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The effect of the aromatase inhibitor, 4-(phenylthio)-4-androstene-3,17-dione, on dimethylbenz(a)anthracene-induced rat mammary tumors
J. steroid B&hem.
Vol.34, Nos IA, pp. 439442, Printedin Great Britain.All rightsreserved
1989 Copyright
0
0022-4731/89 $3.00 + 0.00 1989 PergamonPressplc
THE EFFECT OF THE AROMATASE INHIBITOR, 4-(PHENYLTHIO)-4-ANDROSTENE-3,17-DIONE, ON DIMETHYLBENZ(A)ANTHRACENE-INDUCED RAT MAMMARY TUMORS YUSIJFJ. ABUL-HAJJ Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455, U.S.A. Summary4-(Phenylthio)-4-androstene-3,17-dione (CPTAD), a known inhibitor of human placental aromatase, was examined as a growth inhibitor of DMBA-induced rat mammary tumors. Subcutaneous administration of 4-PTAD at dose levels of 25 or 50 mg/kg/day caused a significant decrease in hormone-dependent tumor growth. Resumption of tumor growth occurred when either the administration of inhibitor was stopped or when inhibitor was coadministered with estradiol indicating that suppression of tumor growth was due to inhibition of estrogen biosynthesis. Additionally, plasma levels of estradiol were found to be lower in the animals treated with 4-PTAD. The major metabolite of 4-PTAD in vitro was identified as 4-(phenylthio)-4-androstene-17fl-ol-3-one and was found to have 60% of the aromatase inhibitory activity of 4-PTAD.
INTRODUCTION Current therapy of breast cancer is by treatment with (a) hormone additive therapy [l], (b) antiestrogens that block the uptake of estradiol binding by estrogen receptors in tumor cells [2] and (c) endocrine ablative therapy that results in removal of circulating estrogens from the system[3]. Although the level of circulating estrogens is greatly reduced following endocrine ablation [3] recent studies have shown that extraglandular estrogens account for nearly all estrogen produced by postmenopausal women [&6]. Furthermore, estrogen synthesis has been shown to occur in some breast tumors [7-lo]. Thus, peripheral estrogen formation could play a significant role, particularly in women with hormonally-dependent metastatic breast cancer. In such patients, effective aromatase inhibitors could be of potential clinical use. Several in vitro studies using aminoglutethimide (AG) [l 1, 13],4-hydroxy-4-androstenedione (4-OHA) [12, 131, 7cr-[(4’aminophenyl)thio]-4-androstenedione (7-APTA) [15, 161 and 4_thiosubstituted4androstenediones [17, 181were found to be effective competitive inhibitors of human placental and mammary tumor aromatase [13, 191. Furthermore, studies in rat mammary tumor models showed that 4-OHA [12], 7-APTA [20] and lo-propargylestr-4-ene-3,17-dione [21] were effective inhibitors of DMBA-induced rat mammary tumors. Currently only two com-
Proceedings of the 9th International Symposium of the Journal of Steroid Biochemistry, Recent Advances in Steroid Biochemistry, Las Palmas, Canary Islands, Spain, 28-31 May 1989.
pounds, AG and A’-testololactone have been used for the treatment of hormone-dependent breast cancer [22,23]. It has been our aim to identify potent and selective steroidal aromatase inhibitors. In addition, it was deemed desirable that any new aromatase inhibitor be free of androgenic and estrogenic activity. 4-PTAD was found to have the desirable profile described above. 4-PTAD was originally identified as an inhibitor of human placental aromatase activity using the tritiated water assay as originally described by Thompson and Siiteri[l l] and later confirmed using the conversion of [4-4C]androstenedione to estrone. To assess the effectiveness of 4-PTAD as an aromatase inhibitor in oivo, studies on the effect of the potent 4-PTAD on reducing the number and size of the DMBA-induced rat mammary tumors is described. Estrogen content in plasma was used as a direct measure of aromatase activity.
EXPERIMENTAL DMBA
tumor induction, growth and treatment
Fifty-five-day-old female Sprague-Dawley rats (obtained from BioLabs, St Paul, Minn.) were given 20 mg of DMBA in 1 ml of sesame oil by gastric intubation. Eighty percent of the rats developed tumors between 9 weeks and 4 months after treatment with DMBA. Rats were palpated for tumors at weekly intervals and the tumors measured in 2 dimensions with a caliper. The size of the tumor was recorded as the mean of 2 perpendicular diameters, one measured across the greatest width, and tumor
439
440
YLISUFJ. ABUL-HAJJ
surface area was plotted on a growth chart. Since it was desired that each treatment group contained about the same tumor size at the initiation of therapy, animals were placed in a treatment group after at least one tumor per rat had reached 2 cm in a single dimension. The average time after appearance of the tumor to initiation of therapy was 3 weeks. Treatment of all groups was continued for an additional 6 weeks. The animals were divided into 4 treatment groups with 10 animals in each group. The following daily injections were given: Group A, 0.2 ml of sesame oil (control); Group B, 25 mg/kg rat/day of 4-PTAD; Group C, 50 mg/kg rat/day of 4-PTAD; Group D, 50 mg/kg/day of 4-PTAD for 4 weeks followed by 50 mg/kg/day of 4-PTAD plus 0.3 pg/ kg/rat/day of estradiol. All injections were given S.C. Mammary carcinomas were considered to be regressing if the tumor size decreased by at least 30% from the beginning to end of treatment period and advancing if the tumor size increased [24].
Incubation, extraction and HPLC analysis
Rat livers were homogenized in a 0.1 M phosphate buffer, pH 7.4 and centrifuged at 2000 g for 15 min to remove cell debris. The incubation mixture consisted of tissue homogenate (30 ml, mg protein/ml), 33 pmol of NADP, 200 pmol glucose-dphosphate, 50 units of glucosed-phosphate dehydrogenase, 250 pmol MgCl, and 5 mg of 4-PTAD. The reaction mixture was incubated for 1 h at 37°C in a constant shaking
bath and terminated
by the addition
of 2 ml
36 32 (1)
20 f v
24
Determination of estradiol plasma IeveI
The measurement of estradiol was carried out using highly specific antiestradiol antisera supplied to us by Dr S. E. Davis, National Institute of Health. Cross-reaction of estrone and 4-PTAD with the antiestradiol antiserum was less than 6% and l%, respectively. Steroids were extracted from plasma (l/4, w/v) with 3 x 5 ml diethyl ether and no purification procedures were incorporated in the assay since results, with and without chromatography, showed no significant difference. Free and bound steroid were separated using dextran-coated charcoal. Recoveries of steroids ranged from 87-102% and duplicate samples agreed to within f 12%.
A
Weeke
’
Fig. 2. Effect of 4-PTAD (50 mg/kg/rat/day) on DMBA tumor growth. Average weekly surface area of 10 control tumors (a), 9 regressing tumors (m), and one advancing tumor (A). Bars, SE. Arrow A indicates time when treatment was begun. Arrow B indicates when 6 rats were continued on 4-PTAD treatment and 3 rats taken off treatment.
36 36 -
32 32 -
28 26
B g I z
24
f v
24
20
I 2
20
I‘:
16
v) ti
12
I I-
:w 0
’
Weeks
Fig. 1. Effect of 4-PTAD (25mg/kg/rat/day) on DMBA tumor growth. Average weekly surface area of 10 control tumors (a), 8 regressing tumors (m) and 2 advancing tumors (A). Bars, SE. Arrow indicates time when treatment was begun.
/I
6
A
Weeks
(10)
B
10
Fig. 3. Effect of coadministration of estradiol and 4-PTAD on DMBA tumor regression. Average weekly surface area of 10 control tumors (O), and 10 regressing tumors (A). Bars, SE. Arrow A indicates time when treatment with 4-PTAD (50mg/kg/day) was begun. Arrow B indicates when both estradiol (0.3 pg/kg/day) and 4-PTAD (50 mg/kg/rat) was begun.
441
Effect of the aromatase inhibitor Table I, Effect of treatment in uiuo of DMBA-induced rat mammary carcinoma with 4-(phenylthio)-4-androstene-3,17-dione (CPTAD)
Group
Treatment
A B C D
Control 4-PTAD 4-PTAD 4-PTAD plus estradiol
Dose (mg/kg/day) 25 50 50
Total no. of animals
Regressing carcinomas ( > 30% size decrease)
IO IO IO IO
0 8 9 0
of 1 N HCl and left at 4°C overnight. The precipitated proteins were centrifuged at 3000 g for 20 min and the supernatant extracted with CHCl,: MeOH (3 : 1). The organic layer was washed twice with water, evaporated to dryness and reconstituted in methanol water. Reverse-phase HPLC was performed with a Beckman system consisting of a single 100 A pump and a single wavelength detector at 254 nm. A 5 p C,, Ultrasphere ODS (Alex Scientific, Berkeley, Calif.) column (4.6 mm x 25 cm) was used, and the separation was achieved with a mixture of MeOH:H,O (8 : 2) as the eluting solvent at a flow rate of 1 ml/min. The identification of the metabolite was determined by comparison of the retention time to that observed for 4-(phenylthio)-4-androstene-l7b-ol-3-one (CPTT). RESULTS AND DISCUSSION
The effects of treatment with 4-PTAD on the growth of DMBA-induced rat mammary carcinomas are shown in Figs l-3 and Table 1. The tumors of control animals grew exponentially. On the other hand, the 4-PTAD treated groups demonstrated a regression in 8 of 10 tumors in the 25 mg/kg/day treatment group (Fig. 1) and 9 of 10 tumors in the 50 mg/kg/day treatment group (Fig. 2). Furthermore, the results in Table 1 show that the number of rats with no evidence of tumors at the end of the treatment period is 5 for Group B and 6 for Group C. In both groups B and C there was a small number
r
Fig. 4. Plasma estradiol concentrations in normal and tumor-bearing rats. At the end of the treatment period, plasma samples were obtained from the tumor bearing rats (n = 46) and estradiol concentrations were determined by radioimmunoassay. Plasma estradiol levels were determined in normal adult rats (N); tumor-bearing rats treated with vehicle only (A); rats treated S.C. with 4-PTAD at 25 mg/ kg/day (B); 4-PTAD at 50mg/kg/day (C); or 4-PTAD at 50 mg/kg/day for the first 3 weeks followed by both 4-PTAD at 50 mg/kg/day and estradiol at 0.3 pg/kg/day for the last 4 weeks (D).
Advancing carcinomas
No evidence of carcinomas at end of 6 week treatment period 0 5 6 0
10 2 I IO
Table 2. Effect of 4-PTAD metabolites on aromatizatiotV Compound None 4-PTAD 4-PT-I Unidentified
% Inhibition 0 91 55 0
‘Human placental microsomes (mg) were incubated with [I/?, 2/l-‘H]4androstene-dione (0.25 PM) in the presence or absence of l.5pM of test compounds. Incubations were carried out in 3 ml of phosphate buffer (pH 7.4) for 30 min at 37°C. The reaction mixture was initiated with cofactors 1 mg NADP, 2mg glucose-6-phosphate and 2.5 units of glucosed-phosphate dehydrogenase.
of tumors which remained unaffected by the treatment. Thus, when rats bearing these tumors were ovariectomized after treatment, the remaining tumors were either unchanged or grew more indicating that these tumors were not hormone dependent. After the 6 week treatment period 3 animals in Group C were left untreated for an additional 4 weeks. By the end of this period all three tumors had resumed growth indicating a resurgence of estrogen biosynthesis. Since CPTAD was found to be an effective inhibitor of tumor growth, experiments were performed to determine if this tumor reduction is due to inhibition of estrogen biosynthesis. Following 3 weeks of treatment (Fig. 3 and Table 1), tumors responded to 4-PTAD by showing a decrease in tumor growth. Coadministration of estradiol and 4-PTAD for an additional 4 weeks resulted in regrowth of the tumor indicating that the inhibitory effect of 4-PTAD is due to aromatase inhibition. To further support that 4-PTAD acts by inhibition of aromatase in viuo, plasma levels of estradiol were determined at the end of the treatment period. As can be seen from Fig. 4, 4-PTAD caused a marked reduction in plasma estradiol levels at both the 25 and 50 mg/kg/day doses to concentrations ranging from 20 to 30 and 8 to 15 pg/ml, respectively. Plasma estradiol levels in the control group ranged from 61 to 79 pg/ml. The studies presented above clearly show that 4-PTAD acts by inhibiting aromatase as evidenced by a decrease in plasma estradiol levels as well as regression of hormone-dependent mammary tumors. However, these studies do not indicate whether the primary in uiuo inhibitory activity of 4-PTAD is
Yusu~ J. ABUL-Hark
442
due to the compound itself or to one or more of its metabolites. To further understand fully the activity of this compound, in vitro incubations of 4-PTAD with rat liver homogenates were carried out. HPLC analysis of the incubation mixture showed that 4-PTAD was metabolized to one major product in 62% yield and was identified as 4-(phenylthio)-4androstene- 17B-ol-3-one (CPTT) by comparison to an authentic sample prepared by chemical synthesis. The remaining peaks obtained from HPLC spectrum showed 20% of substrate remaining and about 20% of unidentifi~ compounds which do not correspond to any of the possible metabolites of 4-PTAD. Incubation of 4-PTT and the unidentified products with human placental microsomes (Table 2) showed that 4-PTT had about 60% of the inhibitory activity of 4-PTAD while the unidentified products showed no inhibition of aromatase. In summary, the evidence indicates that 4-PTAD is an effective inhibitor of DMBA-induced hormone dependent rat mammary tumors which acts by inhibiting estrogen biosynthesis. Furthermore, these studies show that the in oivo effects of 4-PTAD are not only due to the compound itself but also due to its I7-keto reduction product. Acknowledgements-The
author wishes to acknowledge the technical assistance of Y. W. Law and K. D. Leung for
their work on the growth and treatment of DMBA tumors. Thanks aiso to Dr M. A. Ghaffari for the synthesis of several potential 4-PTAD metabolites and HPLC determinations.
REFERENCES 1. McGuire W. L., Chamness G. C., Costlow M. E. and Shepherd R.: Hormone dependence in breast cancer. ~~~~bo~~rn 23 (1974) 75-l& 2. Sutherland R. L. and Murnhv L. C.: Mechanism of estrogen antagonism by nonsteroidal antioestrogens. Molec. cell. Endocr. 25 (1982) S-23. 3. Dao T. and Huggins C.: Bilateral adrenalectomy in the treatment ofcancer of the breast. Arch. Surg. 711(1955) 645-657.
4. Siiteri P. K. and MacDonald P. C. In ~undbook of Physiological Endocrinology (Edited by R. 0. Greep and E. Astwood). American Physiological Society, Washington DC, Vol. 2, Part 1 (1973) pp. 615626. 5. Grodin J. M., Siiteri P. K. and MacDonald P. C.: Source of estrogen production in postmenopausal women. J. clin. Endocr. Metab. 36 (1973) 207-214. 6. Edman C. D., Aiman E. J., Porter J. C. and MacDonald P. C.: Identification of the estrogen product of extraglandular aromatization of plasma androstenedione. Am. J. Obsiet. Gynec. 130 (1978) 439-447.
I. Abul-Hai Y. J.: Metabolism of dehydroepiandrosterone by hormone dependent and hormone independent breast cancer. Steroids 26 (1975) 488-500. 8. Abut-Hajj Y. J., Iverson R. and Kiang D. T.:
Aromatization of androgens by human breast cancer. 33 (1979) 205-222. 9. Li K., Chandra D. P., Foo T. and Adams J. B.: Steroid metabolism by human mammary carcinoma. Steroids Zs (1976) 561-574.
10. Miller W. R. and Forrest A, P. M.: Oestradiol synthesis from C-19 steroids by human breast cancer. Br. J. Cancer 33 (1976) 116118. 11. Thompson E. A. Jr and Siiteri P. K.: The involvement of human placental microsomal P-450 in aromatization. J. biol. Chem. 249 (1974) 5373-5378. 12. Brodie A. M. H., Schwarzel W. C. and Shaikh A. A.: The effect of an aromatase inhibitor. 4-hvdroxv4androstene-3,17-dione, on estrogen-d~~nd~nt processes in reproduction and breast cancer. Endocrinology 6 (1977) 1684-1695. 13. Abul-Hajj Y. J.: Inhibition of androgen aromatization in human breast cancer. J. steroid Biochem. 13 (1980) 1395-1400. 14. Brueggemeier R. W., Floyd E. E. and Counsel1 R. E.: Synthesis and biochemical evaluation of inhibitors of estrogen biosynthesis. J. med. Chem. 21(1978) 1~7-1011. 15. Bellino F. L.. Gilani S. S. H.. Enn S. S.. Osawa Y. and Duax W. L.: Active-site-directed inactivation of aromatase from human placental microsomes by brominated androgen derivatives. Biochemistry 15 w
(1976) 4730-4736.
16. Dao T. L.: Estrogen synthesis in human breast tumor and its inhibition by testololactone and bromoandrostenedione. Cancer Res. 42 (1982) 3338s-3341s. 17. Abul-Hajj Y. J.: Synthesis and evaluation of 4-(substituted thio)-4-androstene-3,17-dione derivatives as potential aromatase inhibitors. J. med. Chem. 29 (1986) 582-584. 18. Abul-Hai Y. .I.: Aromatase inhibition by 4-thiosubstitutu-~androstene-3,17-diones. J. steroid Biochem. 36 (I 990) In press. 19. Abul-Hajj Y. J.: Comparative studies of aromatase inhibitors in relation to the significance of estrogen synthesis in human mammary tumors. Cancer Res. 42 (1982) 3373s-3377s.
20. Brueggemeier R. W. and Li P. K.: Effects of the aroma&e inhibitor 7a-(4’-amino)phenylthio4androstene3,17-dione on 7,12-Dimethyl~nz(a)anthra~ne-undue m~mary carcinoma in rats. Cancer Res. 48 (1988) 680868 10. 21. Ziminski S. J., Brandt M. E., Covey D. F. and Puett D.: Inhibition of aromatase activity and of endocrineresponsive tumor growth by IO-propargylestr-4-ene3,17-dione and its 17-propionate derivative. Steroids 50 (1987) 135-146.
22. Santen R. J., Worgul T. J., Samojhk E., Interrante A., Boucher A. E., Lipton A., Harvey H, A., White D. S., Smart E., Cox C. and Wells S. A.: Randomized trial comparing surgical adrenalectomy with aminoghrtethimide plus hydrocortisone in women with advanced breast cancer: N. Engl. J. Med. 305 (198 1) 545-55 1. 23. Seaaloff A.. Weeth J. 8.. Mever K. K.. Rouaone E. L. and Cunningham M. E. G.: Hormonal therapy in cancer of the breast. XIX. Effect of oral administration of A’-testofolactone on clinical course and hormonal excretion. Cancer 15 (1962) 633635. 24. Heise E., Gorlich M. and Bacigalupo G.: Biochemical studies in advancing and regressing rat mammary carcinoma induced by 7,12-dimethylbenz-a-anthracene (Huggins’ tumors). J. natn. Cancer Inst. 45 (1970) I-12. I
Vol.34, Nos IA, pp. 439442, Printedin Great Britain.All rightsreserved
1989 Copyright
0
0022-4731/89 $3.00 + 0.00 1989 PergamonPressplc
THE EFFECT OF THE AROMATASE INHIBITOR, 4-(PHENYLTHIO)-4-ANDROSTENE-3,17-DIONE, ON DIMETHYLBENZ(A)ANTHRACENE-INDUCED RAT MAMMARY TUMORS YUSIJFJ. ABUL-HAJJ Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455, U.S.A. Summary4-(Phenylthio)-4-androstene-3,17-dione (CPTAD), a known inhibitor of human placental aromatase, was examined as a growth inhibitor of DMBA-induced rat mammary tumors. Subcutaneous administration of 4-PTAD at dose levels of 25 or 50 mg/kg/day caused a significant decrease in hormone-dependent tumor growth. Resumption of tumor growth occurred when either the administration of inhibitor was stopped or when inhibitor was coadministered with estradiol indicating that suppression of tumor growth was due to inhibition of estrogen biosynthesis. Additionally, plasma levels of estradiol were found to be lower in the animals treated with 4-PTAD. The major metabolite of 4-PTAD in vitro was identified as 4-(phenylthio)-4-androstene-17fl-ol-3-one and was found to have 60% of the aromatase inhibitory activity of 4-PTAD.
INTRODUCTION Current therapy of breast cancer is by treatment with (a) hormone additive therapy [l], (b) antiestrogens that block the uptake of estradiol binding by estrogen receptors in tumor cells [2] and (c) endocrine ablative therapy that results in removal of circulating estrogens from the system[3]. Although the level of circulating estrogens is greatly reduced following endocrine ablation [3] recent studies have shown that extraglandular estrogens account for nearly all estrogen produced by postmenopausal women [&6]. Furthermore, estrogen synthesis has been shown to occur in some breast tumors [7-lo]. Thus, peripheral estrogen formation could play a significant role, particularly in women with hormonally-dependent metastatic breast cancer. In such patients, effective aromatase inhibitors could be of potential clinical use. Several in vitro studies using aminoglutethimide (AG) [l 1, 13],4-hydroxy-4-androstenedione (4-OHA) [12, 131, 7cr-[(4’aminophenyl)thio]-4-androstenedione (7-APTA) [15, 161 and 4_thiosubstituted4androstenediones [17, 181were found to be effective competitive inhibitors of human placental and mammary tumor aromatase [13, 191. Furthermore, studies in rat mammary tumor models showed that 4-OHA [12], 7-APTA [20] and lo-propargylestr-4-ene-3,17-dione [21] were effective inhibitors of DMBA-induced rat mammary tumors. Currently only two com-
Proceedings of the 9th International Symposium of the Journal of Steroid Biochemistry, Recent Advances in Steroid Biochemistry, Las Palmas, Canary Islands, Spain, 28-31 May 1989.
pounds, AG and A’-testololactone have been used for the treatment of hormone-dependent breast cancer [22,23]. It has been our aim to identify potent and selective steroidal aromatase inhibitors. In addition, it was deemed desirable that any new aromatase inhibitor be free of androgenic and estrogenic activity. 4-PTAD was found to have the desirable profile described above. 4-PTAD was originally identified as an inhibitor of human placental aromatase activity using the tritiated water assay as originally described by Thompson and Siiteri[l l] and later confirmed using the conversion of [4-4C]androstenedione to estrone. To assess the effectiveness of 4-PTAD as an aromatase inhibitor in oivo, studies on the effect of the potent 4-PTAD on reducing the number and size of the DMBA-induced rat mammary tumors is described. Estrogen content in plasma was used as a direct measure of aromatase activity.
EXPERIMENTAL DMBA
tumor induction, growth and treatment
Fifty-five-day-old female Sprague-Dawley rats (obtained from BioLabs, St Paul, Minn.) were given 20 mg of DMBA in 1 ml of sesame oil by gastric intubation. Eighty percent of the rats developed tumors between 9 weeks and 4 months after treatment with DMBA. Rats were palpated for tumors at weekly intervals and the tumors measured in 2 dimensions with a caliper. The size of the tumor was recorded as the mean of 2 perpendicular diameters, one measured across the greatest width, and tumor
439
440
YLISUFJ. ABUL-HAJJ
surface area was plotted on a growth chart. Since it was desired that each treatment group contained about the same tumor size at the initiation of therapy, animals were placed in a treatment group after at least one tumor per rat had reached 2 cm in a single dimension. The average time after appearance of the tumor to initiation of therapy was 3 weeks. Treatment of all groups was continued for an additional 6 weeks. The animals were divided into 4 treatment groups with 10 animals in each group. The following daily injections were given: Group A, 0.2 ml of sesame oil (control); Group B, 25 mg/kg rat/day of 4-PTAD; Group C, 50 mg/kg rat/day of 4-PTAD; Group D, 50 mg/kg/day of 4-PTAD for 4 weeks followed by 50 mg/kg/day of 4-PTAD plus 0.3 pg/ kg/rat/day of estradiol. All injections were given S.C. Mammary carcinomas were considered to be regressing if the tumor size decreased by at least 30% from the beginning to end of treatment period and advancing if the tumor size increased [24].
Incubation, extraction and HPLC analysis
Rat livers were homogenized in a 0.1 M phosphate buffer, pH 7.4 and centrifuged at 2000 g for 15 min to remove cell debris. The incubation mixture consisted of tissue homogenate (30 ml, mg protein/ml), 33 pmol of NADP, 200 pmol glucose-dphosphate, 50 units of glucosed-phosphate dehydrogenase, 250 pmol MgCl, and 5 mg of 4-PTAD. The reaction mixture was incubated for 1 h at 37°C in a constant shaking
bath and terminated
by the addition
of 2 ml
36 32 (1)
20 f v
24
Determination of estradiol plasma IeveI
The measurement of estradiol was carried out using highly specific antiestradiol antisera supplied to us by Dr S. E. Davis, National Institute of Health. Cross-reaction of estrone and 4-PTAD with the antiestradiol antiserum was less than 6% and l%, respectively. Steroids were extracted from plasma (l/4, w/v) with 3 x 5 ml diethyl ether and no purification procedures were incorporated in the assay since results, with and without chromatography, showed no significant difference. Free and bound steroid were separated using dextran-coated charcoal. Recoveries of steroids ranged from 87-102% and duplicate samples agreed to within f 12%.
A
Weeke
’
Fig. 2. Effect of 4-PTAD (50 mg/kg/rat/day) on DMBA tumor growth. Average weekly surface area of 10 control tumors (a), 9 regressing tumors (m), and one advancing tumor (A). Bars, SE. Arrow A indicates time when treatment was begun. Arrow B indicates when 6 rats were continued on 4-PTAD treatment and 3 rats taken off treatment.
36 36 -
32 32 -
28 26
B g I z
24
f v
24
20
I 2
20
I‘:
16
v) ti
12
I I-
:w 0
’
Weeks
Fig. 1. Effect of 4-PTAD (25mg/kg/rat/day) on DMBA tumor growth. Average weekly surface area of 10 control tumors (a), 8 regressing tumors (m) and 2 advancing tumors (A). Bars, SE. Arrow indicates time when treatment was begun.
/I
6
A
Weeks
(10)
B
10
Fig. 3. Effect of coadministration of estradiol and 4-PTAD on DMBA tumor regression. Average weekly surface area of 10 control tumors (O), and 10 regressing tumors (A). Bars, SE. Arrow A indicates time when treatment with 4-PTAD (50mg/kg/day) was begun. Arrow B indicates when both estradiol (0.3 pg/kg/day) and 4-PTAD (50 mg/kg/rat) was begun.
441
Effect of the aromatase inhibitor Table I, Effect of treatment in uiuo of DMBA-induced rat mammary carcinoma with 4-(phenylthio)-4-androstene-3,17-dione (CPTAD)
Group
Treatment
A B C D
Control 4-PTAD 4-PTAD 4-PTAD plus estradiol
Dose (mg/kg/day) 25 50 50
Total no. of animals
Regressing carcinomas ( > 30% size decrease)
IO IO IO IO
0 8 9 0
of 1 N HCl and left at 4°C overnight. The precipitated proteins were centrifuged at 3000 g for 20 min and the supernatant extracted with CHCl,: MeOH (3 : 1). The organic layer was washed twice with water, evaporated to dryness and reconstituted in methanol water. Reverse-phase HPLC was performed with a Beckman system consisting of a single 100 A pump and a single wavelength detector at 254 nm. A 5 p C,, Ultrasphere ODS (Alex Scientific, Berkeley, Calif.) column (4.6 mm x 25 cm) was used, and the separation was achieved with a mixture of MeOH:H,O (8 : 2) as the eluting solvent at a flow rate of 1 ml/min. The identification of the metabolite was determined by comparison of the retention time to that observed for 4-(phenylthio)-4-androstene-l7b-ol-3-one (CPTT). RESULTS AND DISCUSSION
The effects of treatment with 4-PTAD on the growth of DMBA-induced rat mammary carcinomas are shown in Figs l-3 and Table 1. The tumors of control animals grew exponentially. On the other hand, the 4-PTAD treated groups demonstrated a regression in 8 of 10 tumors in the 25 mg/kg/day treatment group (Fig. 1) and 9 of 10 tumors in the 50 mg/kg/day treatment group (Fig. 2). Furthermore, the results in Table 1 show that the number of rats with no evidence of tumors at the end of the treatment period is 5 for Group B and 6 for Group C. In both groups B and C there was a small number
r
Fig. 4. Plasma estradiol concentrations in normal and tumor-bearing rats. At the end of the treatment period, plasma samples were obtained from the tumor bearing rats (n = 46) and estradiol concentrations were determined by radioimmunoassay. Plasma estradiol levels were determined in normal adult rats (N); tumor-bearing rats treated with vehicle only (A); rats treated S.C. with 4-PTAD at 25 mg/ kg/day (B); 4-PTAD at 50mg/kg/day (C); or 4-PTAD at 50 mg/kg/day for the first 3 weeks followed by both 4-PTAD at 50 mg/kg/day and estradiol at 0.3 pg/kg/day for the last 4 weeks (D).
Advancing carcinomas
No evidence of carcinomas at end of 6 week treatment period 0 5 6 0
10 2 I IO
Table 2. Effect of 4-PTAD metabolites on aromatizatiotV Compound None 4-PTAD 4-PT-I Unidentified
% Inhibition 0 91 55 0
‘Human placental microsomes (mg) were incubated with [I/?, 2/l-‘H]4androstene-dione (0.25 PM) in the presence or absence of l.5pM of test compounds. Incubations were carried out in 3 ml of phosphate buffer (pH 7.4) for 30 min at 37°C. The reaction mixture was initiated with cofactors 1 mg NADP, 2mg glucose-6-phosphate and 2.5 units of glucosed-phosphate dehydrogenase.
of tumors which remained unaffected by the treatment. Thus, when rats bearing these tumors were ovariectomized after treatment, the remaining tumors were either unchanged or grew more indicating that these tumors were not hormone dependent. After the 6 week treatment period 3 animals in Group C were left untreated for an additional 4 weeks. By the end of this period all three tumors had resumed growth indicating a resurgence of estrogen biosynthesis. Since CPTAD was found to be an effective inhibitor of tumor growth, experiments were performed to determine if this tumor reduction is due to inhibition of estrogen biosynthesis. Following 3 weeks of treatment (Fig. 3 and Table 1), tumors responded to 4-PTAD by showing a decrease in tumor growth. Coadministration of estradiol and 4-PTAD for an additional 4 weeks resulted in regrowth of the tumor indicating that the inhibitory effect of 4-PTAD is due to aromatase inhibition. To further support that 4-PTAD acts by inhibition of aromatase in viuo, plasma levels of estradiol were determined at the end of the treatment period. As can be seen from Fig. 4, 4-PTAD caused a marked reduction in plasma estradiol levels at both the 25 and 50 mg/kg/day doses to concentrations ranging from 20 to 30 and 8 to 15 pg/ml, respectively. Plasma estradiol levels in the control group ranged from 61 to 79 pg/ml. The studies presented above clearly show that 4-PTAD acts by inhibiting aromatase as evidenced by a decrease in plasma estradiol levels as well as regression of hormone-dependent mammary tumors. However, these studies do not indicate whether the primary in uiuo inhibitory activity of 4-PTAD is
Yusu~ J. ABUL-Hark
442
due to the compound itself or to one or more of its metabolites. To further understand fully the activity of this compound, in vitro incubations of 4-PTAD with rat liver homogenates were carried out. HPLC analysis of the incubation mixture showed that 4-PTAD was metabolized to one major product in 62% yield and was identified as 4-(phenylthio)-4androstene- 17B-ol-3-one (CPTT) by comparison to an authentic sample prepared by chemical synthesis. The remaining peaks obtained from HPLC spectrum showed 20% of substrate remaining and about 20% of unidentifi~ compounds which do not correspond to any of the possible metabolites of 4-PTAD. Incubation of 4-PTT and the unidentified products with human placental microsomes (Table 2) showed that 4-PTT had about 60% of the inhibitory activity of 4-PTAD while the unidentified products showed no inhibition of aromatase. In summary, the evidence indicates that 4-PTAD is an effective inhibitor of DMBA-induced hormone dependent rat mammary tumors which acts by inhibiting estrogen biosynthesis. Furthermore, these studies show that the in oivo effects of 4-PTAD are not only due to the compound itself but also due to its I7-keto reduction product. Acknowledgements-The
author wishes to acknowledge the technical assistance of Y. W. Law and K. D. Leung for
their work on the growth and treatment of DMBA tumors. Thanks aiso to Dr M. A. Ghaffari for the synthesis of several potential 4-PTAD metabolites and HPLC determinations.
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