From the Hormone Laboratory ( M . Furuhjelm M . D .

) , Department of Ob-
stetrics and Gynaecology (Professor A . Ingelman-Sundberg); Sabbatsberg
Hospital, Karolinska Institutet, Stockholm, Sweden

MECHANISMS OF ACTION OF CLOMIPHENE

BY

KJELL CARLSTROM AND MIRJAM FURUHJELM

For some years clomiphene has been used to induce ovulation in

anovulatory patients ( G r e e n b 1 a t t, 1961). However, the
mechanism of its action is not yet completely clarified.
There are two principal theories about the action of clomiphene.
The first theory ( R o y et al. 1963) claims that clomiphene acts on
the pituitary-hypothalamic axis with resultant release of gonado-
trophins, especially LH. The gonadotrophins will then stimulate the
ovarian steroid biosynthesis. This in turn implies an ovarian enzyme
system, sufficient to react properly on gonadotrophic stimulation.
Increased urinary excretion of gonadotrophins as well as of
steroids has been observed after clomiphene treatment by many
workers. A direct action of clomiphene on the hypothalamus in the
rat was shown by I g a r a s h i and co-workers (1967). After direct
cerebral implantation of clomiphene in the median eminence as well
as in the anterior hypothalamus they found increased plasma and
pituitary L H levels. After injection of tritiated estradiol, E i s e n-
f e l d and A x e l r o d (1967) found lower estradiol levels in the
hypothalamus of clomiphene-treated rats, compared with the con-
trol animals. These results indicate the possibility of a competitive
inhibition of clomiphene against the estrogen feedback inhibition on
the pituitary L H secretion. O n the other hand, such a theory must
include an estrogen production large enough to suppress the LH
release.
The second theory includes a direct action of clomiphene on the
steroidogenic organs. Stimulation of steroid transformations by clomi-

35
 REMAIN1No
TESTOSTERONE
MOLESrlO- 7

‘I CLOMIPHENE

1 2 3 1 5
INCUBATION T I M . HOURS

Fig. 1. Effect of clomiphene and of F 6060 on the conversion of testosterone

into androstenedione by ultrasonic homogenate of P. lilncinum.

phene in vitro has also been shown by some workers.

S m i t h and co-workers (1963, 1966) studied the action of
clomiphene on the aromatization of testosterone by placenta micro-
somes and they found an 1.6-fold increase in the estrogen produc-
tion, caused by clomiphene. The mechanism was shown to be an in-
hibition of the NADPHl-cytochrome c oxido-reductase system with
subsequent increase of the availability of NADPH, which is an
obligatory co-factor for the aromatization.
H a m m e r s t e i n (1967) demonstrated that clomiphene stimu-
lated the 3j?-o1-dehydrogenase-d5-isomerase in slices of human
corpora lutea. This enzyme system, which transforms pregnenolone
into progesterone and dehydroepiandrosterone into androstenedione,
is fundamental for the general steroid biosynthesis.
G o 1 d m a n (1967) has successfully utilized microbial enzymes as
models for human steroid metabolism. I t may therefore be possible
to make some general conclusions from experiments with such
systems.
I n this laboratory ultrasonic homogenates from the fungus Peni-
NADPH =the reduced form of Ni~otinarnide-~4denine-Dinucleotide
Phos-
phate.

36
 mRADloL ESTROM

Fig. 2. Simplified schema over the ovarian steroid biosynthesis.

cillium lilacinum (NRRL 895) have been used. This fungus possesses
a rich collection of steroid transforming enzymes, i.e. 17Jj-ol-de-
hydrogenase. The fungus was cultured on beer wort-distilled water
1 : 1 with addition of testosterone to induce sufficient 17P-ol-de-
hydrogenase activity. The mycelium was washed, resuspended in
0.01 M phosphate buffer pH 6.5 and treated in an ultrasonic dis-
integrator for 30 minutes. Incubations were performed at 30' C for
three and five hours respectively and were run in duplicates. In-
cubation volume was 1.0 ml, added testosterone 6.92 x 10.' moles,
clomiphene citrate 11.80 x moles and F 6060 1 2 . 0 10.'
~ moles
respectively. After termination of the reaction by addition of 3.0 ml
of methanol, the remaining testosterone was determined using thin
layer chromatography on Silica gel G and gas liquid chromatography
on 3 % XE-60. It was tested that neither clomiphene citrate nor
F 6060 interfered with the analytical procedure.
The results showed a clear stimulating effect of clomiphene on the
transformation of testosterone into androstenedione (Fig. 1) . The
compound Ferrosan F 6060, which is the diphenol corresponding to
the ovulation inductor F 6066 (Sexovid), exerted an opposite effect
on this enzyme, i.e. an inhibition.

37
 As is seen from Fig. 2, these three steroid transformations are im-
portant steps in the estrogen biosynthesis. The ability of clomiphene
to increase the available NADPH may cause an increased produc-
tion of Czl-steroids from cholesterol and of C19-steroids from (221-
steroids as well as the previously mentioned increase in the aromati-
zation of ring A. All these reactions are dependent on NADPH.
The stimulation of 3/?-ol-dehydrogenase-d5-isomerase and of
17/3-ol-dehydrogenase may lead to an increased availability of andro-
stenedione from pregnenolone/dehydroepiandrosterone and from
testosterone respectively. As androstenedione indeed is a much better
substrate for the aromatization to estrogens than testosterone itself
( B a u l i e a u et al. 1963, B o l t 6 et al. 1964, M e n i n i and
E n g e 1 1967), this may lead to an increase in the estrogen produc-
tion.
Summing up, it can be said that the action of clomiphene may be
a rather complex one and probably combines the action on the hypo-
thalamus-pituitary axis with a direct action on the steroid trans-
forming enzymes in the ovary.

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