Some comparative endocrinepharmacological aspects and their relevance in the interpretation of adverse drug effects

Pharmac Ther Vol 5, pp 271-286, 1979 PergamonPress Lid

SOME

COMPARATIVE ASPECTS AND INTERPRETATION

Printed m Great Britain

ENDOCRINEPHARMACOLOGICAL THEIR RELEVANCE IN THE OF ADVERSE DRUG EFFECTS* F

NEUMANN

Research Laboratories of Schenng AG, Bedm-Bergkamen, Department o] Endocrine Pharmacology, 1 Berhn 65, Mallerstrasse 170-178, Federal Republic of Germany

and K - J . Gp.AF Free UmversUy Berhn, Khn~um Charlottenburg, Department of Internal Medicine, 1 Berhn 19, Spandauer Datum 130, Federal Republic of Germany

1. INTRODUCTION There are two Important phenomena m female comparative reproducUve physiology. These are" (a) that the same hormones or drugs with hormonal actions can have different effects m different species; and (b) that the same reproducUve processes can m part be regulated by different sexual hormones or different interactions of steroids with pepttde hormones. An added difficulty is the fact that the various hormones are secreted in absolutely different amounts and that the sensitivity of hormone dependent target organs differs from species to species (for review see Neumann and Elger, 1971, 1972). It is the aim of this paper to show, by means of a few selected samples, just how important it is to take differences between species into account when designing toxicity studies and evaluatmg their results. 2. EFFICACY OF F E M A L E SEX HORMONES IN DIFFERENT SPECIES: DOSE SELECTION, DOSE-FINDING, ROUTE OF ADMINISTRATION The problem of dose finding has been discussed in detail very recently (for review see Neumann et al., 1977a). As regards the choice of dosage for rats and mice, st is recommended that twzce or three times the therapeutic human dose calculated on a mg/kg basis be used as the lowest dose. The highest dose should be the one which just fails to shorten the life of the animals, and a third dose should be selected which hes between the other two (Meeting between the Committee on Safety of Drugs and eighteen Pharmaceutical Companies, 1966---personal communication 1970). Similarly, the required dose for long-term toxicity studies m dogs and monkeys are related to multiples of the human dose Dogs should be treated with two, ten, and twenty-five times the human dose (Recommended protocol for chronic oral contraceptlve studies, 1968; Goidenthal, 1969) The route of administration is, of course, the same as m man. With this procedure, however, the biological effect|veness of a certain substance in the species concerned is completely ignored and, even worse, it is frequently not even taken into account, e.g. many steroids do not become effective at all with the route of administration employed We will explain this by means of two examples The slahc acid test, which can be regarded as a parameter of mucm content, has proved its value for determining the ant|oestrogenlc effect of steroids (Nlshino and Neumann, 1974). *Internat,onal W o r k s h o p on Pharmacological Methods m Prechmcal Safety-Evaluatmn of N e w Drugs, Zurich (Sw,tzerland) October 10-13, 1977 271

F NEUMANN and K J

272

GRAF

The bmloglcal principle of this test is that the slahc acid content m the m o u s e vagina is similarly h o r m o n e - d e p e n d e n t as the sIahc acld content m the cervical secreUon of w o m e n It is low in oestrus and high in dloestrus In castrated mice, the administration of oestrogens leads to a d o s e - d e p e n d e n t d e c r e a s e of the slahc acid content in the vagina. The oestrogen-tnduced decrease of the slahc acid content can be c o u n t e r a c t e d d o s e - d e p e n d e n t l y by progestogen treatment. The experimental procedure of this test model Is depicted in Fig. 1. Mice are o v a r i e c t o m l z e d , and beginning on the tenth day thereafter the a m m a l s e a c h receive 0.1 ~zg oestradiol subcutaneously once a day for three d a y s I m m e d i a t e l y a f t e r this, the test substance (progestogen) is administered o v e r 5 days, c o m b i n e d with the s a m e dose of oestradiol The a u t o p s y is p e r f o r m e d on the ninth day, and the siahc acid content is determined in the p r e p a r e d vaginas (further details of this method can be f o u n d in N l s h m o and N e u m a n n , 1974). If the clinical t r a n s f o r m a t o r y d o s e s of progestogens on the h u m a n e n d o m e t n u m are c o m p a r e d with the lowest e f f e c t w e doses in the smhc acid test (antloestrogen test in the mouse), it can be seen that e x t r e m e l y good correlations exist between both test models. T h e S p e a r m a n n - R a n k correlation coefficient is about 0.8, and significance exists at the 95 per cent level (see Fig. 2) A c o m p a r i s o n of the antmestrogenlc efficacy of various p r o g e s t o g e n s in this test model m the m o u s e with the usual oral m m P p d l dose m w o m e n also underhnes the usablhty of this test model for chnical dose finding (see Table 1). The definitmn of a mini-pill effective dose is that dose of a progestogen which prevents migration of the s p e r m a t o z o a through the cervical secretion but which =s usually not high enough to inhibit ovulatmn---or does so only in some women. Unfortunately, we k n o w the mini-pill doses for only a f e w progestogens, so that only four progestogens are listed in Table 1 H o w e v e r , It can be seen clearly that the substances which are m o s t effectzve in the m o u s e test are also a m o n g the chmcally m o s t potent progestogens. H o w e v e r , as regards the route of a d m i m s t r a t m n one should k n o w that m contrast to the h y d r o x y p r o g e s t e r o n e derivatives almost all 19-nortestosterone d e n v a t w e s are ineffective in this test model w h e n given orally It is thus clear that no, or only minor, effects are o b s e r v e d with these substances in toxicity and teratogemclty studies in rodents for which only an oral a d m l m s t r a t m n is required, since even 100 times the h u m a n dose, calculated on a kg/body weight basis, is stdl almost m e f f e c t w e m the m o u s e by this route of a d m m i s t r a t m n . L e t us give a simple mathematlcal example: On s u b c u t a n e o u s administration of d-norgestrel, the dose of 1.5/~g/mouse (this IS a b o u t

Antl~strogen assay m m,ce (Smhc acid assay)

'~stratlon

+ 1

÷ ++++,++++ 10

13

$ Estrogen prtmmg (0,1 /zg E2/animal/day sc ) -qPTest compound I~1 Autopsy (Parameter SJahcac,d content m the vagina)

FIG I

15

18 days

273

The interpretation of adverse drug effects T Endometnal transformation ,n w o m e n (mg/cycle) I 1 = Progesterone 1000 2 = Noreth,sterone acetate 3 = Chlormadmone acetate 4 = Cyproterone acetate 11"13. 5 = Norgestrel ,,~, 6 = D-Norgestrel '~I 7 = Norethmodrel 1008 = Lynestrenol 9 = Medroxyprogesterone ~ (~ ' acetate

®

10-~

1'

i °

rs = 0 , 7 6 2 5 0 +

'

0,0001

0,001

1

I

0,01

0,1

i

M,numal effect=ve dose ~n the s=ahc acid assay in m~ce (mg/anlmat/day sc ) FIG 2

TABLE 1

Gestagen

Stahc actd assay m mice Effective dose range (/~g/ammal/day)

Relative potency (Chlormadmone acetate == 1")

"Mmtpfll" dose/women (rag/day)

Norethtsterone

10-100

8

06

0 3-10 30-1000

83 1

0 03 - 1

RelaUve potency (Chlormadmone acetate = 1") 17

acetate D-Norgestrel Chlormadmone acetate Cyproterone acetate

30-300

1

< 1

33 1 > 1

*Analysts of covarmnce

100 Umes the human oral contraceptive dose) lies within the e f f e c u v e dose range of 0 . 3 - 10.0 ~g, while it is far less than the effective dose on oral administration. The converse can, of course, also occur, that is, that several times the human dose can c o r r e s p o n d to several hundred umes the effective dose in the species c o n c e r n e d in toxicity studies In this case it is only logical that an increased incidence of side effects will be observed which make the particular substance appear dangerous. This is shown by another example: Hill and co-workers (1970) have shown that, in a test p r o c e d u r e in the dog similar to the Clauberg test, c h l o r m a d m o n e acetate was considerably more effecUve than norethisterone, in contrast to Rs efficacy in w o m e n We (Graf et al., 1975) obtained ~ similar results using this less accurate preliminary progestogen test. The canine bmassay for progestogens was later refined, and the effect of different progestogens on the endometrium of the dog can now be tested more accurately (Haase et al., 1977) Figure 3 shows the test model. This progestogen test model, using 2-year-old o v a n e c t o m t z e d female beagle dogs, consists essentially of two treatment phases First of all the dogs received a priming dose of 30 ~ g oestradml per day up to day 15, after which the close was reduced to 3 ~ g per day In addmon, the various progestogen doses were given daily for another 15 days Uterine probe excismns were taken from the right or left u t e n n e horn on the last day of the treatment period and, after histological sections had been prepared,

F

274

NEUMANN a n d K - J

GR~F

Can,ne" B,oassay for Gestagens Procedure

r

J

> 4 weeks rest,rig per,od

_>4 weeks

O

,nvolut,on period

O

O

t

O

!

Ovar,ectomy

!

(B,opsy)

B,opsy

=Esc = Test compound (Gestagen)

I

"Adult beagles (-8-10 kg B W }

FIG 3

were evaluated by means of a modified McPhail scale by at least two investigators. Using this test model, the progestaUonal potencies of four synthetic progestogens were examined and the threshold doses determined. The progestogens investigated first, using this progestogen test model, were two 17a-hydroxy-progesterone derivatwes (cyproterone acetate and chlormadinone acetate) and two 19-nortestosterone derivatives (d-norgestrel and norethisterone acetate). The results of these experiments are given in Table 2. The comparison of the progestational potencies of these compounds after oral administration with that of progesterone given subcutaneously revealed that the 17a-hydroxy progesterone derivatwes were relauvely much more potent---by a ratio of about 30--100--than the 19-nortestosterone derivataves. If one compares these data with the corresponding situation m women, one finds an inverse relatmnship in that the 19-nortestosterone derivaUves are more active in respect of the endometrial transformatory potency than the 17a-hydroxy progesterone derivatives (see Fig. 4). Table 3 shows the lowest dose of d-norgestrel and cyproterone acetate which are effective in the dog m relation to twenty-five times the human dose in the respective OC preparations on a kg/body weight basis. In the case of d-norgestrel, the highest dose used (twenty-five tunes the human dose) is still three times lower than the lowest effective dose in the dog, whereas in the case of cyproterone acetate, the twenty-fiveT ~ L ~ 2. Gesta~remc Endomemal Trans[ormaaon in Dogs Compound (rag/day) 30.0 I00 30 10 03

01 0 03

P s.c.

CPA s c.

p o

+ + -

CMA s c

p o

d-N s c

+

+ +

+

-

+ +

+ +

+

.

+ .

.

p o.

ENTA s.c

p o + + -

+

+

-

.

- = n o transformaUon, + = dmtmct or c o m p l e t e transformation, P = Progesterone, C P A = C y p r o t e r o n e acetate, C M A = Chlorm_~Jmone acetate, d - N = d-Norgestrel, E N T A = N o r e t h m t e r o n e acetate. If progesterone ( s . c ) m c o n s | d e r e d as a reference preparatmn = I c o m p a r a b l e g e s tallem© potencies w e r e then f o u n d to be for s.c a l ~ h c a n o n " x I00 for d - N , C M A ; x 30 for C P A , × I 0 f o r E N T A . p.os. apphcatmn x 30 f o r C P A , C M A ; x 3 f o r d - N ; x I for ENTA

275

The mterpretauon of adverse drug effects

RELATIVE POTENCIES (13o ) xP(sc)-I

Women (13o )

Dog

xP(im)-t CMA

dN

UJ

3C3

- 100

CPA ENTA

-30

CPA C M A

1E)

-10

dN P(sc) 1

3

~ENTA

P = Progesterone CPA - Cyproterone acetate CMA = Chlormadmone acetate

t

dN -- d-Norgestrel ENTA = Norethlsterone acetate

FIG

4

TABLE 3

D-Norgestrel* Effectwe oral dose m dogs Clauberg assay (mg/kg) OC-dose (mg/kg) Human dose/k8 x 25 Relanon of human dose x 25/minimal effecttve dose m dogs

03 0 005 0 125 -0 4

Cyproterone acetatet 0 03 0 04 10 ~30

*Neogynon ® tSH 209 AB

fold human d o s e is already about thirty times higher than the smallest effecUve dose m the dog It can therefore be sa~d that, for the dog, cyproterone acetate Is dosed about 100 Umes h~gher than d-norgestrel. In c o n s e q u e n c e , ~t can almost be presumed that the results of t o x i c o l o g y findings will be in a c c o r d a n c e with this. T o stay with this example, no one ts in a p o s m o n to undertake an estunatmn o f the benefit-risk relationship of d-norgestrel and c y p r o t e r o n e acetate and produce anything but a false mterpretauon unless these facts are taken into account. Thus, with a purely formal procedure, substances which are less e f f e c t w e m a parUcular species wall always appear better, and vice versa In c o n s e q u e n c e to the e x a m p l e s g w e n above, it must be asked h o w the relative risk for man can be estimated based on toxicity m o d e l s in w h i c h even the e f f e c t w e n e s s of a substance, according to the r e c o m m e n d e d route of administration, is ignored as well as without c o n s i d e n n g any species specific differences in biological p o t e n c y In our opinion it w o u l d be n e c e s s a r y either to administer d o s e s m multiples o f the minimal e f f e c t w e dose m the species c o n c e r n e d , w h e r e b y , ff n e c e s s a r y , the route of administration could be different from that used m man, or---and this appears to be the b e s t m t o administer multiples o f e q m e f f e c t i v e d o s e s m the species regarded c o m p a r e d to the e f f e c u v e d o s e in man In our opinion, anything else is unscientific and does not contribute to greater safety In another lecture o f this w o r k s h o p (El Etreby et al., 1977) It is s h o w n that it may depend solely on the bmloglcal efficacy of a progestogen in the dog whether or not m a m m a r y gland nodules will occur in c h r o m c toxicity studies, although s o m e m e c h a n i s m s spectfic to the dog are also certainly revolved. Although there are m a n y more, these e x a m p l e s should suffice for the m o m e n t

276

F NEUMAN~ and K - J GRAF

30ESTROGEN/PROGESTOGEN I N T E R A C T I O N IN D I F F E R E N T S P E C I E S AND S P E C I E S D I F F E R E N C E S IN T H E R E A C T I O N TO PROGESTOGENS WITH OESTROGENIC PARTIAL EFFECTS Th~s aspect becomes ~mportant when ~t Is necessary to test oestrogen/progestogen m~xtures m chromc toxicity studxes or when we are dealing with progestogens which also have inherent oestrogemc partml effects. We should like to give JUst a few examples of th~s If the reproductmn processes in mammals are to take their normal course, certain h o r m o n e s must interact m certain ratios w~th one another at certain times Th~s is true m particular for the interactions of oestrogens and progestogens. Progestogens in g e n e r a l - - a n d th~s goes also for p r o g e s t e r o n e - - a r e almost lnvarmbly only effective after appropriate oestrogen priming or together w~th oestrogens. H o w e v e r , It is very important to know that the optimal oestrogen/progesterone ratm ts d~fferent for each species. Table 4 shows this using the decldual reaction as an example (Dubots et al., 1964; Yochlm and de Feo, 1962; Chambon, 1949; G o o d and Moyer, 1968). The optimum for the rat Is an oestrogen/progestogen ratio of about 1.10,000 to 1 20,000; for the rabbit the ratio ~s 1 : 1000; for the rhesus m o n k e y 1" 50; and for the woman the optimal oestrogen/progesterone ratm hes between 1 50 and I 100. Another example is presented m Table 5, which shows the importance of the oestrogen/progesterone ratm for the mductmn of an e n d o m e t n a l reaction by various oestrogen/progesterone mixtures m the rabbit (Gillman and Stein, 1942). It can be seen that an e n d o m e t n a l reactmn Is reduced only at an oestrogen/progesterone ratm of about 600.1. T h e r e ~s pracUcally no progestational effect at ratios less than 100: 1. In most OC preparations, the ratm of ethlnyl oestradiol to a syntheuc progestogen is between about 1 • 5 and 1 80 The ratm is narrower with more potent progestogens than with less effective compounds (see Table 6) Now, when such c o m b m a t m n s are tested in the rat, for example, virtually only the oestrogen c o m p o n e n t exerts an effect at this raUo--quite m contrast to the s~tuatmn in primates All changes which o c c u r with such a formal procedure of simple transference of the oestrogen/progestogen ratm to kg/rat or to a multiple of ~t are oestrogen-mduced. We do not wish to go into further detail here e x c e p t to say that furthermore we know that, depending on the target organs regarded and the species used, both steroids can act synergistically (e.g TAI3LE4 0 p t l m u m E s t r o g e n / P r o g e s t e r o n e R a t z o a n d 4 b s o l u t e D o s e s ( A m m a l / D a y ,gwen

Subcutaneously) for Producing the Decutuai Reaction m Vanous Species Specms Hamster Rat Rabbit Rhesus monkey

Estrachol- 17l/ (~tgJ

Progesterone (ttg)

not essential 0 1 1 0-1 3 20-40

2000 2000 1000 1000-2000

E/P ratio I -1 -1 ~ i'

® 20,000 1000 50

Reference Dubols et al, 1964 Yochlm and de Feo, 1962 Chambon, 1949'o Good and Moyer, 1968

TABLE 5 Elect of Varymg Amounts of Oestrogen m Combmatwn with Progesterone on the Endometnal Reaction of Ovanectomlzed Rabbits (7 Days of Pretreatment with a Total of 0 07 rag of Oestrone or 0 00588 mg

of Estradlol Benzoate, then Progesterone + Oestrone Twice a Day for 4 days ) (After GUlman and Stein, 1942) Dosage (mg) Oestrone Progesterone 02 001 008 004 002 00025

075 075 15 15 15 15

Endometnai reaction 0 0 0 0 + +++

Progesterone/Oestrone rauo 375 7.5 185 375 75 600

1 1 1 1 ! 1

The mterpretatmn of adverse drug effects

277

TABLE 6 ProgestagenlOestrogen Ratzo m s o m e Combination Contraceptwes

ProgestagenlOestrogen ratso Noreth~sterone acetate + E E * Cyproterone acetate + EE'~ D-Norgestrel + EE:~

80 1 40 1 5 I

*Anovlar ® i'Dmne ® ~;Neogynon ®

as regards the antlgonadotrophlc effect) or antagonistically (e g as regards the antloestrogen~c effect on the e n d o m e t r m m ) . This, of course, ceases to a p p l y w h e n the ratio ~s no longer correct for the p a m c u l a r species W e are then deahng wRh an Inadequate model which permits no estimate of the benefit-risk ratio. It is t h e r e f o r e our o p m m n that o e s t r o g e n / p r o g e s t o g e n mixtures m u s t be investigated at a ratio which Is a p p r o x i m a t e l y physiological for the species concerned. T h e s~tuatlon b e c o m e s e v e n m o r e problematical m the case of p r o g e s t o g e n s with o e s t r o g e m c partial effect, for e x a m p l e norethlsterone or n o r e t h y n o d r e l (for r e w e w see N e u m a n n , 1968) It ~s known, f o r m s t a n c e , that almost all p r o g e s t o g e n s are capable of developing the rabbit e n d o m e t n u m (Clauberg test) Various 19-nortestosterone derivatives, on the other hand, are not able to m a m t a m p r e g n a n c y m castrated rats. T e n years ago this fact was the c a u s e of a sc~enttfic argument, w h e n various m v e s U g a t o r s m a d e a disuncUon b e t w e e n 'real g e s t a g e n s ' - - i e p r o g e s t o g e n s which are effective not only m the rabbR, but are also able to m a m t m n p r e g n a n c y m the c a s t r a t e d rat and to reduce dec~dual r e a c u o n s , and ' p r e - g e s t a g e n s ' - - l . e . p r o g e s t o g e n s which, although e f f e c u v e m the rabbR, are not effecUve m the rat and m o u s e ( M a d j e r e k et al., 1960). This was a quarrel a b o u t nothing, s m c e the optimal progest e r o n e / o e s t r o g e n raUo is m u c h n a r r o w e r m the rabb~t than in the rat and mouse, which explains w h y p r o g e s t o g e n s wRh o e s t r o g e m c partial effects are effective m the rabbit but not m the rat Since the optimal p r o g e s t e r o n e / o e s t r o g e n raUo In m a n is in turn ten u m e s lower than m the rabbR, oestrogenlc partml effects (with the possible exception of norethynodrel) play no further role at all It is thus certmn at least that findings such as those which o c c u r w~th such c o m p o u n d s m c h r o m e toxicity studies in rats h a v e no r e l e v a n c e for man---or at least c a n n o t be easily e x t r a p o l a t e d We h a v e already d e m o n s t r a t e d th~s in detail using noreth~sterone o e n a n t h a t e as an e x a m p l e ( N e u m a n n et al., 1974) In s u m m a r y , s m c t l y speaking, p r o g e s t o g e n s with o e s t r o g e m c potencies should be tested m species m which the o e s t r o g e m c parual effect does not, or only to a hmRed extent, interfere wRh the progestatlonal effect as, f o r example, in m o n k e y s ff one wishes to m~m~c the sltuatmn m man.

4 I N T E R A C T I O N B E T W E E N S T E R O I D (SEX) H O R M O N E S OTHER DRUGS AND PEPTIDE HORMONES

OR

Besides s p e c i e s - d e p e n d e n t differences m biological p o t e n c y and differences m strength of c e r t a m partial effects, such as the oestrogenlclty of certain progestogens, the Interaction of steroid (sex) h o r m o n e s with peptide h o r m o n e s also differs m a r k e d l y f r o m species to species After the p n n c l p l e interactions of sex h o r m o n e s with the anterior pituitary as well as with certain h y p o t h a l a m l c centers forming a functional unit had been discovered, various species-specflic pecuharlUes within these m e c h a n i s m were f o u n d which further c o m p h c a t e the m t e r p r e t a t l o n of pharmacological effects f r o m the v~ew of c o m p a r a t i v e e n d g c n n o l o g y . Thxs, of course, is especially true for the interpretation of tOXiCological findings relevant to m a n as s h o w n in the following e x a m p l e s JP'r ~ l/3--s

278

F NEUMANN and K - J

GP.AF

In most speczes ~t =s Impossible to stimulate tubulo-alveolar growth of the m a m m a r y gland with oestrogens and progesterone or oestrogen/progesterone combmaUons m the absence of the p~tuitary (for revmw see Cowle and Tmdal, 1971, N e u m a n n and Elger, 1972). It has been shown that, m rats, oestrogens st:mulate, in a dose dependent manner, prolactm synthesis and secreUon by md~rect hypothalamlc actmns, as well as directly at the level of the antermr p~tmtary It =s also known that progesterone may act synerg=sUcally or antagonisUcally to the prolactm stlmulatory effect of oestrogens, again depending on the doses used (for review see Meltes and Clemens, 1972) Since m rats m a m m a r y gland and hypophyseal tumors were reduced by oestrogen treatment, we mztmted several experiments to mvesUgate the interaction of oestrogens and progestogens on rat m a m m a r y gland prohferaUon w~th special r e f e r e n c e to the role of prolactm As parameters for the extent of m a m m a r y gland prohferatlon the D N A content m the m a m m a r y gland Ussue was esUmated as well as the standard histology and whole mount preparations. Figure 5 summarizes the effect of increasing doses of oestrone, progesterone or oestron-progesterone combmaUons, each g=ven s.c for 18 days to ovanectom=zed rats. Whde m the doses used, oestrone and progesterone g~ven alone were meffecUve on the rat m a m m a r y gland D N A content, the combined' o e s t r o n e - p r o g e s t e r o n e treatment led to a dose-dependent increase. The histology as well as the mvesugatmns of whole mount preparations have been found to parallel these findings exactly (see Fig. 6). In Fig. 7 the prolactin concentrations m the various experimental groups are shown m comparison to the related D N A contents and indicate that rat m a m m a r y gland

IJg ONA m a m m a r y gland complex

12250"

i

mean +S E M () No of animals

2000-

FIG 5 Effect of increasing doses of oestrone, progesterone and o e s t r o n e - p r o g e s t e r o n e c o m b m a u o n s each g~ven s c for 18 d a y s to ovanectom~zed rats on D N A c o n t e n t / m g m n a l m a m m a r y gland c o m p l e x

The mterpretataon of adverse drug effects

279

p r o h f e r a t m n does not depend on increasing s e r u m p r o l a c t m levels but rather on the relatwe ratio of oestrone, p r o g e s t e r o n e and p r o l a c t m concentrations In a further e x p e r i m e n t the i m p o r t a n c e of p r o l a c t m within the m a m m o t r o p h l c h o r m o n e c o m p l e x m the rat was investigated m m o r e detatl, using h y p o p h y s e c t o m l z e d female rats bearing four p~tmtary grafts transplanted beneath the kidney capsules to secure p r o l a c t m release T h e s e rats r e c e w e d a c o m b i n e d t r e a t m e n t of oestradlol b e n z o a t e (5/zg/kg) and p r o g e s t e r o n e (50mg/kg) g w e n dally for 22 days wRh or wmthout CB-154 (2 mg/kg) or h s u n d e hydrogen maleate ( L H M ) (0 1 mg/kg), two potent prolactln mhlbRors (Graf et al, 1976; 1977b) As shown m Fig 8, the c o m b i n e d t r e a t m e n t with oestradlol b e n z o a t e and p r o g e s t e r o n e led to a m a r k e d stimulation of prolactln secreUon as well as to p r o l i f e r a u o n of the rat m a m m a r y gland Due to the a d d m o n a l t r e a t m e n t with CB-154 or L H M the r a d l o l m m u n o a s s a y a b l e p r o l a c t m as well as the D N A concentrations w e r e lowered distinctly, u n d e r h n m g the a s s u m e d role of prolactm m p r o h f e r a u v e changes at the rat m a m m a r y gland This has been further substantiated by an e x p e r i m e n t using intact f e m a l e rats treated daily for 6 w e e k s with either o e s t r a d m l - b e n z o a t e (5 mg/rat) or norethlsterone o e n a n t h a t e (4 mg/rat), a progestogen with o e s t r o g e m c potencies m the rat, or by a c o m b i n e d t r e a t m e n t regime of norethlsterone o e n a n t h a t e (4 mg/rat) together wRh CB-154 (0.3 mg/rat) (see Fig 9) As a result of this study it b e c a m e evident that oestradlol b e n z o a t e itself, or p r o l a c t m sUmulated by oestradlol benzoate, led only to a hmRed p r o h f e r a t l o n of the rat m a m m a r y gland w h e r e a s noreth~sterone o e n a n t h a t e was m u c h more e f f e c t w e m this r e s p e c t H o w e v e r , the m a r k e d sttmulatory effect of. norethisterone o e n a n t h a t e could be c o u n t e r a c t e d only m part by CB-154 This fact might suggest that CB-154 a n t a g o m z e d the oestrogen partml effect of norethlsterone o e n a n t h a t e by inhibiting the o e s t r o g e n / p r o g e s t o g e n st~mulatory action on p r o l a c t m secretion, ~mplying that the action of oestrogens at the m a m m a r y gland of the rat might be mediated via enhanced p r o l a c t m secreUon Because, m the rat, p r o l a c t m is k n o w n to be luteotroph~c and thus capable of maintaining functioning c o r p o r a lutea, the effect of p r o l a c t m in turn might be mediated by p r o g e s t e r o n e T h e results p r e s e n t e d can be shortly s u m m a r i z e d as follows (a) The action of oestrogens and p r o g e s t o g e n s at the rat m a m m a r y gland should be m t e r p r e t e d on the basis of synerglsUc acUons b e t w e e n both h o r m o n e s with special regard to their interactions w~th p r o l a c t m secretion (b) T h e m d u c u o n of m a m m a r y gland t u m o r s m rats due to long-term oestrogen treatment is m o s t p r o b a b l y not related to a & r e c t oestrogen effect but to a s u m u l a t o r y action at the p r o l a c t m producing cells (c) The effect of prolactm m rats should be discussed also m view of its luteotrophlc action m this species Coming b a c k to c o m p a r a t w e endocrinology the q u e s u o n must be a n s w e r e d as to whether these m e c h a m s m s also a p p h e s m other species In this context it is of interest that m the dog, p r o g e s t o g e n s rather than oestrogens are recriminated m the mductmon of m a m m a r y gland prohferaUon C o m p a r i n g the effects of p r o g e s t o g e n s and oestrogens m relation to their interaction with h y p o p h y s e a l h o r m o n e s m rats and dogs m a r k e d differences between the species b e c o m e evident W h d e in the rat prolactm m addition to p r o g e s t e r o n e is tmportant for the mducUon of m a m m a r y gland prohferatlon, growth h o r m o n e s e e m s to be the c o m p l e m e n t o r y m a m m a t o r p h l c factor m dogs (Graf et al, 1977a, G r a f and El Etreby, 1977) This e x a m p l e m a y indicate already that species-specific differences m the mteracUon of o v a r m n steroids and h y p o p h y s e a l h o r m o n e s have also to be taken into a c c o u n t when toxicological findings are e x t r a p o l a t e d f r o m one species to another, especially to man Only hmlted reformation Is a~adable on the m e c h a m s m s revolved In m a m m a r y gland prohferaUon, and especially m a m m a r y tumorogeneszs, m m a n H o w e v e r , ~t is interesting that, m contrast to the dog and rat, m w o m e n using oral contracepUves for a prolonged duraUon no enhanced risk m the d e v e l o p m e n t of m a m m a r y gland tumors could be s u b s t a n u a t e d and, on the contrary, progest o g e n / o e s t r o g e n c o m b m a U o n s have even b e e n regarded as being protective m the

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The interpretation of adverse drug effects

285

d e v e l o p m e n t of prolfferaUve b r e a s t diseases (Fasal and Paffenbarger, 1975; V e s s e y e t a l , 1975) Besides stermd h o r m o n e s , of course, several other drugs are k n o w n to influence dn.ectly or indirectly steroid- or peptlde-hortnone synthesis and/or secretion. Excellent e x a m p l e s are substances k n o w n to stimulate p r o l a c t m b o t h tn a m m a l s and in man, such as reserpine, which is c o m m o n l y used m the t h e r a p y of h y p e r t e n s m n , or the v a r m u s p s y c h o p h a r m a c o l o g l c a l drugs such as c l o m d m e (Dogmatfl®), ptmozlde (Orap ®) or c h l o r p r o m a z m e (Megaphen®), to m e n u o n just a f e w W e are far f r o m knowing all the m e c h a m s m s of a c t m n of these s u b s t a n c e s H o w e v e r , we do k n o w that they interfere mainly with n e u r o t r a n s m l t t e r s y s t e m s whtch are also regarded as of central i m p o r t a n c e m the m e c h a m s m s regulating synthesis and s e c r e t m n of h y p o thalamlc and h y p o p h y s e a l h o r m o n e s F o r e x a m p l e , m the case of prolactm, the v e r y tmportant role of dopamlnerglc s y s t e m s has been well e s t a b h s h e d ( H o r o w s k l and Graf, 1976, L ' H e r m l t e et al., 1976, Meltes and Clemens, 1972) Thus, the p r o l a c t m s u m u l a t o r y effects of the s u b s t a n c e s menUoned above, which are all k n o w n to be dopammerg~c agomsts, can be interpreted on the basts of then" m t e r a c t t o n s w~th central d o p a m m e r g l c s y s t e m s To c o m e b a c k to the classical field of endocrinology, It ~s k n o w n that increased p r o l a c t m s e c r e t m n m a y cause mfertflzty m man, which e m p h a s t z e s once m o r e the c o m p l e x i t y of the various n e u r o e n d o c r i n e p r o c e s s e s Finally, we should hke to show, b y m e a n s of a last e x a m p l e , h o w certain partml effects of a h o r m o n e or drug can give rise to toxicological changes m long-term toxicity studms which can only be interpreted w h e n the endocrmologlcal f u n d a m e n t a l s - - m this case specml f e e d - b a c k m e c h a m s m s - - a r e t a k e n into a c c o u n t We h a v e heard f r o m unotticml sources that tesUcular t u m o r s d e v e l o p e d m rats during the c h r o m c tOXiCity study of flutamlde, a non-stermdal antt-androgen ( N e n , 1977) We w e r e able to show that these were p r o b a b l y not g e n u m e tumors, but rather c o m p e n s a t o r y h y p e r p l a s t a of the mtersUUal cells ( N e u m a n n e t a l , 1977b). Flutamlde, p r o b a b l y hke all a n t m n d r o g e n s wzthout additional inherent anttgonadotrophlc propert~es, leads to s u m u l a t m n of androgen secretion w a mhibltmn of the negative androgen f e e d - b a c k , which c o n s e q u e n t l y results m stlmulatmn of the Leydlg cells The administration of spn.onolactone possibly has a slmtlar effect and certain s~de-effects of spn.onolactone can possibly be explained on th~s basts W~th th~s last e x a m p l e we m e r e l y w~shed to show just how ~mportant ~t ~s to think of such m e c h a m s m s w h e n designing toxicity studies and, e v e n m o r e so, w h e n evaluating the findings 5 SUMMARY We h a v e tried to d e h n e a t e with several e x a m p l e s the value of toxicity studies, as they are r e c o m m e n d e d at present, m the hght of c o m p a r a U v e e n d o c n n o l o g y . It b e c a m e clear that knowledge a b o u t the species-specific m e c h a m s m s responsible for the o c c u r r e n c e or n o n - o c c u r r e n c e of certain drug effects are to be interpreted correctly m then" r e l e v a n c e to man Without regarding this, tox~clty studies m a y cause risks to be predicted w h e r e there are none, o r - - w h a t ~s e v e n w o r s e m n s k s m a y not be r e c o g m z e d b e c a u s e of the msutticlency of the toxicological test model used. REFERENCES CHAMBON, Y (1949) Besoms endocnmens quahtaufs et quant,tatffs de I'ovolmplantauon chez la Lapme C R Soc B w l 143 1172-1175

COWIE,A T and TINDAt.,J S (1971) Prolactm and related mammotrophlc hormones (Chap 2), Mammary growth (Chap 3), The neuroendocnne regulation of mammary gland function (Chap 6), In Monographs of the Physloiobncal Society, No 22 The Physiology of Lactation, DAWSON,H et al (eds) Edward Arnold, London DUBOlS, P, CzYv^, J C and DUMONT,L (1964) Les premiers stades de ia d~clduahsauon de l'endom~tre chez ie Hamster dor~ C R Soc Biol 158 745-748 EL ETREBY,M F, G~F, K - J , NEUMANN,F and GONZEL,P (1979) Effect of contraceptive steroids on mammary gland of Beagle dog and tts relevance to human carcmogemcity Internatmnal Workshop on Pharmacological Methods m Preclmacal Safety Evaluauon of New Drugs, Zurich Pharmac Ther 5 369--402

286

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GRAF

FASAL, E and PAFFENBARGER, R S (1975) Oral contraceptives as related to cancer and benign lesions of the breast J Natn Cancer lnst 54 767 GILLMAN, J and STEIN, H B (1942) Quantitative study of antagonism of estrogen and progesterone m castrate rabbit Endocnnology 31 167-171 GOLDENTHAL, E V (1969) Contraceptives, estrogens and progestogens a new FDA policy on animal studies FDA papers, p 15 GOOD, R G and MOYER, D L (1968) Estrogen*progesterone relationship m the development of secretory endometnum Fert Stenl 19 37--49 GR~F, K -J and EL ETREnY, M F (1977) Endocnnology of reproduction m the female Beagle dog and Its slgmficance m mammary gland turmoungenes~s (In preparation) GRgF, K - J , EL ETREBV, M F and NEUMANN, F (1977a) Contraceptive hormones The problem of the reduction of mammary gland nodules m Beagle dogs given progestogens First Int Cong Toxzcology, Toronto G ~ F , K - J , EL ETREBY, M F A., RXCtrrER, K - D , GONZEL, P and NEUMANN, F (1975) The progestogenlc potencies of different progestogens in the beagle bitch Contraception 12 529--540 GRAF, K - J , HOROWSKI. R and EL ETREBY, M F (1977b) Effect of prolactm inhibitory agents on the ectoplc anterior pituitary and the mammary gland in rats Acta Endocnnol (Copenhagen) 8S 267-278 GRAF, K -J, NEUMANN, F and HOROWSKI, R (1976) Effect of the ergot derivative hsurlde hydrogen maleate on serum prolactln concentrations In female rats Endocnnology 98 598 HAASE, F , BEIER, S , HARTMANN, D and ELGER, W (1977) Development of a qualitative canine bloassay for gestagens Acta Endocnnoi (Copenhagen) Suppl 208 122 HILL, R , AVERKIN, E , BROWN, W , GAGNE, W E and SEOP.~, E (1970) Progestational potency of chlormadtnone acetate m the immature Beagle bitch prehmmary report Contraception 2 381-390 I-IoRoWSKI, R and G ~ , K -J (1976) Influence of dopammerglc agomsts on serum prolactm concentraUons tn the rat Neuroendocnnology 22 273 L'HERMn'E, M , ROeYN, C , VANgAELS'r, L , LECLERCQ, R , GOLSTEIN, J , VIRASORO, C , VEKEMANS,[~, DENAYER, P and COPINSCHI, G (1976) Control of pituitary prolactm secretion In humans, In Cellular and Molecular Bases o[ Neuroendocnne Processes, ENDROCZl, E (ed), pp 151-173 Akad6mtal Ksad6, Budapest MADJEREK, Z , DE VISSER, J , VAN DER VIES, J and OVERSEEK, G A (1960) Allylestrenol, a pregnancy mamtalmng oral gestagen Acta Endocnnol (Copenhagen) 35 8-19 MEITES, J and CLEMENS, J A (1972) I-lypothalamxc control of prolactm secretion Vitam Harm (New York) 30 165 NERI, R O (1977) studies on the biology and mechanism of action of non-steroidal antlandrogens, In Androgens and Antlandrogens, MARTINI, L and MOYrA, M (eds), pp 179-189 Raven Press, New York NEUMANN, F (1968) Chemlsche Konstltution und pharmakoiogqsche Wirkung, In Gestagene, Handbuch der Expenmentelle Phannakohgte, Vol 22/1, Chap VI, pp 680-1025, JUNKMANN, K (ed) Springer, Berhn-Heldelberg-New York NEUMANN, F and ELGER, W (1971) Kntlsche Uberlegnngen zu den blologqschen Grundlagen van Toxszltatsstudlen mlt Steroid- (Sexual-) hormonen, In Methodlk der Steroldtoxlkologle--fur Forschung und kllmsche Anwendung der Sterolde, pp 6-48. PLOTZ, E - J and HALLEit, J ('eds) Georg Thleme, Stuttgart NEUMANN, F and ELGER, W (1972) Critical considerations of the biological basis of toxicity studies with steroid (sex) hormones, In Methods in Steroid Toxicology Report of a workshop held sn Bonn, Dec 1970, pp 10-91, PLOT'Z, E -J and HALLER, J (eds) Geron-X, Los Altos, Cahforma NEUMANN, F . ELGER, W , NISFIINO, Y and S'rEINgECK, H (1977a) Probleme der Doslsfindung Sexualhormone Arznetm Forsch 27 296-318 NEUMANN, F , GRAF, K - J , HASAN, S H , SCHENCK, B and STEINSECg, H (1977b) Central actions of antlandrogens, In Androgens and Antmndrogens, pp 163-177, MARTINI, L and MO'I'rA, M (eds) Raven Press, New York NEUMANN, F , BERSWORDT-WALLRABE, R vON, ELGER, W , GR,/~, K - J , HASAN, S H , MEHRING, M , NISHINO, Y and STEINBECK, H (1974) Special problems m toxicity testing of long acting depot contraceptives WHO Meeting on Pharmacological Models to Assess Toxicity and Side Effects of Fertility Regulating Agents Acta Endocnnol (Copenhagen) Suppl 185 315-354 NISHINO, Y and NEUMANN, F (1974) The sJahc acid content m mouse female reproductive organs as a quantitative parameter for testing the estrogemc and antlestrogemc effect, antlestrogenlc depot effect, and dissociated effect of estrogens on the uterus and vagina Acta Endocnnol (Copenhagen) Suppi 187, 76 1-62 VESSEY, M P , DOLL, R and JONES, K (1975) Oral contraceptives and breast cancer Lancet i 941 YOCHIM. J M and DE FEO, V J (1962) Control of declduai growth m the rat by steroid hormones of the ovary Endocnnology 71 134--142