- Email: [email protected]
The progestogenic potencies of different progestogens in the Beagle bitch
THE PROGESTOGENIC
POTENCIES
OF DIFFERENT
IN THE BEAGLE
K.-J. K.-D.
Grtif, M.D.,
Richter,
M.F.A.
D.V.M.,
D.V.M. and
D.V.M.
Laboratories
SCHERING Berlin
El Etreby,
P. Ciiinzel,D.V.M.,
F. Neumann,
Research
PROGESTOGENS
BITCH
AG
- Bergkamen Germany
ABSTRACT Progesterone, d-norgestrel, norethisterone acetate, and norethisterone enanthate were tested for their progestogenic potencies using the Clauberg assay in the immature Beagle bitch. Because of the limited response of the Beagle-endometrium to estrogens and it was found that this method - in the progestogens, way used - waa not very sensitive and therefore only of limited value for testing the relative progestogenie potencies of different progestogene. The data on the progestogenic potencies of the progestogens tested corresponded fairly well, but not completely, with the corresponding data for the Clauberg assay in rabbits and the transformation doses in women. In this context it is discussed that, in contrast to the hydroxyprogesterone-derivative chlormadinone acetate where a wide discrepancy of the progestogenic potency in various species has been described, those proAccepted
JWVEMBER
for publication
September
1975 VOL. 12 NO. 5
17, 1975
529
CONTRACEPTION
gestogens of the 19-nor-testosterone type we have tested, failed to show this enormous discrepancy at least when their progestogenic potencies in man, rabbits and dogs were compared.
INTRODUCTION This study was undertaken in view of the perennial discussion about biological or pharmacological progestogenic activities in different animal species and using different test models and especially their relevance in man. These discussions resulted in the WBeagle affair", and the intervention of the Food and Drug Administration (FDA), as it was found that in long term toxicity studies some progestogens, e.g. chlormadinone acetate, induced mammary gland nodules in this species. We tested the progestogenic potencies of progesterone (P) and of different progestogens of the l+nortestosterone-type using the Clauberg assay in the dog as described by Hill et al. (1) with slight modifications.
MATERIAL
AND METHODS
In the first experiment (I) 76 female Beagles, 8 to IO weeks of age, were randomized and arranged in 13 groups of 5 or 6 animals each (see Table I). All Beagles received daily doses of 5 pg/kg body weight estradiol beneoate (E ) subcutaneously for 5 consecutive days in order to ca 1 se a proliferation of the endometrium. After this estrogen-priming, the Beagles were treated daily for 5 consecutive days with the different progestogens, i.e. progesterone (PI, d-norgestrol (N), norethisterone acetate (NA) and norethisterone enanthate (NE), each in 3 difforent doses (see Table I). In a second experiment (II) using 25 other female Beagles, again 8 to 10 weeks of age, NE was further tested with different doses using principally the same model as described above. Here NE was injected only once on day 6 of the experiment except in one group were the substance was given for 5 consecutive days following estrogen priming (see Table I). On day 11 the Beagles were killed by Evipan -anaesthesia and the following investigations were done. The uteri and cervices were removed and weighed. In addition the uterine/body weight ratios were calculated (mg/lOO g body weight). One portion of one uterine horn
NOVEMBER
1975 VOL. 12 NO. 5
::: 3.3
1
5.3
i ;I:
4.3
.. .
Norethisterone enanthate*'
Controls
Norethisterone enanthate**
acetate.**
Norethisterone
d-Norgestrel***
Progesterone-*
Controls
Treatment*
3.2 0.8 0.2 0.2
Solvents only
12. 3.28 0.8
;$j . 0.2
0.32 0.02 0.08
12.8 3.2 0.8
Solvents only
Dose mg/kg/d
d6
- 10
6 - IO
d6 d6 d6 d 6 - 10
d 6
/d
i
6 - 10
6 - 10
d 6 - 10
1d I I j I'd
’
i.m.
/
1
pi:
/
i.m.
i.m.
S.C.
p.0.
P.O.
e.c.
5-c.
Route of administration
1
weight ratio with different
Duration of treatment
Design of experiment, uterine/body weight ratio after treatment
:
5
z 6
:
6
6 z
5
2
5
n
dry/wet
2
8.9
61.5 + 37.8 + 39.7 5 34.8 z
29.9 +
6.0 3.0 3.7 2.3
1.2
102.5 77.7 5+ 29.4 12.6 77.7 T 16.0
80.1 + 21.4 81.6 T 11.1 69.7 F 14.1
83.7 + 13.7 57.8 57.7 z 5 12.3 6.3
76.2 + 21.5 68.4 ‘; 13.4 86.2 i 19.3
43.2
Uterine/body weight ratio mg/100 g (mean + SEMI
and uterine progestogens
1
0.15 0.16 0.17 0.16
0.16
0.14 0.15 0.13
0.14 0.14 0.14
0.14 0.16 0.14
0.15 0.15 0.14
0.16
l 0.01 7 0.00 5 0.01 7 0.00
+ 0.00
5+ 0.02 0.01 T 0.01
+ 0.01 5 0.02 f 0.01
+ 0.01 f 7 0.02 0.01
+ 0.01 5 0.00 IO.01
2 0.01
(mean + SEM)
Uterine dry/wet weight ratio
ill animals Including the control groups received a priming dose of 5 @kg estradiol benzonte for 5 consecutive days before the progestogen wes given These substances were dissolved in benzyl-benzoate : castor oil in the ratio 11 : 6 v/v; 0.1 ml of solution was inJeCted per kg body weight These substances were administered as a microcrystalline suspension
z / 6 Y e .,E 7.1 5 c 0"
..
.
: i 1
2.1 2.2 2.3
I l
L' wx I ::::
.z
I
H c,
I.
Group
Tsble
CONTRACEPTION
from each animal was taken for the determination of the dry weight/wet weight ratio5 and in addition from each animal another part of the uterine horn was taken, transverse sections prepared and histologically assessed. The sections had A mean thickness of 5 p and were stained with haematoxylin and eosin. To estimate the progestational sections, the
potency by McPhail-type
evaluation scoring
of
the
histOlOgica
WAS
not useful. For this reason we made transparencies of each section which were than projected onto tracing paper. The sections were copied completely and thereafter the projected proliferated together with the proliferated and transformed endometrium (UE) WAS separated from the whole projected stroma (US) in one transverse section for each animal by cutting the tracing paper. The share of the projected, copied and separated stroma AS well AS that of the corresponding endometrium of each transverae section weighed. This wa5 done for all animals in all groups separately. Thereafter the weight-ratios UE/(US + UE) were calculated and used as a relative objective parameter for the estimation of the progeatogenic potencies, as this quotient reflected the degree of endometrial proliferation. WAS
RESULTS In all group5 treated with different progestogens, the uterine/body weight ratios were mostly dramatically higher when compared with the control groups (see Table There WAS no statistical dose relationship within I). However in all groups, except in the prothe groups. gesterone group, the highest applied dose was always most effective. In contrast, all treated groups showed no Changes of the uterine dry/wet weight ratios when compared with the controls (see Table I). The evaluation of the histological sections showed only slight effects on the degree of endometrial stimulation. Glandular proliferation was not very extensive, as is known from the Clauberg assay in the rabbit. Because of this we were not able to classify these sections with certainty by using the McPhail-type scoring (see Figure 1). Therefore, we used the rather complicated but - compared with the McPhail-type scoring - more objective method as described above. The quotient UE/(US + UE) reflects the degree of glandular proliferation and of endometrial transformation.
532
NOVEMBER
1975 VOL. 12 NO. 5
CONTRACEPTION
Figure
1.
Cross-sections of uteri horns in (see also material and methods).
Beagles
(x 16)
benzoate treated immature Beagle a) Eatradiol (group 1). proliferation of the endob) Physiological metrium at the end of the oestrus stage of an mature Beagle. (12.8 mg/kg, group 2.1) treated cl Progesterone immature Beagle. (0.02 mg/kg, group 3.3) treated d) d-Norgestrel immature Beagle.
NOVEMBER
1975 VOL. 12 NO. 5
533
CONTRACEPTION
As it can be seen from Figures 2 and 3 in nearly all groups primed with estradiol benzoate with a following treatment with different progestogens, the endometrial glands were stimulated to a different degree, as is indicated by an increase in the absolute value of the The effect on the endometrium, quotient UE/(US + UE). as it was calculated by this quotient, seemed to inThis crease using higher doses of the same progestogen. Statistically was valid for all the progestogens used. significant increases (Dunnett test) compared with the that were only primed with estrogen, control groups, were shown by the doses of 12.8 and 3.2 mg P, 0.32 mg N, 3.2 and 0.8 mg NA and all the doses of NE in experiment I (see Table II). In experiment II all the doses of NE used were not significantly different from the corresponding control group (see Table II).
DISCUSSION The results of this study indicate that the model of the Clauberg assay in the Beagle bitch we used was not The extent as sensitive as is the case in the rabbit. of proliferation as well as of transformation of the endometrial glands seemed to be very limited after estrogen-priming and treatment with progestogens in different doses for 5 or only I day by different routes of administrations. The calculation of the uterine dry/wet weight ratios showed no significant differences neither within the From this one can suggroups nor between the groups. discussed by Hill et al. cl), gest, as has been already that the increase in uterine weight represents a true proliferation of the uterine tissue. The estimation of both parameters concerning the progestogenic potencies of the substances tested, firstly the uterine/body weight ratios and secondly the endometrial stimulation calculated on the basis of the did not permit any statistical UE/(US + UE) quotient, analysis of the relative progestogenic potencies of the progestogens tested. This might be explained by several possibilities. Firstly, the Clauberg assay - in the way used - is not a very sensitive method. The ability of the Beagle endometrium to proliferate and transform is very poor when compared with the endometrium of the rabbit, at least when the estrogen-priming and the progestogen treatment lasted each for only 5 days. Secondly, the 3 doses used of each of the progestogens tested were probably not all within the dose interval where a dose
NOVEMBER
1975
VOL. 12 NO. 5
Figure
_
2-
4-
6-
8-
lo-
12-
14-
16-
18-
,I iZ;Z?
-2
1
lzrof
J
1 i;?rfl
T T
2.
Evaluation of the Clauberg assay using different progestogens.
No:
isty;e
T
!
4.3
Itate
;$iro;;
T
1
T
in the
immature
Beagle
of the experiment, following
bitch
US = weight of the projected uterine stromakection
1
All the progestogens were given for 5 consecutive days from day 6-10 estrogen-priming with 5 )lg estradiol benzoate from day 1 - 5 .
UE = weight of the projected uterine endometrium/section
i;;mg
Group
x 10
-1 T
r
1
Control
6
x 10-2
3.2 mg
0.8 mg
7.2
0.2 mg
I,
1.
Evaluation of the Clauberg assay using norethisterone enanthate.
UE = weight of the projected uterine endometriumkction US = weight of the projected uterine stromakction.
l
in
the
immature
Beagle
These doses were given onlv on dav 6 of the experiment, except in group 7.4 where the treatment lasted from da>s 6-lO,~follow/ng estrogen priming with 5’ug es&ad;01 benzoate from day 1-5.
kg/d*
Dose/
(tiroup
Figure
2
4
6
8
10
12
14 1
S+UE
UE
L 1
bitch
0.2 mg
1
.. ...
.
5
/ 12.8
5
3.2
5
0.8
5
IO.32
5
0.08
d-Norgestrel** (p.0.)
5
0.02
1
5
+
+
0.8
5
3.2
-
5
0.2
Norethimterone acetate** (D.O.1
1
5
+
+
3.2
5
12.8
+
5
0.8
Norethiatarona enanthatee* (s.c.1
1
_
_
0.2
angle transformed. control group. control group.
-
-
.0.8
llli
3.2
5
0.2
Norethimterone enanthate*** (i.m. 3
Statistical evaluation using the Dunn&t tomto of the endoretrial transformation in the iramature Beagle bitch after traatmant with different progastogana in different domam, compared with the group that was only primed with oemtrogans (Clauberg ammay).
Progesterone** (i.m. 1
II.
the calculated parameter UE/(US + UE) was Llefore analyses in the Dunnett test, These substances were tested in experiment I and compared with the corresponding This substance was tested in experiment II and compared with the corresponding
of treatment
Days
[Bose/mg&g/d
Substance
Table
I
I
I
1
CONTRACEPTION
Furthermore, it might be posrelated response exists. sible from our experiments that the maximal response is not uniform but different according to the progestogen Our results are principally in accordance with used. between the Hill et al. (1) who found wide differences progestogenic potencies of chlormadinone acetate (CAP) and norethisterone (NET) depending on the parameter The estimated progestational potency of CAP measured. related to NET was 225:l using the uterine/body weight whereas the McPhail-type scoring ratio as the parameter, revealed that CAP was only 62 times more potent than Principally one would not expect a general agreeNET. ment between these parameters because the transformatory effect on the endometrium seems to be dependent on the the uterine weight progestogenic potency only, whereas may be influenced by other partial effects the progepotencies as with stogens might have, e.g. by androgenic Therefore, we N or by estrogenic potencies as with NE. would suggest that the estimation of the endometrial stimulation is a more sensitive parameter concerning the progestogenic effect whereas by calculation of the uterine/ body weight ratios other effects might interfere. Despite the fact that our investigations did not show clear dose relations, it is possible to draw several conclusions. According to Hill et al. (1) who found marked interspecies difference-the relationship of CAP and NET with respect to their relative progestogenic potencies, it was surprising that similar differences could not be found for the substances we tested (see Table III). Even if one takes into account the limited interpretation it is evident that comparing possible for our results, the transformation dose in women (4) with the results of potencies the Clauberg assay in dogs, the progestational of N and NA are about 5 to 20 times and of P and NE about 1 to 6 times more potent in man than in dogs when calThese differences are culated on the basis of mg/kg. about in the same range as described by Hill et al. (1) for NET, and in marked contrast to the reported related When one compares the threshold doses results for CAP. in the acute Clauberg assay in rabbits and the transformaall the tested progestogens were tion doses in women, about 10 times more potent in rabbits than in women. Therefrom one can conclude that the species might be ranked as follows: rabbits
women
dogs9
according to their degree of susceptibility to progestoCAP seems to be a much gens in this type of test model. more potent progestogen in dogs than NET (1) or the other 19-nortestosterone derivatives we have tested, when one compares their potencies in dogs and man.
538
NOVEMBER
1975
VOL.
12 NO.
5
l
See
Ref.
,Voretllisterone enanthate
Norethisterone / acetate 1 (micronized)
I
I
Progesterone
Substance
(4)
’ j ;
Tested
Approximate
i.m.
i ~0.0-100.0
i m.
D.O.
S.C.
l
/ 12.0-14.0
4.6-6.0
200.0
i.m. S.C. p.o.
p.0.
S.C.
in
the
dog
potencies
1.6
c-0
1.0
mg/kg
16.0
dose
effective
0.7-1.4 I
0.4
4.0
mg/kg
4.0
1
I
I
/
I
/ dose
effective
assay) / (Beagle-Clauberg ! highest in- lowest,
Tested
of progestogenic
0.17-0.2
0.06-0.09
2.9
w/kg
dose
in women*
comparison
Route of w adminisTransformation tration mg/cycle
Ta,ble III.
;
I
and women
1.4
5.0
4.4
-
-
-
x.4 -
5.4
23.5
26.7
5.6
:
:
:
:
1
1
1
I
Approximate progestogenic potency in dogs (Claubetg assay) relative to the transformation dose in women dks
in dogs
CONTRACEPTION
The hypothesis ha% still not been proved that those progestogens which are capable of inducing nodules in ma%n%ary glands of Beagles may exert their effect due to their chemical configuration together.with their progestogenic potency (3). Regarding our result% and the fact that mammary gland nodules of Beagles might be induced by both 19-nortestosterone or hydroxyprogesterone derivatives (21, it might be possible or likely that alone the progestogenic potency plays the significant role in the expression of the tumorigenic potential of these compounds. To answer these questions it seems to be necessary that more progestogens of different structures be tested in a more optimal Clauberg assay in the dog, although this still has to be developed. However, so far we also believe "that for clinical uses or for toxicity studies, dose selection must be based on the comparative responsiveness of man and the experimental animal to the drug" (1). Until now there is no method and no species known from whom the data from the testing of different substances for different purposes can be in general easily extrapolated to reveal relevant data for their clinical assessment or use in man.
REFERENCES
540
1.
Hill, R., Averkin, E., Brown, W., Gagne, W.E., and Progestational potency of chlormadinone Segre, E.: acetate in the immature Beagle bitch: Preliminary Contraception, Vol. 2, 6 : 381-390 (19i’O) report.
2.
Hill, R., and Dumas, K.: The use of dog% for studies of toxicity of contraceptive hormones. In: Pharmacological Models in Contraceptive Development (M.H. Briggs & E. Diczfalusy, Editors). WHO Research and Training Centre on Human Reproduction, Karolinska Institutet, Stockholm 1974, p. 74-89.
3.
Berliner, V.R.: U.S. Food and drug administration requirements for toxicity testing of contraceptive products. In: Pharmacological Models in Contraceptive Development (M.H. Briggs & E. Diczfalusy, WHO Research and Training Centre on Human Editors). Reproduction, Karolinska Institutet, Stockholm 1974, p. 240-253.
4.
Hammerstein, J.: Die als Kontrazeptiva gebrguchlithen Oestrogene und Gestagene. In: Nebenwirkungen contraceptiver Steroide (H. Kewitz, Editor). Westkreuz Verlag, Berlin 1971, p. 10-17.
NOVEMBER
1975
VOL. 12 NO. 5
POTENCIES
OF DIFFERENT
IN THE BEAGLE
K.-J. K.-D.
Grtif, M.D.,
Richter,
M.F.A.
D.V.M.,
D.V.M. and
D.V.M.
Laboratories
SCHERING Berlin
El Etreby,
P. Ciiinzel,D.V.M.,
F. Neumann,
Research
PROGESTOGENS
BITCH
AG
- Bergkamen Germany
ABSTRACT Progesterone, d-norgestrel, norethisterone acetate, and norethisterone enanthate were tested for their progestogenic potencies using the Clauberg assay in the immature Beagle bitch. Because of the limited response of the Beagle-endometrium to estrogens and it was found that this method - in the progestogens, way used - waa not very sensitive and therefore only of limited value for testing the relative progestogenie potencies of different progestogene. The data on the progestogenic potencies of the progestogens tested corresponded fairly well, but not completely, with the corresponding data for the Clauberg assay in rabbits and the transformation doses in women. In this context it is discussed that, in contrast to the hydroxyprogesterone-derivative chlormadinone acetate where a wide discrepancy of the progestogenic potency in various species has been described, those proAccepted
JWVEMBER
for publication
September
1975 VOL. 12 NO. 5
17, 1975
529
CONTRACEPTION
gestogens of the 19-nor-testosterone type we have tested, failed to show this enormous discrepancy at least when their progestogenic potencies in man, rabbits and dogs were compared.
INTRODUCTION This study was undertaken in view of the perennial discussion about biological or pharmacological progestogenic activities in different animal species and using different test models and especially their relevance in man. These discussions resulted in the WBeagle affair", and the intervention of the Food and Drug Administration (FDA), as it was found that in long term toxicity studies some progestogens, e.g. chlormadinone acetate, induced mammary gland nodules in this species. We tested the progestogenic potencies of progesterone (P) and of different progestogens of the l+nortestosterone-type using the Clauberg assay in the dog as described by Hill et al. (1) with slight modifications.
MATERIAL
AND METHODS
In the first experiment (I) 76 female Beagles, 8 to IO weeks of age, were randomized and arranged in 13 groups of 5 or 6 animals each (see Table I). All Beagles received daily doses of 5 pg/kg body weight estradiol beneoate (E ) subcutaneously for 5 consecutive days in order to ca 1 se a proliferation of the endometrium. After this estrogen-priming, the Beagles were treated daily for 5 consecutive days with the different progestogens, i.e. progesterone (PI, d-norgestrol (N), norethisterone acetate (NA) and norethisterone enanthate (NE), each in 3 difforent doses (see Table I). In a second experiment (II) using 25 other female Beagles, again 8 to 10 weeks of age, NE was further tested with different doses using principally the same model as described above. Here NE was injected only once on day 6 of the experiment except in one group were the substance was given for 5 consecutive days following estrogen priming (see Table I). On day 11 the Beagles were killed by Evipan -anaesthesia and the following investigations were done. The uteri and cervices were removed and weighed. In addition the uterine/body weight ratios were calculated (mg/lOO g body weight). One portion of one uterine horn
NOVEMBER
1975 VOL. 12 NO. 5
::: 3.3
1
5.3
i ;I:
4.3
.. .
Norethisterone enanthate*'
Controls
Norethisterone enanthate**
acetate.**
Norethisterone
d-Norgestrel***
Progesterone-*
Controls
Treatment*
3.2 0.8 0.2 0.2
Solvents only
12. 3.28 0.8
;$j . 0.2
0.32 0.02 0.08
12.8 3.2 0.8
Solvents only
Dose mg/kg/d
d6
- 10
6 - IO
d6 d6 d6 d 6 - 10
d 6
/d
i
6 - 10
6 - 10
d 6 - 10
1d I I j I'd
’
i.m.
/
1
pi:
/
i.m.
i.m.
S.C.
p.0.
P.O.
e.c.
5-c.
Route of administration
1
weight ratio with different
Duration of treatment
Design of experiment, uterine/body weight ratio after treatment
:
5
z 6
:
6
6 z
5
2
5
n
dry/wet
2
8.9
61.5 + 37.8 + 39.7 5 34.8 z
29.9 +
6.0 3.0 3.7 2.3
1.2
102.5 77.7 5+ 29.4 12.6 77.7 T 16.0
80.1 + 21.4 81.6 T 11.1 69.7 F 14.1
83.7 + 13.7 57.8 57.7 z 5 12.3 6.3
76.2 + 21.5 68.4 ‘; 13.4 86.2 i 19.3
43.2
Uterine/body weight ratio mg/100 g (mean + SEMI
and uterine progestogens
1
0.15 0.16 0.17 0.16
0.16
0.14 0.15 0.13
0.14 0.14 0.14
0.14 0.16 0.14
0.15 0.15 0.14
0.16
l 0.01 7 0.00 5 0.01 7 0.00
+ 0.00
5+ 0.02 0.01 T 0.01
+ 0.01 5 0.02 f 0.01
+ 0.01 f 7 0.02 0.01
+ 0.01 5 0.00 IO.01
2 0.01
(mean + SEM)
Uterine dry/wet weight ratio
ill animals Including the control groups received a priming dose of 5 @kg estradiol benzonte for 5 consecutive days before the progestogen wes given These substances were dissolved in benzyl-benzoate : castor oil in the ratio 11 : 6 v/v; 0.1 ml of solution was inJeCted per kg body weight These substances were administered as a microcrystalline suspension
z / 6 Y e .,E 7.1 5 c 0"
..
.
: i 1
2.1 2.2 2.3
I l
L' wx I ::::
.z
I
H c,
I.
Group
Tsble
CONTRACEPTION
from each animal was taken for the determination of the dry weight/wet weight ratio5 and in addition from each animal another part of the uterine horn was taken, transverse sections prepared and histologically assessed. The sections had A mean thickness of 5 p and were stained with haematoxylin and eosin. To estimate the progestational sections, the
potency by McPhail-type
evaluation scoring
of
the
histOlOgica
WAS
not useful. For this reason we made transparencies of each section which were than projected onto tracing paper. The sections were copied completely and thereafter the projected proliferated together with the proliferated and transformed endometrium (UE) WAS separated from the whole projected stroma (US) in one transverse section for each animal by cutting the tracing paper. The share of the projected, copied and separated stroma AS well AS that of the corresponding endometrium of each transverae section weighed. This wa5 done for all animals in all groups separately. Thereafter the weight-ratios UE/(US + UE) were calculated and used as a relative objective parameter for the estimation of the progeatogenic potencies, as this quotient reflected the degree of endometrial proliferation. WAS
RESULTS In all group5 treated with different progestogens, the uterine/body weight ratios were mostly dramatically higher when compared with the control groups (see Table There WAS no statistical dose relationship within I). However in all groups, except in the prothe groups. gesterone group, the highest applied dose was always most effective. In contrast, all treated groups showed no Changes of the uterine dry/wet weight ratios when compared with the controls (see Table I). The evaluation of the histological sections showed only slight effects on the degree of endometrial stimulation. Glandular proliferation was not very extensive, as is known from the Clauberg assay in the rabbit. Because of this we were not able to classify these sections with certainty by using the McPhail-type scoring (see Figure 1). Therefore, we used the rather complicated but - compared with the McPhail-type scoring - more objective method as described above. The quotient UE/(US + UE) reflects the degree of glandular proliferation and of endometrial transformation.
532
NOVEMBER
1975 VOL. 12 NO. 5
CONTRACEPTION
Figure
1.
Cross-sections of uteri horns in (see also material and methods).
Beagles
(x 16)
benzoate treated immature Beagle a) Eatradiol (group 1). proliferation of the endob) Physiological metrium at the end of the oestrus stage of an mature Beagle. (12.8 mg/kg, group 2.1) treated cl Progesterone immature Beagle. (0.02 mg/kg, group 3.3) treated d) d-Norgestrel immature Beagle.
NOVEMBER
1975 VOL. 12 NO. 5
533
CONTRACEPTION
As it can be seen from Figures 2 and 3 in nearly all groups primed with estradiol benzoate with a following treatment with different progestogens, the endometrial glands were stimulated to a different degree, as is indicated by an increase in the absolute value of the The effect on the endometrium, quotient UE/(US + UE). as it was calculated by this quotient, seemed to inThis crease using higher doses of the same progestogen. Statistically was valid for all the progestogens used. significant increases (Dunnett test) compared with the that were only primed with estrogen, control groups, were shown by the doses of 12.8 and 3.2 mg P, 0.32 mg N, 3.2 and 0.8 mg NA and all the doses of NE in experiment I (see Table II). In experiment II all the doses of NE used were not significantly different from the corresponding control group (see Table II).
DISCUSSION The results of this study indicate that the model of the Clauberg assay in the Beagle bitch we used was not The extent as sensitive as is the case in the rabbit. of proliferation as well as of transformation of the endometrial glands seemed to be very limited after estrogen-priming and treatment with progestogens in different doses for 5 or only I day by different routes of administrations. The calculation of the uterine dry/wet weight ratios showed no significant differences neither within the From this one can suggroups nor between the groups. discussed by Hill et al. cl), gest, as has been already that the increase in uterine weight represents a true proliferation of the uterine tissue. The estimation of both parameters concerning the progestogenic potencies of the substances tested, firstly the uterine/body weight ratios and secondly the endometrial stimulation calculated on the basis of the did not permit any statistical UE/(US + UE) quotient, analysis of the relative progestogenic potencies of the progestogens tested. This might be explained by several possibilities. Firstly, the Clauberg assay - in the way used - is not a very sensitive method. The ability of the Beagle endometrium to proliferate and transform is very poor when compared with the endometrium of the rabbit, at least when the estrogen-priming and the progestogen treatment lasted each for only 5 days. Secondly, the 3 doses used of each of the progestogens tested were probably not all within the dose interval where a dose
NOVEMBER
1975
VOL. 12 NO. 5
Figure
_
2-
4-
6-
8-
lo-
12-
14-
16-
18-
,I iZ;Z?
-2
1
lzrof
J
1 i;?rfl
T T
2.
Evaluation of the Clauberg assay using different progestogens.
No:
isty;e
T
!
4.3
Itate
;$iro;;
T
1
T
in the
immature
Beagle
of the experiment, following
bitch
US = weight of the projected uterine stromakection
1
All the progestogens were given for 5 consecutive days from day 6-10 estrogen-priming with 5 )lg estradiol benzoate from day 1 - 5 .
UE = weight of the projected uterine endometrium/section
i;;mg
Group
x 10
-1 T
r
1
Control
6
x 10-2
3.2 mg
0.8 mg
7.2
0.2 mg
I,
1.
Evaluation of the Clauberg assay using norethisterone enanthate.
UE = weight of the projected uterine endometriumkction US = weight of the projected uterine stromakction.
l
in
the
immature
Beagle
These doses were given onlv on dav 6 of the experiment, except in group 7.4 where the treatment lasted from da>s 6-lO,~follow/ng estrogen priming with 5’ug es&ad;01 benzoate from day 1-5.
kg/d*
Dose/
(tiroup
Figure
2
4
6
8
10
12
14 1
S+UE
UE
L 1
bitch
0.2 mg
1
.. ...
.
5
/ 12.8
5
3.2
5
0.8
5
IO.32
5
0.08
d-Norgestrel** (p.0.)
5
0.02
1
5
+
+
0.8
5
3.2
-
5
0.2
Norethimterone acetate** (D.O.1
1
5
+
+
3.2
5
12.8
+
5
0.8
Norethiatarona enanthatee* (s.c.1
1
_
_
0.2
angle transformed. control group. control group.
-
-
.0.8
llli
3.2
5
0.2
Norethimterone enanthate*** (i.m. 3
Statistical evaluation using the Dunn&t tomto of the endoretrial transformation in the iramature Beagle bitch after traatmant with different progastogana in different domam, compared with the group that was only primed with oemtrogans (Clauberg ammay).
Progesterone** (i.m. 1
II.
the calculated parameter UE/(US + UE) was Llefore analyses in the Dunnett test, These substances were tested in experiment I and compared with the corresponding This substance was tested in experiment II and compared with the corresponding
of treatment
Days
[Bose/mg&g/d
Substance
Table
I
I
I
1
CONTRACEPTION
Furthermore, it might be posrelated response exists. sible from our experiments that the maximal response is not uniform but different according to the progestogen Our results are principally in accordance with used. between the Hill et al. (1) who found wide differences progestogenic potencies of chlormadinone acetate (CAP) and norethisterone (NET) depending on the parameter The estimated progestational potency of CAP measured. related to NET was 225:l using the uterine/body weight whereas the McPhail-type scoring ratio as the parameter, revealed that CAP was only 62 times more potent than Principally one would not expect a general agreeNET. ment between these parameters because the transformatory effect on the endometrium seems to be dependent on the the uterine weight progestogenic potency only, whereas may be influenced by other partial effects the progepotencies as with stogens might have, e.g. by androgenic Therefore, we N or by estrogenic potencies as with NE. would suggest that the estimation of the endometrial stimulation is a more sensitive parameter concerning the progestogenic effect whereas by calculation of the uterine/ body weight ratios other effects might interfere. Despite the fact that our investigations did not show clear dose relations, it is possible to draw several conclusions. According to Hill et al. (1) who found marked interspecies difference-the relationship of CAP and NET with respect to their relative progestogenic potencies, it was surprising that similar differences could not be found for the substances we tested (see Table III). Even if one takes into account the limited interpretation it is evident that comparing possible for our results, the transformation dose in women (4) with the results of potencies the Clauberg assay in dogs, the progestational of N and NA are about 5 to 20 times and of P and NE about 1 to 6 times more potent in man than in dogs when calThese differences are culated on the basis of mg/kg. about in the same range as described by Hill et al. (1) for NET, and in marked contrast to the reported related When one compares the threshold doses results for CAP. in the acute Clauberg assay in rabbits and the transformaall the tested progestogens were tion doses in women, about 10 times more potent in rabbits than in women. Therefrom one can conclude that the species might be ranked as follows: rabbits
women
dogs9
according to their degree of susceptibility to progestoCAP seems to be a much gens in this type of test model. more potent progestogen in dogs than NET (1) or the other 19-nortestosterone derivatives we have tested, when one compares their potencies in dogs and man.
538
NOVEMBER
1975
VOL.
12 NO.
5
l
See
Ref.
,Voretllisterone enanthate
Norethisterone / acetate 1 (micronized)
I
I
Progesterone
Substance
(4)
’ j ;
Tested
Approximate
i.m.
i ~0.0-100.0
i m.
D.O.
S.C.
l
/ 12.0-14.0
4.6-6.0
200.0
i.m. S.C. p.o.
p.0.
S.C.
in
the
dog
potencies
1.6
c-0
1.0
mg/kg
16.0
dose
effective
0.7-1.4 I
0.4
4.0
mg/kg
4.0
1
I
I
/
I
/ dose
effective
assay) / (Beagle-Clauberg ! highest in- lowest,
Tested
of progestogenic
0.17-0.2
0.06-0.09
2.9
w/kg
dose
in women*
comparison
Route of w adminisTransformation tration mg/cycle
Ta,ble III.
;
I
and women
1.4
5.0
4.4
-
-
-
x.4 -
5.4
23.5
26.7
5.6
:
:
:
:
1
1
1
I
Approximate progestogenic potency in dogs (Claubetg assay) relative to the transformation dose in women dks
in dogs
CONTRACEPTION
The hypothesis ha% still not been proved that those progestogens which are capable of inducing nodules in ma%n%ary glands of Beagles may exert their effect due to their chemical configuration together.with their progestogenic potency (3). Regarding our result% and the fact that mammary gland nodules of Beagles might be induced by both 19-nortestosterone or hydroxyprogesterone derivatives (21, it might be possible or likely that alone the progestogenic potency plays the significant role in the expression of the tumorigenic potential of these compounds. To answer these questions it seems to be necessary that more progestogens of different structures be tested in a more optimal Clauberg assay in the dog, although this still has to be developed. However, so far we also believe "that for clinical uses or for toxicity studies, dose selection must be based on the comparative responsiveness of man and the experimental animal to the drug" (1). Until now there is no method and no species known from whom the data from the testing of different substances for different purposes can be in general easily extrapolated to reveal relevant data for their clinical assessment or use in man.
REFERENCES
540
1.
Hill, R., Averkin, E., Brown, W., Gagne, W.E., and Progestational potency of chlormadinone Segre, E.: acetate in the immature Beagle bitch: Preliminary Contraception, Vol. 2, 6 : 381-390 (19i’O) report.
2.
Hill, R., and Dumas, K.: The use of dog% for studies of toxicity of contraceptive hormones. In: Pharmacological Models in Contraceptive Development (M.H. Briggs & E. Diczfalusy, Editors). WHO Research and Training Centre on Human Reproduction, Karolinska Institutet, Stockholm 1974, p. 74-89.
3.
Berliner, V.R.: U.S. Food and drug administration requirements for toxicity testing of contraceptive products. In: Pharmacological Models in Contraceptive Development (M.H. Briggs & E. Diczfalusy, WHO Research and Training Centre on Human Editors). Reproduction, Karolinska Institutet, Stockholm 1974, p. 240-253.
4.
Hammerstein, J.: Die als Kontrazeptiva gebrguchlithen Oestrogene und Gestagene. In: Nebenwirkungen contraceptiver Steroide (H. Kewitz, Editor). Westkreuz Verlag, Berlin 1971, p. 10-17.
NOVEMBER
1975
VOL. 12 NO. 5