Anti-tumor and endocrine effects of non-steroidal aromatase inhibitors on estrogen-dependent rat mammary tumors

J. Steroid Biochem. Molec. Biol. Vol.44, No. 4-6, pp. 633~i36, 1993

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ANTI-TUMOR AND ENDOCRINE EFFECTS OF NON-STEROIDAL AROMATASE INHIBITORS ON ESTROGEN-DEPENDENT RAT MAMMARY TUMORS K. SCHIEWECK,*A. S. BHATNAGAR,CH. BATZLand M. LANG Research Department, Pharmaceuticals Division, CIBA-GEIGY Limited, 4002 Basel, Switzerland

Summary--The non-steroidal aromatase inhibitor CGS 20 267, at maximally effective doses in non-tumor bearing adult female rats, elicits endocrine effects mimicking those seen after surgical ovariectomy and thus induces a "medical" ovariectomy. We now report on studies characterizing the anti-tumor and endocrine effects of three orally active non-steroidal aromatase inhibitors, CGS 20 267, CGP 45 688 and CGP 47 645, in adult female rats bearing estrogen-dependent DMBA-induced mammary tumors. Doses ranging from 3 to 3000 gg/kg were given by gavage once daily for 6 weeks. After 6 weeks of treatment, the EDs0 for suppression of tumor volume was 10-30, 100 and 3-10/tg/kg for CGS 20 267, CGP 45 688 and CGP 47 645, respectively. The maximally effective dose for anti-tumor efficacy was 300, 1000 and 100/~g/kg for each of the three inhibitors, respectively. The observed potent anti-tumor efficacy was accompanied by potent endocrine effects. Thus, disruption of ovarian cyclicity (at maximal doses rats remained in constant diestrus) was observed in all animals from the 2nd or 3rd week of treatment to the end of the 6-week treatment period. Uterine weight, at the maximally effective doses for each of the three inhibitors, was suppressed to between 42 and 28% of pre-treatment levels. This suppression was similar to the suppression of uterine weight (27% of pre-treatment) seen after ovariectomy. Serum estradiol concentrations in rats treated with 300 #g/kg CGS 20 267 were significantly suppressed to 12% of pre-treatment levels and serum luteinizing hormone (LH) concentrations were elevated 3 to 4-fold over pre-treatment levels. Thus the potent anti-tumor efficacy seen with each of the three non-steroidal aromatase inhibitors was accompanied in each case by a variety of endocrine effects corresponding to those seen after ovariectomy.

INTRODUCTION Aromatase, a microsomal cytochrome /45odependent enzyme is a key enzyme in the biosynthesis of estrogens [1]. Thus inhibition of this enzyme should lead to an attenuation of estrogen biosynthesis and consequently to an inhibition of estrogen-dependent effects. In non-tumor bearing animals these effects, amongst others, would be characterized by a reduction in uterine weight, a disruption of ovarian cyclicity and an increase in serum luteinizing hormone (LH) concentrations. In rats bearing estrogen-dependent mammary tumors induced by the chemical carcinogen 7,12dimethylbenz[a]anthracene (DMBA), an inhibition of estrogen biosynthesis should lead a suppression in tumor volume. We have reported previously on the anti-tumor effects of Fadrozole hydrochloride (CGS 16 949A) [2]. We have Proceedings of the Third International Aromatase Conference. Basic and Clinical Aspects of Aromatase, Bologna,

Italy, 14-17 June 1992. *To whom correspondence should be addressed.

also reported previously on the endocrine efficacy of CGS 20 267 and have presented preliminary data on its anti-tumor efficacy [3]. In this report, we present data on the anti-tumor efficacy and the associated endocrine effects of CGS 20 267 and two new non-steroidal aromatase inhibitors, C G P 45 688 and C G P 47 645. The chemical structures of these three compounds are shown in Fig. 1.

ANTI-TUMOR EFFICACY The tumor model used for these studies was the estrogen-dependent DMBA-induced mammary carcinoma in adult female Sprague-Dawley rats. Details of the experimental methodology have been reported previously [2]. In brief, animals bearing 1-3 tumors of 10-12 mm dia are divided into groups of 15 animals. They are treated with aromatase inhibitor orally, once daily for 42 days (6 weeks). Tumor size, numbers of tumors and bodyweight are determined once weekly. During the 6-week treatment schedule with each of the three

633

POSTER PRESENTATIONS

634

K . SCHIEWECK et al.

N N

N.N,~

"N s

NC ~~~L~

CN

NC" ~ ' ~

CN

CGP 45 688

CGS 20 267

N.N,~ NC~

CN CGP 47 645

Fig. I. C h e m i c a l s t r u c t u r e s o f C G S 20 267, C G P 45 688 a n d C G P 47 645.

aromatase inhibitors, vaginal smears were taken from all animals to assess ovarian cyclicity. At the end of the 6-week treatment period a group of 5 animals was sacrificed, and the uteri were excised and weighed. Blood was collected and serum separated by centrifugation for the measurement of serum concentrations of estradiol and L H by specific radioimmunoassays.

Suppression of tumor volume A summary of the results obtained with CGS 20 267, CGP 45 688 and C G P 47 645 is presented in Table 1. The ED~0s with which the three compounds suppress mean tumor volume show that C G P 47 645 is the most potent of the three followed by CGS 20 267 and then CGP 45 688. This order of potency is also reflected in their potencies for inhibiting aromatase in vivo in non-tumor bearing rats (EDs0 for in vivo inhibition of aromatase is 0.1-0.3, 1-3 and 30-100 #g/kg for CGP 47 645, CGS 20 267 and C G P 45 688, respectively). All three compounds exhibit maximal efficacy in that they all almost completely suppress mean tumor volume at maximally effective doses listed in Table 1. In Table I. Suppression of tumor volume ~ #/kg, p.o. Compound

EDs0

EDgs

CGS 20 267 C G P 45 688 C G P 47 645

10-30 100 3-10

300 1000 100

"For details of methodology see [2]

their maximal efficacy they also show the same rank order of potency seen with the EDs0.

Regression of established tumors All three aromatase inhibitors are very potent in causing the regression of tumors present at the start of treatment. The results are summarized in Table 2. This effect is dose-dependent for all three compounds with CGS 20 267 being the most potent of the three followed by CGP 47 645 and CGP 45 688. Maximal efficacy was reached for CGS 20 267 and CGP 45 688 at doses of 300 and 3000/~g/kg, respectively, whereas the highest dose of CGP 47 645 used did not reach maximal efficacy.

Suppression of newly emerging tumors All three compounds are also very potent in suppressing the appearance of new tumors Table 2. Regression of established tumors ~ % Complete and partial regression~ Dose ,ug/kg, p.o. Control 3 10 30 100 300 1000 3000

CGS 20 267 0 26 17 81 95 94

C G P 45 688 0 3 10 7 25 71 98 100

C G P 47 645 0 16 37 48 62 63

"For details of methodology see [2]. bpercent regression is related to tumors existing at start of treatment (N = 20-50/group). C R = c o m p l e t e regressions, tumors not palpable. PR = partial regressions, decrease of 50-99% in tumor size.

A n t i - t u m o r effects o f a r o m a t a s e i n h i b i t o r s Table 5. Disruption of ovarian cyclieity~

Table 3. Suppression of newly emerging tumors'

Onset b

Dose /~g/kg

#g/kg, p.o. Compound

EDso

CGS 20 267 C G P 45 688 C G P 47 645

10-30 100 3-10

635

ED95

30 100 300 1000 3000

100-300 1000 100-300

aFor details of methodology see [2].

during the course of the 6-week treatment with the individual potencies following the rank order seen with the suppression of mean tumor volume. This data is summarized in Table 3. Another way to assess the potency with which these compounds inhibit the appearance of new tumors during the course of treatment is to examine the number of animals which were free of palpable tumors. This data is summarized in Table 4. At the maximally effective doses of the three compounds for suppression of newly emerging tumors, over 50% of the animals are totally free of any palpable tumors. Thus in terms of suppression of mean tumor volume, regression of tumors existing at the start of treatment and suppression of the appearance of newly emerging tumors, all three non-steroidal aromatase inhibitors were both very potent and efficacious in their anti-tumor efficacy.

CGS 20 267 2 2 2 2

C G P 45 688

C G P 47 645 3 3 2

4 2 2

'All rats (N = 15/group) in constant diestrus as evaluated by vaginal smears. bWeeks after start of treatment.

doses as low as 30 #g/kg daily, both CGS 20 267 and CGP 47 645 were able to disrupt ovarian cyclicity maximally in that all the animals in the dose groups of 30/~g/kg and above showed vaginal smears indicating continuous diestrus starting from the 2nd or 3rd week of treatment. With CGP 45 688, this effect was first seen with the 300/~g/kg daily dose in the 4th week of treatment. The maximally effective doses of 1000 and 3000/~g/kg CGP 45 688 disrupted ovarian cyclicity in the 2nd week of treatment. All three aromatase inhibitors showed maximal efficacy in that at their maximally effective doses each inhibitor disrupted ovarian cyclicity to the extent that all animals showed continuous diestrus smears to the end of the treatment period of 6 weeks.

Suppression of uterine weight ENDOCRINE EFFICACY Administration of aromatase inhibitors to adult, cycling female rats should result in an inhibition of estrogen biosynthesis which would consequently cause a disruption of ovarian cyclicity, a reduction in uterine weight and an increase in serum LH. The maximal attenuation of estrogen biosynthesis should result in effects equivalent to those seen after ovariectomy in adult female rats.

Disruption of ovarian cyclicity

Serum estradiol and luteinizing hormone (LH)

The results obtained with the three aromatase inhibitors are summarized in Table 5. With Table 4. Rats free of palpable

The effect of treatment with each of the aromatase inhibitors on uterine weight is shown in Table 6. All three inhibitors caused a dose-dependent reduction in uterine weight. CGS 20 267 was the most potent in reducing uterine weight followed by CGP 47 645 and CGP 45 688. Both CGS 20 267 and CGP 45 688 reduced uterine weight to the level seen in ovariectomized animals, whereas CGP 47 645, at the highest dose tested had not reached maximal efficacy.

The effects of CGS 20 267 on serum estradiol and LH are shown in Fig. 2. Determinations of

tumors a

Table 6. Suppression of uterine weight

% Rats without tumors b Dose /~g/kg

% of Controls CGS 20 267

C G P 45 688

C G P 47 645

0 0 13 60 67 79

0 0 0 0 7 30 67 57

0 0 7 27 20 54

Control 3 10 30 100 300 1000 3000 aAt cessation of bN ~ 15/group.

treatment.

Dose /~g/kg 3 10 30 100 300 1000 3000 Ovex

CGS 20 267 64* 53** 45*** 45** 31"** 28***

C G P 45 688

C G P 47 645

120 119 112 97 72* 49** 30*** 27***

*2P < 0.05, **2P < 0.01, ***2P < 0.001, N = 5/group.

123 83 74* 44*** 42***

636

K. SCHIEWECKet al.

Estradiol ~ LH Levels in % of Controls

mg/kg, x 42 p.o. Control

100

100

0.003

25

164

0.010

210

0.030

14

0.100

14

0.300

12

420

I

1.000 120

100

80

60

40

20

0

100

200

300

400

500

Fig. 2. Serum estradiol and luteinizing hormone (LH) concentrations in adult female rats bearing DMBA-induced mammary tumors treated orally, once daily for 6 weeks with CGS 20 267. Hormone concentrations are expressed as percent of the placebo-treated control group. Serum estradiol was measured using a highly specific radioimmunoassay described previously [4]. Serum LH was measured using a commercially available radioimmunoassay kit for rat-LH (Code RPA 552) obtained from Amersham Int. (England). serum estradiol and L H were not performed in the experiments using C G P 47 645 and C G P 45 688. D a t a have been normalized and expressed as percent o f the serum concentrations o f estradiol and L H measured in the placebo-treated control g r o u p (100%). C G S 20 267 very potently and dose-dependently reduces serum estradiol to 1 2 - 1 5 % o f control with doses o f 10/~g/kg and higher. This reduction in serum estradiol is a c c o m p a n i e d by a dose-dependent increase in serum L H which is maximal at doses o f 3 0 # g / k g and above and is 3- to 4-fold elevated as c o m p a r e d to the placebo-treated control group.

Thus all three inhibitors completely disrupt ovarian cyclicity and significantly reduce uterine weight. In addition, C G S 20 267 significantly reduces serum estradiol and elevates serum L H concentrations as c o m p a r e d with placebotreated controls. These endocrine effects at maximally effective doses o f the three inhibitors are equivalent to those seen after ovariectomy. In conclusion, we have reported here on C G S 20 267, C G P 45 688 and C G P 47 645, three new orally active non-steroidal aromatase inhibitors which show anti-tumor and endocrine efficacy equivalent to that seen after ovariectomy in D M B A - i n d u c e d tumor-bearing adult female rats.

DISCUSSION AND CONCLUSIONS REFERENCES

We have reported here on the anti-tumor efficacy o f C G S 20 267 and on the a n t i - t u m o r efficacy o f two other new non-steroidal a r o m a tase inhibitors, C G P 45 688 and C G P 47 645 in the D M B A - i n d u c e d estrogen-dependent m a m m a r y c a r c i n o m a model. All three inhibitors show potent a n t i - t u m o r efficacy and in most cases maximally suppress t u m o r volume, cause almost total regression o f existing t u m o r s and inhibit the appearance o f new tumors. These potent a n t i - t u m o r effects are a c c o m p a n i e d by endocrine effects resulting f r o m the potent inhibition o f estrogen biosynthesis in these animals.

1. Fishman J.: Biochemical mechanism of aromatization. Cancer Res. 42 (Suppl.) (1982) 3277s-3280s. 2. Schieweck K., Bhatnagar A. S. and Matter A.: CGS 16949A, a new nonsteroidal aromatase inhibitor: effects on hormone-dependent and -independent tumors in vivo. Cancer Res. 48 (1988) 834-838. 3. Bhatnagar A. S., H~iusler A., Schieweck K., Lang M. and Bowman R.: Highly selective inhibition of estrogen biosynthesis by CGS 20267, a new non-steroidal aromatase inhibitor. J. Steroid Biochem. Molec. Biol. 37 (1990) 1021-1027. 4. Trunet P. F., Miiller Ph., Girard F., Aupetit B., Bhatnagar A. S., Zognbi F., Ezzet F. and Menard J.: The effects of fadrozole hydrochloride on aldosterone secretion in healthy male subjects. J. Clin. Endocr, Metab. 74 (1992) 571 576.