Comparative teratological study of stobadin in vivo and in vitro

Toxic. in Vitro Vol. 7, No. 6, pp. 803-807, 1993 Printed in Great Britain. All rights reserved

0887-2333/93 $6.00 + 0.00 Copyright © 1993 Pergamon Press Ltd

COMPARATIVE TERATOLOGICAL STUDY OF STOBADIN IN VIVO A N D IN VITRO K. MIH,~,LIKOV~t*,E. UJH,~ZYt,E. BRAXATORISOV.~*and P. KU~ERA~ *Institute of Preventive and Clinical Medicine, Bratislava, tInstitute of Experimental Pharmacology, SAS Bratislava, CSFR and :~Institute of Physiology, University of Lausanne, Switzerland Abstract--Stobadin (STO) is a prospective cardioprotective drug with antiarrhythmic and antihypoxic effects on the myocardium. Single iv injections of stobadin administered to rats on days 3, 6, 9 or 12 of gestation at doses of 2 and 6 mg/kg had no teratogenic effect. Slight foetal toxicity was manifested by decreased foetal weight (day 3 of gestation, 6 mg/kg) and increased incidence of delayed ossification of the skull (day 12 of gestation, 6 mg/kg). In vitro studies were performed on chick embryos explanted at Hamburger and Hamilton (HH) stages 4-5 and cultivated in a medium with stobadin concentration ranging from 10-3 to I0 -8 mol/litre under standard conditions. Concentrations of 10-3 and 10-4 mol/litre were lethal. Embryos treated with concentrations from 10 -8 to 10 -5 mol/litre were comparable to those of the control group. The results of in vivo and in vitro tests showed that the antiarrhythmic agent stobadin at concentrations up to the maximal iv therapeutic dose had no overt effects on different developmental stages of the rat embryo and early chick embryogenesis.

INTRODUCTION Stobadin, cis - ( - ) - 2, 3,4,4a, 5, 9b - hexahydro-2, 8- dimethyl-1H-pyrido-[4,3-b] indole (STO) is an effective cardioprotective drug with antiarrhythmic and antihypoxic effects on the myocardium (Beneg and gtolc, 1989). The pharmacology, pharmacokinetics and biotransformation of this drug were described in several studies (Kfillay et al., 1990; ~oltfs et al., 1991; ~tefek et al., 1987; ~tolc et al., 1985). Preclinical toxicological studies on rats (Babinskfi et al., 1984) and beagle dogs (Majerfik et al., 1984) showed a low order of toxicity and did not reveal any unusual toxicological properties of STO. A teratological study of STO in mice (Zeljenkovfi et al., 1986) and reproductive toxicity studies in rats (Balonovfi et al., 1991) showed no overt effects on embryogenesis in these species. Some evidence of adult toxicity in rats (reduction of body weight gain, sedated behaviour) and decreased body weight of the young on day 21 post p a r t u m were evident in a general reproductive study of STO, at the dose of 50 mg/kg. Administration of STO during organogenesis had no overt effects on rat embryofoetal development except for an increased incidence of some skeletal variations (skull, sternebrae, ribs) in all the groups studied (Balonovfi et al., 1991). Because the above-mentioned studies indicated slight maternal and foetal toxicity in the mammalian embryos, we wanted to compare these findings with direct effects of STO on early chick embryo development in vitro. For the evaluation of potential

adverse effects of STO on chick embryos in vitro the standardized method of Kufera and Burnard (1987) was used. MATERIALS AND METHODS

Animals

Virgin female SPF Wistar rats (220-240 g, aged 12-14 wk) obtained from IEP (SAS Breeding Laboratories, Dobrfi Voda, CSFR), were housed in an airconditioned room with a 12-hr photoperiod (22-24°C, 50-60% relative humidity). F o o d and tap water were provided ad lib. They were mated by housing three females with one male. Pregnancy was confirmed by the presence of sperm in a vaginal smear and this was considered to be day 0 of gestation. The fertilized eggs of Rhode Island hens were obtained from the Poultry Research Institute (Ivanka pri Dunaji, CSFR). The eggs, weighing up to 60 g, were washed in distilled water and preincubated for 20 hr at 37.5°C and 60% relative humidity. Drug

STO, as a dihydrochloride salt (DH 1011, tool. wt 202.3) was synthesized at the Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague, CSFR. Experimental procedure In vivo study. STO, dissolved in physiological saline, was given in a single iv injection to rats on days 3, 6, 9 or 12 of gestation at 2 or 6 m g / k g at a constant volume of 0.1 ml/100g body weight. Control animals were dosed with vehicle only. The dose levels represent approximately two and six times the anticipated maximal iv therapeutic dose (1 mg/kg). The general conditions of the dams as well

Abbreviations: HH = stages of Hamburger and Hamilton; STO = stobadin, cis-(-)-2,3,4,4a,5,9b-hexahydro-2,8-

dimethyl-lH-pyrido-[4,3-b] salt).

indole

(dihydrochloride 803

K . MIFL/~LIKOV,~ et al.

804

a s the symptomatology of the toxic effects of STO were checked daily. The females were killed on day 20 of gestation. The corpora lutea in both ovaries were counted. The uteri were examined for the sites of implantation and for the number of resorptions, live and dead foetuses. The live foetuses were removed and their wet weight recorded. All foetuses were inspected for external malformations. One-third of the foetuses from each litter were used for assessment of skeletal defects (Lorke, 1977) and the remainder were fixed for soft tissue examination (Wilson, 1965). In vitro study. Chick blastoderms at stages 4-5 HH (Hamburger and Hamilton, 1951) were explanted from eggs together with a large part of the vitelline membrane and transferred to transparent silicone chambers. The vitelline membrane with blastoderm was spread over the ring protruding from the bottom of the chamber. Culture medium (modified Tyrode solution with thin egg albumen) was injected below and dropped over the membrane in constant volumes of 1 ml and 501~1, respectively. The chambers were closed with plexiglass lids and the embryos were then incubated at 37.5°C and 60% relative humidity. The whole procedure was carried out under sterile conditions. STO was dissolved in the culture medium at

concentrations

of

10 - 3 , 10 - 4 , 10 - 5 , 10 - 6 , 5 x 10 - 7 ,

10 -7 and 10 -8 mol/litre. For each concentration, 9-17 embryos were used. A total of 19 control embryos were used. The distribution of the concentrations and the controls among all explanted embryos was carried out randomly. We also incubated 29 controls in ovo under the same conditions. The development of embryos was observed under a stereoscopic microscope after 24, 48 and 72 hr of culture in vitro. The morphological characteristics and the developmental stage attained by each embryo were recorded. Finally, all embryos were classified as live normal, live abnormal, or dead; the group of live abnormal embryos included those that were malformed and

developmentally retarded. The development and survival of in ovo controls were evaluated on day 7 of incubation. Statistical evaluation

The data obtained were statistically evaluated using Student's t-test for reproductive data and litter values in rats, chi-square test in two-by-two tables and one-tailed Fisher's exact test for numbers of normal and damaged rat and chick embryos (Sachs, 1978). RESULTS

In vivo study

No female died throughout the study. On any day of medication, immediately after injection of the higher dose (6mg/kg) of STO to pregnant rats, saccade abdominal respiration, hindlimb tremor and sedated behaviour persisting for about 15 min were observed as the major clinical symptoms. A summary of the reproductive parameters measured in pregnant rats is presented in Table 1. No significant differences were recorded in litter size or in pre- and postimplantation losses. The mean foetal weight was significantly decreased only after treatment on day 3 of gestation in the 6 mg STO/kg group; however, a significant increase in mean foetal weight was observed after treatment on day 12 of gestation in both STO dose groups. The types of skeletal anomalies of foetuses and their frequency are listed in Table 2. Significantly increased incidences of delayed ossification of the parietal and supraoccipital bones occurred only after treatment on day 12 of gestation in the 6 mg STO/kg group. A dose-dependent increase in sternebral anomalies was seen after treatment on day 3 or 12 and in thoracolumbar rudimentary ribs on day 6 of gestation in comparison with controls. Compared with the controls, the incidence of other skeletal

Table I. Influence of single intravenous injection of dihydrochloride salt of stobadin (STO) on embryofoetal development of rats Foetal loss (%) Day of treatment

Dose (rag STO/kg)

No. of dams

Litter size1"

3

Control 2 6

9 9 9

10.10 _+0.61 10.40 ± 1.44 9.89 + 1.53

20.17 _+ 3.15 14.05 + 4.29 25.99 + I 1.49

12.33 _+4.43 6.82 ± 2.70 6.18 + 2.66

3.30 ± 0.03 3.24 ±. 0.02 3.15 ± 0.04*

6

Control 2 6

8 9 8

10.00 _ 1.62 9.78 -i- 1.23 11.10 ± 1.26

23.42 _+ 10.29 17.73 _ 8.31 9.75 + 4.40

9.34 _+ 3.98 13.52 + 5.56 10.74 ± 7.01

3.33 ± 0.03 3.35 ± 0.04 3.42 ± 0.03

9

Control 2 6

9 l0 11

7.55 ± 1.49 9.50 +_ 1.10 8.45 ± 1.26

32.60 _+ 10.30 21.08 + 6.12 26.36 _+ 8.95

23.42 _+ 7.61 12.59 ± 4.76 13.06 ± 4.20

3.21 ± 0.07 3.07 ± 0.04 3.23 ± 0.07

12

Control 2 6

9 8 l0

10.40 + 0.83 10.90 + 0.85 9.70 +_ 1.30

I 1,42 _+ 3.72 7,67 ± 3.35 23,68 _+ 9.10

17.72 +_ 3.92 13.96 ± 4.77 7.60 ± 2.39

3.18 ± 0.03 3.28 ± 0.03* 3.29 ± 0.03*

+ SE. tTerm foetuses. :[:Corpora lutea--implant sites/corpora lutea ( × I00). §Implant sites--viable foetuses/implant sites ( x I00). *Significantly different frorr control: P ~<0.05 (Student's t-test). V a l u e s are m e a n s

Preimplantation:~ Postimplantation§

Foetal weight (g)

Teratological study of stobadin

805

Table 2. Skeletal anomalies of rat foetuses after intravenous injection of stobadin dihydrochloride (STO) to dams on different days of gestation Skeletal anomaly No. of Day of Dose foetuses/ Pelvic treatment (mg STO/kg) litters Skull~" Sternebrae:l: R i b s ~ Forefootll Vertebrae¶ bone'M" Hindfoot:l::l: 3 Control 60/9 0 13/8 30/9 0 0 0 0 2 61/9 1/1 20/6 30/7 0 0 0 0 6 60/9 0 24/8 36/6 0 1/1 0 0 6 Control 52/8 2/1 12/5 29/7 1/1 0 0 0 2 57/9 0 13/7 36/9 0 0 0 0 6 58/8 3/1 5/4 40/7 0 0 0 0 9 Control 43/9 1/1 14/6 36/9 0 0 0 0 2 63/10 7/5 27/9 30/8 0 l/I 0 0 6 63/11 5/3 15/5 37/10 1/1 0 3/I I/I 12 Control 64/9 I/I 24/7 50/9 0 0 0 0 2 59/8 3/I 26/8 30/8 0 0 0 0 6 65/10 9/4"* 27/9 33/8 0 1/1 0 0 '['Delayed ossifcation of the parietal and supraoecipital bone. :~Unossified and retarded sternebrae. §14th thoracolumbar rudimentary and wavy fibs. I[Retarded ossification of the metacarpals. ¶Split vertebral centra of the thoracoabdominal region. ttlneomplete ossification of the pelvic bone. :l::l:Retarded ossification of the metatarsals. **Significantlydifferent from control: P ~<0.01 (Fisher's exact test).

anomalies gave no significant indication o f teratogenicity in any o f the treated groups. A s u m m a r y of visceral anomalies is presented in Table 3. N o significant increase in the incidence o f any a n o m a l y (dilatation o f the cerebral ventricle a n d renal pelvis, testicular malposition) was observed in either t r e a t m e n t g r o u p c o m p a r e d with controls.

In vitro study The n u m b e r s o f live normal, live a b n o r m a l a n d dead e m b r y o s for each c o n c e n t r a t i o n are s h o w n in Table 4. The c o n c e n t r a t i o n s of STO from 10 -3 to 10-Smol/litre were considered to be toxic, as the survival a n d n o r m a l d e v e l o p m e n t o f e m b r y o s were significantly decreased at c o n c e n t r a t i o n s o f 10 -3 a n d 10 -4 mol/litre. A t 10 -5 mol/litre, the survival was also decreased b u t n o t to a statistically significant extent. There were n o significant differences in the n u m b e r s o f n o r m a l a n d d a m a g e d e m b r y o s in the remaining c o n c e n t r a t i o n groups c o m p a r e d with the control.

Different degrees o f d a m a g e were observed in the m a l f o r m e d e m b r y o s a n d these were c o n c e n t r a t i o n dependent, ranging from complete decomposition to slight growth retardation. Complete decomposition o f the e m b r y o occurred at c o n c e n t r a t i o n s o f 10 -3 a n d 10 -4 mol STO/litre, H e a r t enlargement, decreased somite n u m b e r a n d lack o f d e v e l o p m e n t o f the caudal part o f the embryonic body were o b s e r v e d at 10-5 mol STO/litre. E m b r y o s w i t h o u t cervical flexure a n d body torsion were f o u n d after 48 hr o f culture'in the c o n c e n t r a t i o n range from 10 -6 to 10-Stool STO/litre. N o r m a l d e v e l o p m e n t a n d survival o f embryos in the 10 -6 moi/litre g r o u p was even better t h a n in the control g r o u p b u t this difference also was n o t statistically significant. DISCUSSION Evaluation o f the safety o f cardiovascular drugs during pregnancy becomes m o r e i m p o r t a n t with their

Table 3. Visceralanomalies of rat foetuses after intravenous injection of stobadin dihydrochloride to dams on different days of gestation No. of Visceral anomaly Day of Dose foetuses/ treatment (mg/kg) litters DCV DRP TM 3 Control 31/9 0 1/I 0 2 33/9 0 0 0 6 29/9 0 3/3 0 6 Control 28/8 0 0 0 2 31/9 0 I/1 0 6 31/8 0 0 1/1 9 Control 25/9 0 l/I I/I 2 32/10 I/1 2/2 0 6 30/11 1/1 3/3 0 12 Control 30/9 0 0 0 2

28/8

0

0

0

6 32/10 0 I/1 0 DCV = dilatation of the cerebral centficles DRP ffi dilatation of the renal pelvis, hydronephrosis TM = testicular malposition

K. MIHALIKOVAet al.

806

Table 4. In vitro developmentof chick embryosat differentconcentrations of stobadin dihydrochloride(STO) 24 hr in vitro 48 hr in vitro 72 hr in vitro Treatment No. of (mol STO/litre) embryos LN LA D LN LA D LN LA D (Control in vitro) 19 6 9 4 3 9 3 2 3 7 10 s 10 2 3 5 1 2 2 0 3 0 10 7 10 1 3 6 0 2 2 0 0 I 5×10 7 9 1 7 1 I 6 1 0 2 I 10 6 17 7 8 2 4 8 3 2 1 0 10 s 11 I 8 2 1 3 5 0 0 4 *10 4

I1

0

7

4

0

I

6

0

0

1

*10 3 9 0 2 7 0 0 2 0 0 0 (Control in ovo)~ 29 19 6 4(NS) LN = number of live normal embryos LA = number of live abnormal embryos D = number of dead embryos *Significantly different from in vitro control P < 0.05: (chi-square test in two-by-two tables, Fisher's exact test). tln ovo control after 7 days of incubation. NS = not significant (compared with in vitro control).

increased therapeutic use. Nevertheless, relatively little is known about their possible adverse effects on human reproduction. Clinical studies have, in the main, been restricted to investigation of the direct actions and side effects of, for example, quinidine, procainamid, disopyramide and phenytoin (foetal hydantoin syndrome; Tamari et al., 1982, Witter et al., 1981). In animal studies, after administration of antiarrhythmic drugs [aetmozine in rats (Lyubimov et aL, 1976) and pirmenol hydrochloride in rats and rabbits] no particular reproductive or teratogenic findings were recorded (Anderson et al., 1986; Martin and de la Iglesia, 1987; Schardein et al., 1980). N o teratogenicity, but decreased foetal weight, was reported with another antiarrhythmic drug, disopyramide (Jequier et al., 1970). In our teratological study in vivo single iv injections of STO (6 mg/kg) to pregnant rats on different gestational days caused acute clinical symptoms that may have been attributable to the hypotensive and sedative effects of iv injection of STO, previously described in rats, guinea pigs, cats and dogs (Bene~ and ~tolc, 1989). A reduction in foetal weight (95.6% of control weight) was recorded only on day 3 at 6 mg STO/kg. The minor skeletal anomalies detected in our study may be regarded as c o m m o n skeletal variations that represent transient developmental changes correlating with inhibition of foetal growth (Schardein, 1987). STO caused no malformations of the main visceral organs in the thoracic and abdominal cavities. The chick embryo in vitro assay allows close observation of the early developmental period (Kucera and Burnard, 1987 and 1988). The results of this in vitro study of the effects of STO at different concentrations showed no difference between treated and control groups, except for concentrations of 10 3 and 10-4 mol/litre, which were lethal. The therapeutic dose of STO (1 mg/kg) represents approximately 5 × 10 -6 mol/litre, which corresponds to the levels of 1000 ng/g found in rat placentas and foetuses 60 min after oral administration of 5 mg STO/kg (Kri~tofovfi

et al., 1991). Reduced survival in the control group

was due to the poor viability of the fertilized eggs, as shown by the survival of the in ovo control group (no significant difference between the two control groups). The results of a previous investigation of the effects of STO in ovo (0.4, 0.8 mg/egg) also showed no adverse effects on the late stages of chick embryogenesis. The decreased survival of embryos was recorded only after the dose of 1.6 mg/egg injected on day 5 of incubation (Ujhfizy et al., 1988). The comparison of mammalian and avian models can help to elucidate potential adverse effects of substances under test. In addition mice, rats and chicks share a similar period of development, about 21 days of gestation or incubation. In the early stages of development through the period of major organogenesis, the rates of their development are fairly uniform (Schneider and Norton, 1979). The results of in vivo and in vitro tests showed that the antiarrhythmic agent stobadin at concentrations up to the maximal iv therapeutic dose had no overt effects on different developmental stages of the rat embryo and early chick embryogenesis. Acknowledgements--The authors are grateful to Dr Tomfi~

Trnovec, DSc, for critical review of the manuscript. We are also indebted to the European Teratology Society for support enabling the poster presentation of this paper at the 3rd Vertebrate Whole Embryo Culture Symposium, 8 I1 November 1992, Basel, Switzerland.

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