the histological investiga- tions, both forelimbs could not always be analyzed in com- parison. Thus, the sectional plane was not always optimum to allow a clear-cut statement on the differences in limb size between the fight and left side. Histologically the evaluated limbs were either normal or they showed the described mesenchymal cavities (Fig. 4 d, 7 a). Since these cavities occurred quite often, this pathological effect may have been responsible for the disturbances in limb develop- ment.

Histologically, the somites did not show any lesions. Mesenchymal portions were also missing in the yolk

sac. Light microscopic inspection showed large lacunae. Electron microscopy revealed that these were bordered by vascular endothelium. The lacunae appeared instead of normal vessels which are embedded in the mesoderm be- tween the yolk sac epithelium and the mesothelium (Figs. 4b, c, e, 6a).

Electron microscopic findings. The yolk sacs of the ex- plants exposed to 10 ~g NIT/ml were also investigated electron microscopically. The following abnormalities were found:

- the first abnormality concerned the cylindrical cells of the yolk sac epithelium. Almost all types of inclusions, such as electron-dense granules, were missing, and in the

cells that still contained inclusions there were decreases in number and size (Figs. 8 b, 9 b).

- The second abnormality concerned the mesenchy- real portions of the yolk sac wall. The ultrastructural obser- vations confirmed the light microscopic findings of large cavities between the yolk sac epithelium and the me- sothelium of the exocoelomic cavity due to the absence of the mesenchymal cells normally seen surrounding the ves- sels. The typical endothelial cells were still present.

Although not studied in detail, we assume that the electron microscopical appearance of the tissues exposed to the other five drugs will be identical or at least very similar.

Discussion

Can our data help evaluate an embryofeto-toxic risk assess- ment with relevance to man? What is the relevance of the results obtained in our studies for interpreting results ob- tained from in vivo studies (segment II) in other laborato- ries? Pharmacokinetic data will have to be considered in order to answer both of these questions.

The present study illustrates the difficulties that we are faced with when attempting to interpret the significance and applicability of data from in vitro studies in developmental toxicology for human risk assessment.

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Fig. 3. Abnormalities of embryo and yolk sac after exposure to 1, 3, 4 and 5 = 10 lag VER/ml; 2 = I pg NIM/ml; 6 = 10 lag GAIdml; 7 = 30 lag NIF/ml; 8 = 10 lag Dlldml. For explanation of the types of abnormalities see legend to Table 4, The black arrows in 3 indicate the size difference in the right and left hand side of the embryo (limb abnormality type Q). The black arrows in 4 indicate a wavy neural wall (= neural tube abnormality type N). The black arrows in 5 indicate the head abnormality type O

Possible mode of action and primary target

The six calcium channel blockers tested belong in three different chemical classes:

- nifedipine (NIF), nimodipine (NIM) and nitren- dipine (NIT) are 1,4-dihydropyridines,

- gallopamil HC1 (GAL) and verapamil HC1 (VER) are phenylalkylamines, and

- diltiazem HC1 (DIL) is a 1,5-benzothiazepine.

The aim of these comparative in vitro studies was to investigate whether (a) each drug may give rise to distinct, compound-specific effects on embryonic development, (b) the induction of specific effects is related to one of the basic chemical structures, or (c) there is a common ability to influence embryonic development perhaps due to the calcium channel antagonistic properties of all of these agents.

The results presented here permit us to conclude that despite differences in chemical structure all the calcium channel blockers exhibit an identical (or at least very simi- lar) effect on embryonic development in vitro, particularly

on the yolk sac. The differences observed with the various agents were merely quantitative. The swelling of the heart tube and the absence of heartbeat were only observed after exposure to VER and GAL, but since the histological ex- aminations showed similarities with the other four drugs, this could also just be an especially pronounced effect.

These findings suggest a common mechanism of action for the six calcium channel blockers for inducing distur- bances on embryonic development. Since the observed adverse effects occurred independently of the chemical structure of the agents tested, we conclude that these altera- tions are probably due to the calcium channel blocking properties. This provides the opportunity to study the pos- sible mode of action with more sophisticated biochemical techniques.

As with many embryofeto-toxic agents a steep concen- tration-response curve (with less than a factor of 10 be- tween the NOEL and the 100%EL) was observed.

Although it is obvious from the results of our studies that the yolk sac function (circulation as well as endocy- totic properties) was affected, it is difficult to decide from the information now available whether the effects on the

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Table 4. Type and frequency of abnormalities and observations unusual for day 11.5: effects of calcium channel antagonists at the lowest tested concentration level that induced 100% abnormalities

Yolk sac Heart Heart beat Shape Neural tube Head Forelimb Ear Eye Tail Somites

3 x D+E+F Verapamil- 6 x D+E HC1 10 gg/ml 6 x D+F 3 x Q n = 2 1 5 x D 1 6 x K 8 x V

2 1 x A l x E 8 x : - l x l I l 2 x N 1 8 x O (100%) (100%) (38%) (76%) (10%) (86%) (14%)

2 x O 8 x Q Gallopamil- 17 x E 18 x P 1 x Q+R HC1 10 I.tg/ml 1 2 x A 3 x E + F 11 x K 1 x O + P 7 x V n = 2 2 1 0 x B 2 x E + D 2 I x : - 11 x I I I 1 3 x N I x V

(100%) (100%) (95%) (50%) (59%) (95%) (41%)

1 3 x D Diltiazem- 6 x A 5 x D+F 9 x K HCI 30 gg/ml 13 x B 1 x D+E+F 8 x III n = 2 0 l x I l x E +

( 9 5 % ) (100%) (45%)

9 x A 9 x D Nifedipine 11 x B 1 x D+H 30 gg/ml 1 x D+E+F 3 x K n = 2 1 l x C 4 x I I + 1 6 x I I I

(100%) (52%) (14%)

1 6 x D 2 x K Nitrendipine 1 x E 2 x L 10 gg/ml 19 x A 1 x D+F+G n = 2 2 3 x B 2 x I I + 17 x l I I

(100%) (82%) (23%)

Nimodipine 4 x D 1 gg/ml 15 x A 1 x D+F n = 19 3 x B 3 x I I + 14 x l I l

( 9 5 % ) (26%)

21 x V I

22 x VI

16 x O + P 7 • 2 x P 2 x Q + R 2 0 x V I l x T 1 x l V 5 x V 1 x U

(90%) (45%) (10%)

3 • l x Q 2 0 x V I 11 x P 1 x Q + R

1 5 x N 6 x l V 12 x V I I 1 • 8 • (71%) (67%) (10%) (38%)

1 x K + M 8 x P 7 x Q 2 2 x V l 2 1 x N 1 3 x l V 1 2 x V 5 x S (95%) (36%) (32%) (23%)

6 x O 1 8 x 10 x P 3 x V VIII

15 x N 1 x l V 1 x V I I (80%) (63%)

2 x W

Letters indicate the different abnormalities within one variable. Roman numerals indicate variables (mostly retardations). Arabic numerals indi- cate the frequency of an observation. In brackets the frequency of ab- normalities within one variable is given in per cent Abnormalities: Yolk sac: A = overall form is flattened; swollen vessels; main vessels

do not form branch-like pattern; no circulation to be ob- served (Fig. 3.2) / B = form of yolk sac round; swollen vessels like A (Fig. 3.1); C = yolk sac surface ruffled; no vessels or circulation. I = Thin main vessels developed, no circulation to be observed

Heart: D = bulge in the region of prospective left ventricle (Fig. 3.7) / E = swollen cardiac tube (Fig. 3.6) / F = swollen pericardium (Fig. 2.3 b) / G = slight constriction in the region of the atrium commune / H = swollen atrium com- mune. II = stricture between atrium commune and prospec- tive left ventricle still missing

Heart-beat: : - = no heart beat detectable Shape: K = kinking in lower rump region (Fig. 3.8) / L = kinking

of the tail / M = kinking in region of rhombencephalon. III = incomplete flexion, telencephalon not yet approaching heart

Neural tube: N = walls of neural tube show wavy appearance (Fig. 3.4). (Note: all neural tubes in this study were closed)

Head: O = telencephalic vesicles have grown beyond the former prosencephalic region (Fig. 3.5) / P = other abnormalities, including very small heads, irregular shapes, etc. IV = fis- sura telodiencephalica still miss ing

Forelimb: Q = left limb >>>>>right limb (Fig. 3.3) / R = swollen limb. V = limbs still missing

Ear: VI = otic anlage still forms a pit / VII = closed vesicle with recessus dorsalis

Eye: S = irregular borderlines of eye vesicle Tail: T = Bleb at tail tip / U = kinking in tail region Somites: W = irregular arrangement

Table 5. Yolk sac morphology and function at LOEL concentrations

Normal Deviating Abnormal

Nifedipine 10 gg/ml 91% 9% 0% Verapamil HCI 3 gg/ml 20% 80% 0% Gallopamil HCI 1 gg/ml 9% 82% 9% Diltiazem HCI 10 gg/ml 33% 67% 0%

In the specimens summarized under deviating, circulation was not clearly detectable, due to rather thin main vessels and a not very transparent tissue color but it could not be categorized as abnormal

embryo should be considered direct effects or are the in- direct result of disturbed yolk sac function. The breakdown of the yolk sac circulation must drastically affect embry- onic development. However, according to the histological examinations the adverse effects observed within the tis- sues of the embryo proper cannot only be considered "un- specific". The very pronounced effect on the embryonic mesenchyme appears to be the result of a direct effect of the drugs.

631

Fig. 4. Rat e m b r y o s af ter 48 h o f culture: (a) = Con- trol: Sagi t ta l sect ion s o m e w h a t lateral to the med ian plane, h = heart an lage; m = mesencepha lon ; n = neural tube; p = prosencepha lon ; r = rhomben- cephalon; * = otic ves ic le ; a r row = somites ; ar row- head = first branchia l arch. (b) = 10 I.tg GAL/mI : �9 = absence of normal s ized vesse l s in the yolk sac wal l ; smal l a r r o w = the outer contour of the head is de fo rmed; a r r o w h e a d = empty spaces in the mesen- chyme ; a r r o w = prosencepha lon ; h = heart anlage; e = ec toplacenta l cone wi th placenta anlage. (c) = 10 ~g NIT/ml : * = Large lacunae be tween the yo lk sac ep i the l ium and the meso the l ium. A r r o w = prosencepha lon ; h = heart anlage.

(d) = 30 lag DIL/ml : a r r o w = open opt ic vesicle; �9 = empty spaces in the m e s e n c h y m e of the l imb bud; c = coe lomic cavi t ies ; r = rhombenecepha lon . (e) = 30 lag NIF/ml : * = large lacunae in the yo lk sac wal l ; h = heart anlage; n = neural tube; p = pros- encepha lon

Comparison with in vivo embryotoxicity studies in the rat

The histological inspection indicates that the embryos can- not be regarded as dead. However, it is extremely unlikely that embryos that did not establish a functioning circulation on gestational day 11.5 would survive under in vivo condi- tions. Therefore, the present observations may explain the increase in resorptions recorded in the in vivo studies with some of the calcium channel blocking drugs. The altera- tions observed in the embryo proper at all effective concen- trations always correlated with drastic morphological and probably functional changes within the yolk sac. For this reason, our data do not provide good evidence for a possi- ble direct teratogenic action of this class of agents at con- centrations not affecting the circulation. However, addi- tional teratogenic effects (possibly induced through effects on the mesenchyme) for single substances of this group are still possible.

Published data on the embryotoxic potential of calcium channel blockers in vivo are only available for NIF and DIL. Besides an increased rate of embryomortality, NIF induced teratogenic effects in the cardiovascular system (Cabov and Palka 1984), defects of the finger skeleton, i.e. hyperphalangy (Yoshida et al. 1988), short limbs, oligo- dactyly, short tail, and hematomas (Fukunishi et al. 1980). Some of these defects could be induced by single oral

administrations of 20-150 mg NIF/kg body wt on 1 day between days 10 and 16 of pregnancy.

For NIF additional data from the drug manufacturer in the form of a report to the regulatory agencies (information given to physicians), point to a teratogenic potential of this substance and it is mentioned that disturbances in postnatal development occur at daily doses of 30 mg NIF/kg body wt or higher.

Reproductive toxicity has furthermore been studied with DIL in mice, rats and rabbits (Ariyuki 1975) using i.p. injections. While treatment of mice during days 7 - ! 2 of pregnancy induced embryomortality only (with concentra- tions of or exceeding 12.5 mg DIL/kg body wt) various teratogenic effects were observed subsequent to single doses of 25 or 50 mg DIL/kg body wt given on days 9 or 13. In rats (treated during days 9 - 14 of pregnancy) 80 mg DIL/kg body wt was found to be teratogenic, as was the case following a single dose on day 13 or 14; the resorption rate was clearly increased at this dose. Studies with rabbits revealed an increased resorption rate (70%) at 12.5 mg DIL/kg body wt (when applied on days 7-16) and an apparent increase in abnormalities in the few (approxi- mately 20) surviving fetuses.

Unpublished data from studies on developmental tox- icity represent the only (but not readily available) informa- tion for the other calcium channel blockers. Summaries of

632

Fig. 5. Rat embryos with and without treatment after a 48 h culture period: (a) = Control: o = otic vesicle; * = first branchial arch; a = part of the atrium and v = ventricle of the heart anlage with the myoepithelium proliferating in a cone-shaped fash- ion into the cardiac jelly (arrow head). Myo- epithelium and endothelium are linked. (b) = 10 lag GAL/mI: arrow = empty spaces in the mesenchyme of the head; p = prosencephalon. No thickening of the myoepithelium in the ventricular region (v) of the heart anlage. * = clear-cut dilatation of the space between myoepithelium and endothelium. Missing linkage between the two layers. (c) = I pg NIM/ml: * = large empty spaces in the region of the mesenchyme of the head. Missing thickening of the myoepithelium in the region of the ventricle anlage (v); a = atrium; o = optic process; r = rhomben- cephalon. (d) = 10 lag VER/ml: * = empty spaces in the region of the mesenchyme of the head. Distinct swellings in the region of the heart anlage (v). o = transition of the eye process into the prosen- cephalon; r = rhombencephalon

Table 6. Segment II studies in rat

Substance Smallest dose showing: (in mg/kg body wt per day)

Teratogenic Increased effects embryofetal mortality

NIF 30 a 10a? NIT n.t.e, at 100 b no info NIM n.t.e, at 100 a§ 100a?

VER n.t.e, at 3 x 20 a 3 x 20 a GAL n.t.e, at 2 x 7.5 d 2 x 4.75 d

DIL 25 e,f 12,5e,f,g, h 80~.g

a Drug information; b Hoffman 1984; c Schltiter 1986; d personal com- munication Knoll AG; e Ariyuki 1975 Studies with: f = mice; g -- rats; h = rabbits no info = no information available n.t.e, at 100 = no teratogenic effects recorded in segment II studies in rats with oral doses up to 100 mg/kg body wt per day

these results (segment II studies in the rat, oral administra- tion) are accessible from the basic scientific information provided with the marketing of these drugs. It is difficult to judge whether all of these studies were performed accord- ing to todays' standards (Table 6).

Comparison of in vitro data with plasma levels in the rat

Pharmacokinetic data have to be considered in order to compare the results obtained in our in vitro studies with the situation probably existing in vivo. Plasma concentrations achieved in vivo under defined exposure/treatment condi- tions are the most reasonable for comparison with in vitro culture medium concentrations.

A comparison of our in vitro data with the results of segment II studies is difficult, since no pharmacokinetic studies have been concurrently performed with such ex- periments, and data on plasma levels have to be taken from other studies conducted under different experimental con- ditions. Furthermore, comparing only plasma levels of the

633

Fig. 6. Rat embryos alter 48 h of culture: (a) = 10 lag VER/mI: * = empty spaces in the mes- enchyme of the head. Dilatation of the space be- tween yolk sac epithelium and mesothelium of the yolk sac wall (y). p = prosencephalon; m = mesen- cephalon. (b) = 10 lag NIT/mI: prosencephalon (p) with eye process (arrow); numerous necroses in the neuroepithelium; * = large empty spaces in the mesenchyme. (c) = 10 lag GAL/mI: huge cavities (*) next to the neural tube (n), which is slightly deformed. Some cell debris in the cavity (arrow head). (d) = 10/ag VER/mI: like Fig. 6c. o = deformed otic vesicle

parent drug will neglect other important pharmacokinetic aspects such as protein-binding, or the presence of differ- ent isomers with different biological activities, or the role which metabolites may play for the induction of embryo- toxic effects in vivo, and concentrations in maternal plasma may not reflect the actual concentrations reached in the embryo.

Therefore, only rough estimates can be made. Some of the pharmacokinetic data obtained in rats after oral admin- istration are compiled in Table 7. The following tentative conclusions may be drawn: one might assume that the in vivo teratogenic dose (see above) of 30 mg NIF/kg body wt could produce plasma peak levels that at least approach the in vitro LOEL of 10000 ng/ml. The in vivo application of 3 • 20 mg VER/kg body wt per day caused increased prenatal mortality in rats and led to plasma concentrations

about two to three times lower than the in vitro LOEL of about 3000 ng/ml. For GAL, even the repeated in vivo application of 2 • 7.5 mg GAL/kg body wt per day ap- parently does not lead to plasma concentrations equivalent to the in vitro LOEL of 1000 ng/ml. After in vivo applica- tion of 100 mg NIT/kg body wt plasma levels might ap- proach concentrations effective in vitro, but there is no information about in vivo effects from studies on reproduc- tive toxicity. There are no corresponding pharmacokinetic data in the rat available for NIM or DIL.

These very rough estimates indicate that in the segment II studies in rats plasma concentrations may have been reached that are able to interfere with embryonic develop- ment in vitro. This may explain the embryotoxic effects seen, e.g. with NIF.

634

Fig. 7. Rat embryos after 48 h of culture: (a) = 30 jag DIL/ml: oblique section of an embryo with neural tube (n). The limb bud anlagen with thickened epithelium (arrow) and large empty spaces (*) in the mesen- chyme. (b) = 3 ~g VER/ml: Vena cava (*) with defined endothelial proliferations (arrow)

Comparison of in vitro data with plasma levels in human therapy

For human therapy the maximum recommended daily doses (MRDD) in mg are: NIF (6 x 20 p . o . / 2 x 40 RR p.o./ 30/24 h infusion); NIT ( 2 x 2 0 p.o.); NIM ( 4 x 6 0 p .o . /48 /24 h infusion); VER ( 4 x 120/ 3 x 160/ 2 x 2 4 0 RR p.o./ 100/24 h infusion); GAL ( 4 x 5 0 p.o.); DIL ( 4 x 9 0 RR/ 3 x 1 2 0 p.o./ 300/24 h infusion). Levels reached under steady-state conditions during oral therapy or after constant infusion may be expected to mimic best an in vitro situation with the drug present at the same concen- tration over the entire culture period. Unfortunately, such data for man are only available for some of the investigated agents. For this reason rough estimates can only be made using other available data, less convenient for risk assess- ment (cf Table 7).

These data suggest that for NIM (the drug leading to abnormal development in vitro at the lowest concentration) plasma levels achieved in man are very close to a NOEL

for the in vitro effect. As far as suitable data are available for the other compounds, the therapeutic plasma concentra- tions may be assumed to be at least one order of magnitude from the concentrations which interfere with embryonic development in vitro, when one regards applications pro- ducing rather constant plasma levels. Single individual peak levels may lead to a difference of less than one order of magnitude.

Unsolved problems and drawbacks when attempting extrapolations from in vitro data

There are a number of problems which could not be ade- quately dealt with in the present study. It is, for example, generally accepted that drug effects are better related to the free (unbound) rather than to the total plasma concentra- tion. In order to evaluate data obtained with different drugs or to predict effects from in vitro to in vivo the free concen- trations of the drugs or the tissue levels should be corn-

635

Fig. 8. Yolk sac epithelium after a 48 h culture peri- od: (a) = Control: Numerous electron-dense inclu- sions (arrow) in the apical cellular region (protein- resorbing apparatus). (b) = 10 ~tg NIT/ml. Missing inclusions, some lipid droplets (*). m = mesenchy- mal cell

pared. However, in the case of the calcium channel block- ers there are several factors which complicate such com- parisons or extrapolations. These include:

Protein binding. The six calcium channel antagonists stud- ied here are all extensively bound to human plasma pro- teins: NIF 99% (Otto and Lesko 1986), NIT 98% (Eichel- baum et al. 1986), NIM 99% (drug information), VER about 90% (Keefe et al. 1981; McGowan et al. 1983), GAL 93% (Rutledge and Pieper 1987), DIL about 80% (Morselli et al. 1979; Bloedow et al. 1980; Kwong et al. 1985).

Concentration dependence. The degree of protein binding has been shown to be concentration dependent: e. g. protein binding of 98% is documented for NIF in human serum in the presence of 200 ng/ml, but of only 92% with 20000 ng/ml (Rosenkranz et al. 1974), and for DIL, bind- ing to rat plasma proteins, a decrease in protein binding has been found between the concentrations of 300 and 30000 ng/ml from 84% to 66%, respectively (Nakamura et al. 1987).

Species differences. Such differences have to be considered since we want to compare free concentrations in humans

and rats in vivo with that in cow serum in vitro. Species differences in protein binding have been documented for NIF at least between man and dog (Rosenkranz et al. 1974), but not between rat and man (cited in Downing and Hollingsworth 1988), and for DIL, i.e. with 300 ng/ml, between rat plasma (84%) and human plasma (63%) and human serum albumin (40%) and bovine serum albumin (66%) (Nakamura et al. 1987). Also for VER species differences seem to occur, since binding to rat plasma proteins (Todd and Abernethy 1986, 1987) is about 98%, but to human plasma protein only about 90%. No data are available for bovine serum.

pH dependence. Differences in protein binding due to var- ious pH values may also be of significance for comparative assessments since the pH of the culture medium shifts during the culture period from about 7.8 to 7.0. A reduction in protein binding is described for a decrease in pH in the case of NIF (Rosenkranz et al. 1974) and GAL (90% with pH = 7.0/95% with pH = 8.0; Rutledge and Szlaczky 1988).

Isomers. NIT, NIM, VER and GAL are administered as a racemic mixture. Since normal measuring methods for

636

Fig. 9. Apical part of the yolk sac epithelium after 48 h of culture. Tannic acid fixation: (a) = Control: (arrows) Numerous tannic acid-positive vesicles and tubuli (coated vesicles). (b) = 10 gg NIT/ml: numer- ous coated pits, but only a few invaginations (arrows)

Table 7. Synopsis of the concentrations with the six calcium channel antagonists. Culture medium concentrations in vitro versus plasma concentrations in vivo in rat segment II studies and under therapeutic conditions in man.

Concentrations (ng/ml)

NIF NIT NIM VER GAL DIL

In vitro NOEL 1 000 100 LOEL 10 000 100%EL 30 0 0 0 10 0 0 0 1 000

In vivo 1 7 - 7 2 l at max imum 9 9 recommended human dose

at lower than max imum recommended (35 - 2805 ) 40 - 606 human dose

in segment H study >2500 a >4006 9 in rats

1 000 1 000 3000 1 000 10000

10 0 0 0 10 000 30 000

119-1372 ?(63 - 800) ( 167 - 4094)

(50-90)(3613~

687

( 4 0 - 125)

1000 ~ 391N - 1500 - 390

t Drug information, range of mean steady-state plasma concentrations (max. recommended dose) after p.o. or infusion (for similar data see R~imsch et al. 1984) 2 Biihler et al. 1984, after 2 x 2 4 0 mg (sustained release formula) 3 Frishmann et al. 1982, after 3 x 160 mg. VER plasma levels after infusion are lower than the levels obtained after p. o. (Reiter et al. 1982) 4 Hung et al. 1988, after 3 x 120 mg. During infusion about 200 ng/ml are reached (drug information) 5 Cmax after single dose application of 10 mg p.o. (Kleinbloesem et al. 1987; Donnelly et al. 1988; Menke et al. 1988)

6 Steady-state concentration after 20 mg (Kirch et al. 1984; R~imsch and Sommer 1984) 7 Stieren et al. 1983, after 3 x 50 mg

The plasma levels of a Ct=3o min = 2500; b C m a x = 400; c C m a x = 1000- 1 5 0 0 ; d C m a x = 250 were reached after a.b a single dose of 5 mg/kg body

wt of NIF and NIT; c repeated doses of 3 x 20 mg VER/kg body wt per day, and d repeated doses of 2 x 7.5 mg GAL/kg body wt per day

a Patzschke et al. 1975; b Krause et al. 1988; c,d personal communica- tion, Knoll AG

concentration determination do not discriminate between the different optical isomers, a comparison of in vivo and in vitro levels is difficult if the isomers possess different biological properties and if the ratio of the enantiomers changes in vivo. It has been shown that the (-)-isomers of NIM and NIT (Bellemann et al. 1982) and VER (Eichel- baum et al. 1984) are biologically more effective than the (+)-isomer. Furthermore, in the case of VER it is known that the ratio of (+)/(-)-isomer changes in vivo because the (+)-isomer is metabolically more stable (Eichelbaum et al. 1984).

Concluding remarks

The concentrations of the calcium channel antagonists ex- hibiting an adverse effect in the whole-embryo culture appear high (IaM range) when compared with data publish- ed (e. g. Bolger et al. 1983; Godfraind and Wibo 1985; Qar et al. 1988) for receptor binding (nM range). However, up till now, no clue on the possible concentrations reached at the targets under our in vitro conditions exists.

This study clearly indicates the difficulties we are faced with at the present time when attempting to extrapolate results from in vitro studies to the situation possibly ex- isting in man (or even in experimental animals in vivo).

In order to integrate data obtained with in vitro tech- niques into risk assessment studies, new strategies have to be established. This especially concerns the planning of specially designed pharmacokinetic studies to provide rel- evant information for the interpretation of in vitro data.

Acknowledgements. The fellowship granted to Mithilesh Kumar Srivastava by the German Academic Exchange Programm (DAAD) making his stay in Berlin possible is thankfully acknowledged. The calcium channel blockers were generously provided by Bayer AG, Knoll AG, and GOdecke AG. We appreciate the many fruitful discussions with the Knoll AG and the advice given by the Bayer AG with respect to the handling of the light-sensitive substances. These studies were supported by grants from the Bundesministerium fiir Forschung und Technologie and the Deutsche Forschungsgemeinschaft. Jane Klein-Friedrich is grate- fully acknowledged for her help in preparing the manuscript.

References

Ariyuki F (1975) Effects of diltizem hydrochloride on embryonic development - species differences in the susceptibility and stage specificity in mice, rats and rabbits. Folio Anat Jap 52: 103- 117

Bellemann P, Ferry D, L/ibbecke F, Glossmann H (1982) (3H)-ni- modipine and (3H)-nitrendipine as to61s to directly identify the sites of action of 1,4-dihydropyridine calcium antagonists in guinea-pig tissues. Arzneimittelforschung 32:361 -363

Bloedow DC, Piepho RW, Nies AS, Gal J (1980) Serum binding of diltiazem in humans. Pharmacologist 22:192

Bolger GT, Gengo P, Klockowski R, Luchowski E, Siegel H, Janis RA, Triggle AM, Triggle DJ (1983) Characterization of binding of the Ca++ channel antagonist, [3H]-nitrendipine, to guinea-pig ileal smooth muscle. J Pharmacol Exp Ther 225:291 -309

Bfihler V, Einig H, Stieren B, Hollman M (1984) Pharmacokinetics of verapamil sustained release formulation with multiple applications. Naunyn-Schmiedeberg's Arch Pharmacol [Suppl] 325:R86

Cabov AN, Palka E (1984) Some effects of Cordipin R (nifedipine) ad- ministered during pregnancy in the rat. Teratology 29:21 a

637

Constantine G, Beevers DG, Reynolds AL, Luesley DM (1987) Nifedipine as a second line antihypertensive drug in pregnancy. Br J Obstet Gynaecol 94:1136- 1142

Donnelly R, Elliott HL, Meredith PA, Kelmann AW, Reid JL (1988) Nifedipine: individual responses and concentration-effect relation- ships. Hypertension 12:443-450

Downing S J, Hollingsworth M (1988) Nifedipine kinetics in the rat and relationship between its serum concentrations and uterine and car- diovascular effects. Br J Pharmacol 95:23-32

Eichelbaum M, Mikus G, Vogelgesang B (1984) Pharmacokinetics of (+)-, (-)- and ( + )-verapamil after intravenous administration. Br J Clin Pharmacol 17:453--458

Eichelbaum M, Fischer C, Heuer B, Mikus G (1986) Pharmakokinetik und Metabolismus von Nitrendipin. In: Distler A (ed) Calcium-An- tagonisten in der Hochdrucktherapie. Europ~iisches Bayotensin- Symposium 1985, Schattauer, Stuttgart, New York, pp 35-42

Frishman W, Kirsten E, Klein M, Pine M, Johnson SM, Hillis LD, Packer M, Kates R (1982) Clinical relevance of verapamil plasma levels in stable angina pectoris. Am J Cardiol 50: 1180- 1184

Fukunishi K, Yokoi Y, Yoshida H, Nose T (1980) Effects of nifedipine on rat fetuses. Med Consult New Remed 17:2245-2256 (Japanese)

Godfraind T, Wibo M (1985) Subcellular localization of [3H]-nitren- dipine binding sites in guinea-pig ileal smooth muscle. BrJ Pharma- col 85:335-340

Hoffman K (1984) Toxicological studies with nitrendipine. In: Scriabine A e t al. (eds) Nitrendipine. Urban & Schwarzenberg, Baltimore, Munich, pp 25- 32

Hung J, Hackett PL, Stephen PF, Gordon SPF, llett KF (1988) Pharma- cokinetics of diltiazem in patients with unstable angina pectoris. Clin Pharmacol Ther 43:466-470

Keefe DL, Yee YG, Kates RE (1981) Verapamil protein binding in patients and in normal subjects. Clin Pharmacol Ther 29 :21-26

Kirch W, Hutt H J, Heidemann H, Rfimsch K, Janisch HD, Ohnhaus EE (1984) Drug interactions with nitrendipine. J Cardiovasc Pharmacol 6: $982- $985

Kleinbloesem CH, van Brummelen P, Breimer DD (1987) Nifedipine. Relationship between pharmacokinetics and pharmacodynamics. Clin Pharmacokinet 12:12-29

Klug S, Lewandowski C, Neubert D (1985) Modification and standard- ization of the culture of early postimplantation embryos for toxico- logical studies. Arch Toxicol 58:84-88

Krause HP, Ahr HJ, Beermann D, Siefert HM, Suwelack D, Weber H (1988) The pharmacokinetics of nitrendipine. I. Absorption, plasma concentrations, and excretion after single administration of [~4C]nitrendipine to rats and dogs. Arzneimittelforschung 38: 1593- 1599

Kwong TC, Sparks JD, Sparks CE (1985) Lipoprotein and protein bind- ing of the calcium channel blocker diltiazem. Proc Soc Exp Biol Med 178:313-316

Laragh JH (1987) Round table discussions. J Cardiovasc Pharmacol [Suppl] 10:203-212

Lilja H, Karlsson K, Lindecrantz K, Sabel KG (1984) Treatment of intrauterine supraventricular tachycardia with digoxin and vera- pamil. J Perinat Med 12:151 - 154

McGowan FX, Reiter MJ, Pritchett ELC, Shand DG (1983) Verapamil plasma binding: relationship to alpha-l-acid glycoprotein and drug efficacy. Clin Pharmacol Ther 33:485-490

Menke G, Kausch M, Rietbrock N (1988) Eine Model|untersuchung zur Bioaquivalenz von gel6stem Nifedipin aus Weichgelantinekapseln. Arzneimittelforschung 38: 300- 304

Morselli PL, Rovei V, Mitchard M, Durand A, Gomeni R, Larribaud J (1979) Pharmacokinetics and metabolism of diltiazem in man (ob- servations on healthy volunteers and angina pectoris patients). In: Bing RJ (ed) New drug therapy with a calcium antagonist. Diltiazem Hakone Symposium 1978, Excerpta Medica, Amsterdam, Princeton, pp 152- 167

Nakamura S, Suzuki T, Sugawara Y, Usuki S, Ito Y, Kume T, Yo- shikawa M, Endo H, Ohashi M, Harigaya S (1987) Metabolic fate of diltiazem. Distribution, excretion and protein binding in rat and dog. Arzneimittelforschung 37: 1244- 1252

638

Otto J, Lesko LJ (1986) Protein binding of nifedipine. J Pharmacol 38: 399-400

Orlandi C, Marlettini MG, Cassani A, Trabatti M, Agostini D, Salomone T, Testoni N (1986) Treatment of hypertension during pregnancy with the calcium antagonist verapamil. Curr Ther Res 39: 884- 893

Patzschke K, Schlossmann K, Horster FA (1975) Nifedipin (AdalatR) - Pharmakokinetik und Biotransformation. Ther Ber 47:197-203

Pipkin FB, Lawrence MR (1987) Preliminary studies on the cardiovascu- lar effects of nitrendipine in human pregnancy. J Cardiovasc Phar- macol 9:$286-$289

Qar J, Barhanin J, Romey G, Henning R, Lerch U, Oekonomopulos R, Urbvach H, Lazdunski M (1988) A novel high affinity class of Ca 2+ channel blockers. Mol Pharmaco133: 363-369

Riirnsch KD, Sommer J (1984) Pharmacokinetics and metabolism of nitrendipine. In: Scriabine A, Vanov S, Deck K (eds) Nitrendipine. Urban & Schwarzenberg, Baltimore, Munich, pp 409-421

R~nsch KD, Graefe KH, Sommer J (1984) Pharmacokinetics and metab- olism of nimodipine. In: Betz E, Deck K, Hoffmeister F (eds) Ni- modipine. Pharmacological and clinical properties. Schattauer Vet- lag, Stuttgart, New York, pp 147-159

Read MD, Wellby DE (1986) The use of a calcium antagonist (nifedipine) to suppress preterm labour. Br J Obstet Gynaecol 93: 933-937

Reiter M J, Shand DG, Aanonsen LM, Wagoner R, McCarthy E, Pritchett ELC (1982) Pharmacokinetics of verapamil: experience with a sus- tained intravenous infusion regimen. Am J Cardiol 50:716-721

Rosenkranz H, Schlossmann K, Scholtan W (1974) Die Bindung von 4-(26-Nitrophenyl)-2,6-dimethyl-l,4-dihydropyridin-3.5-dicarbon- s~iuredimethylester (Nifedipin) sowie von anderen koronarwirksa-

men Stoffen an die Eiweil3k6rper des Serums. Arzneimittelfor- schung 24:455-466

Rutledge DR, Pieper JA (1987) Gallopamil binding to human serum proteins. Eur J Clin Pharmacol 33:375 -380

Rutledge DR, Szlaezky M (1988) The effect ofpH on gallopamil protein binding. Pharm Res 5:619-620

Schliiter G (1986) Toxicological investigations with nimodipine. Arz- neimittelforschung 36: 1733-1735

Stieren B, Biihler V, Hege HG, Hollmann M, Neuss H, Schlepper M, Weymann J (1983) Pharmakokinetic und Metabolismus yon Gal- lopamil. In: Kaltenbach M, Hopf R (eds) Gallopamil. Springer-Ver- lag, Berlin, Heidelberg, New York, Tokyo, pp 90-96

Strigl R, Gastroph G, Hege HG, D6ring P, Mehring W (1980) Nachweis yon Verapamil im mfitterlichen und fetalen Blut des Menschen. Geburtsh u Frauenheilk 40:496-499

Todd EL, Abernethy DR (1986) Pharmacokinetics and dynamics of ( +)-verapamil in lean and obese zucker rats. J Pharmacol Exp Ther 238:642-647

Todd EL, Abernethy DR (1987) Physiological phannacokinetics and pharmacodynamics of (~e)-verapamil in female rats. Biopharm Drug Disp 8:285-297

WolffF, Breuker KH, Schlensker KH, Bolte A (1980) Prenatal diagnosis and therapy of fetal heart rate anomalies: with a contribution on the placental transfer of verapamil. J Perinat Med 8:203 -208

Trnccone N, Mariona F (1985) Intrauterine conversion of fetal su- praventricular tachycardia with combination of digoxin and vera- pamil. Pediatr Pharmacol 5: 149-153

Yoshida T, Kanamori S, Hasegawa Y (1988) Hyperphalangeal bones induced in rat pups by maternal treatment with nifedipine. Toxicol Lett 40: 127-132