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Intracellular aromatase and its relevance to the pharmacological efficacy of aromatase inhibitors
Journal of Steroid Biochemistry & Molecular Biology 76 (2001) 199– 202 www.elsevier.com/locate/jsbmb
Intracellular aromatase and its relevance to the pharmacological efficacy of aromatase inhibitors A.S. Bhatnagar a, A.M.H. Brodie b, B.J. Long b, D.B. Evans a,*, W.R. Miller c b
a No6artis Pharma AG, CH-4002, Basel, Switzerland Department of Pharmacology and Experimental Therapeutics, School of Medicine, Uni6ersity of Maryland, Baltimore, MD 21201, USA c Breast Research Unit, Western General Hospital, Edinburgh EH4 2XU, Scotland, UK
Abstract An important feature of the pharmacological profile of aromatase inhibitors is the ability of the various inhibitors to inhibit intracellular aromatase. It is now well documented that a large proportion of breast tumors express their own aromatase. This intratumoral aromatase produces estrogen in situ and therefore may contribute significantly to the amount of estrogen to which the cell is exposed. Thus it is not only important that aromatase inhibitors potently inhibit the peripheral production of estrogen and eliminate the external supply of estrogen to the tumor cell, but that they in addition potently inhibit intratumoral aromatase and prevent the tumor cell from making its own estrogen within the cell. To study the inhibition of intracellular aromatase we have compared the aromatase-inhibiting potency of the non-steroidal aromatase inhibitors, letrozole, anastrozole and fadrozole in a variety of model cellular endocrine and tumor systems which contain aromatase. We have used hamsters ovarian tissue fragments, adipose tissue fibroblasts from normal human breast, the MCF-7Ca human breast cancer cell line transfected with the human aromatase gene and the JEG-3 human choriocarcinoma cell line. Although letrozole and anastrozole are approximately equipotent in a cell-free aromatase system (human placental microsomes), letrozole is consistently 10 – 30 times more potent than anastrozole in inhibiting intracellular aromatase in intact rodent cells, normal human adipose fibroblasts and human cancer cell lines. Whether these differences between letrozole and anastrozole are seen in the clinical setting will have to await the results of clinical trials which are currently in progress. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Fibroblasts; Genes; Letrozole
1. Introduction Estrogen deprivation is effective therapy for the treatment of hormone dependent breast cancer [1,2]. Since the final step in estrogen biosynthesis is mediated by the enzyme aromatase, aromatase inhibitors offer the best opportunity of potently and selectively inhibiting estrogen biosynthesis. Two such non-steroidal aromatase inhibitors (AIs) which are available commercially today for the treatment of advanced
Proceedings of the 14th International Symposium of the Journal of Steroid Biochemistry and Molecular Biology ‘‘Recent Advances in Steroid Biochemistry and Molecular Biology’’ (Quebec, Canada 24 – 27 June 2000). * Corresponding author. Tel.: + 41-61-6965605; fax: + 41-616963835. E-mail address: [email protected] (D.B. Evans).
breast cancer are letrozole (Femara®) and anastrozole (Arimidex®) [3]. In pharmacologically profiling non-steroidal AIs, we have used both endocrine and anti-tumor effects in assessing efficacy [4]. We have used suppression of uterine weight as a key endocrine parameter and defined the consequences of ovariectomy as the maximal effects which could be obtained. To assess anti-tumor efficacy, we have used carcinogen-induced (DMBA) mammary carcinoma models in rodents. We have reported previously that letrozole when administered once-daily orally, is a highly effective and selective aromatase inhibitor [4]. Further, it has also been shown that treatment with letrozole can achieve total estrogen deprivation in normal adult cycling female rats by mimicking the endocrine sequelae of ovariectomy [5]. We have also reported previously the presence of the aromatase enzyme in human breast cancer tissue and
0960-0760/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0960-0760(01)00050-4
A.S. Bhatnagar et al. / Journal of Steroid Biochemistry & Molecular Biology 76 (2001) 199–202
200
that the enzymatic characteristics of the aromatase found in breast cancer tissue are generally similar to those of aromatase found in other tissues like placenta or ovary [6–9]. In postmenopausal women, intracellular aromatase is present not only in peripheral adipose tissue but also in the breast tumor itself, thus providing the breast tumor cell with two different sources of estrogen (Fig. 1). The first being from the peripheral conversion of androgens to estrogens in adipose tissue which are then transported by the circulation to the tumor cell and the second being the in situ production of estrogens within the tumor cell. Several clinical studies have been performed in the past demonstrating the endocrine effects of aromatase inhibitors on the levels of serum estrogen which are derived from peripheral conversion in adipose tissue in postmenopausal women [10,11]. Here we report on studies performed in vitro where we have taken various sources of aromatase, both cellular and non-cellular, and have compared the inhibition of this aromatase by the two non-steroidal third generation aromatase inhibitors, letrozole and anastrozole and the second generation non-steroidal aromatase inhibitor, fadrozole. With the intent of studying the ability of aromatase inhibitors to inhibit intratumoral
aromatase and as a consequence the in situ production of estrogens, the cellular sources of aromatase that were used included human breast and choriocarcinoma cancer cell lines.
2. Experimental Letrozole, anastrozole and fadrozole used in these studies were all synthesized in the laboratories of Novartis Pharma AG, Basel, Switzerland. Preparations of aromatase were obtained from the following sources, human placental microsomes, hamster ovarian tissue, human adipose fibroblasts obtained by normal female breast tissue obtained at reduction mammoplasty, MCF-7Ca cells which are transfected with the aromatase gene and the human choriocarcinoma cell line JEG-3. The experimental conditions under which aromatase inhibition was measured in human placental microsomes [12], hamster ovarian tissue [13], human adipose fibroblasts [9], MCF-7Ca cells transfected with the aromatase gene and the human choriocarcinoma cell line JEG 3 [14], have been described previously.
Fig. 1. Peripheral and intratumoral production of estrogen in postmenopausal women with breast cancer. Table 1 IC50 (concentration for 50% inhibition) for the inhibition of aromatase Inhibitor
Letrozole Anastrozole Fadrozole
IC50 (nM) Human placental aromatase
Hamster ovarian tissue
Human breast fibroblasts
MCF-7Ca cancer cells
JEG-3 cancer cells
11 23 5
20 600 30
0.8 14 1
0.07 0.82 0.05
0.07 0.99 0.07
A.S. Bhatnagar et al. / Journal of Steroid Biochemistry & Molecular Biology 76 (2001) 199–202
201
Fig. 2. Relative potencies with which letrozole, anastrozole and fadrozole inhibit aromatase from non-cellular and intracellular sources.
3. Results The results obtained from the incubation studies with the three non-steroidal inhibitors in the non-cellular and cellular preparations are shown in Table 1 and Fig. 2. Table 1 lists the IC50s (the concentration of inhibitor at which 50% inhibition of enzyme activity was seen) for inhibition of estrogen production by each of the inhibitors in each of the systems. Using the IC50s from Table 1, the results are plotted as relative potency in Fig. 2, with the potency of anastrozole being set arbitrarily as one. From the results, it is apparent that in a non-cellular preparation of aromatase such as microsomal aromatase prepared from human placenta, all three inhibitors inhibit aromatase equally well. However, in all of the cellular systems used in this study, letrozole and fadrozole are at least 10 fold more effective at inhibiting aromatase compared with anastrozole. This increased potency of letrozole and fadrozole over anastrozole is seen both in non-tumor as well as tumor cells and in cells of either human or rodent origin. Thus both letrozole and fadrozole more effectively inhibit the in situ production of estrogens through the aromatase enzyme than does anastrozole.
4. Discussion In the past we have reported on the differences between letrozole, anastrozole and fadrozole seen in a large variety of in vitro and in vivo preclinical experimental studies [15,16]. Letrozole was demonstrated as being the most effective of the three aromatase inhibitors with an endocrine efficacy equivalent to that of ovariectomy in the rat. Surprisingly, neither fadrozole
nor anastrozole were effective in reducing uterine weight in normal cyclic female rats. One of the explanations offered to explain this finding was the relatively short half lives of both aromatase inhibitors compared to that of letrozole [17]. The results described here offer an additional explanation for the previously reported difference between anastrozole and letrozole. Recently, results from a clinical study in postmenopausal women with breast cancer were reported. In this study, the authors measured effects on circulating estrogens [18] and also determined the effects on in vivo aromatization [19] using the commercially available aromatase inhibitors Femara® (letrozole) and Arimidex® (anastrozole). The authors showed that Femara® was able to reduce circulating levels of serum estrone and estrone sulfate significantly better than Arimidex®. Furthermore, Femara® was able to inhibit in vivo aromatisation significantly more effectively than Arimidex®. One could speculate that these results may be a consequence of the differences in the in situ inhibition of aromatase by the two aromatase inhibitors reported in this publication.
References [1] G.T. Beatson, On the treatment of inoperable cases of carcinoma of the mamma: suggestions for a new method of treatment with illustrative cases, Lancet 2 (1896) 104 – 107. [2] C.K. Osborne, Aromatase inhibitors in relation to other forms of endocrine therapy for breast cancer, Endocrine-Related Cancer 6 (1999) 271 – 276. [3] I.E. Smith, Clinical overview of aromatase inhibitors, in: W.R. Miller, R.J. Santen (Eds.), Aromatase Inhibition and Breast Cancer, Marcel Dekker, New York, 2001, pp. 51 – 62. [4] A.S. Bhatnagar, C. Batzl, A. Ha¨ usler, K. Schieweck, M. Lang, P.F. Trunet, Pharmacology of non-steroidal aromatase inhibitors, in: J.R. Pasqualini, B.S. Katzenellenbogen (Eds.), Hor-
202
[5]
[6]
[7] [8] [9]
[10]
[11]
[12]
[13]
A.S. Bhatnagar et al. / Journal of Steroid Biochemistry & Molecular Biology 76 (2001) 199–202 mone-Dependent Cancer, Marcel Dekker, New York, 1996, pp. 155 – 168. A.S. Bhatnagar, C. Batzl, A. Ha¨ usler, V. Nogue´ s, The role of estrogen in the feedback regulation of follicle-stimulating hormone secretion in the female rat, J. Steroid Biochem. Molec. Biol. 47 (1 – 6) (1993) 161 –166. W.R. Miller, Steroid metabolism in breast cancer, in: B.A. Stoll (Ed.), Breast Cancer: Treatment and Progress, Blackwell Scientific Publications, Oxford, 1986, pp. 156 – 172. W.R. Miller, J. O’Neill, The importance of local synthesis of estrogen within the breast, Steroids 50 (1987) 537 – 540. W.R. Miller, Uptake and synthesis of steroid hormones by the breast, Endocrine-Related Cancer 4 (1997) 307 – 311. W.R. Miller, Biology of aromatase inhibitors: pharmacology/endocrinology within the breast, Endocrine-Related Cancer 6 (1999) 187 – 195. J. Geisler, N. King, M. Dowsett, L. Ouestad, S. Lundgren, P. Walton, P.O. Kormeset, P.E. Lønning, Influence of anastrozole (Arimidex®) a non-steroidal aromatase inhibitor, on in vivo aromatisation and plasma estrogen levels in postmenopausal women with breast cancer, Br. J. Cancer 74 (1996) 1286 – 1291. M. Dowsett, A. Jones, S. Johnston, S. Jacobs, P. Trunet, I.E. Smith, In vivo measurements of aromatase inhibition by letrozole (CGS 20267) in potmenopausal patients with breast cancer, Clin. Cancer Res. 1 (1995) 1511 –1515. A.S. Bhatnagar, A. Ha¨ usler, K. Schieweck, Inhibition of aromatase in vitro and in vivo by aromatase inhibitors, J. Enzym. Inhib. 4 (1990) 1021 –1027. A. Ha¨ usler, L. Schenkel, C. Kra¨ henbu¨ hl, G. Monnet, A.S. Bhatnagar, An in vitro method to determine the selective inhibi-
.
[14]
[15]
[16]
[17]
[18]
[19]
tion of estrogen biosynthesis by aromatase inhibitors, J. Steroid Biochem. 33 (1989) 125 – 131. B.J. Long, S.L. Tilghman, W. Yue, A. Thiantanawat, D.N. Grigoryev, A.M.H. Brodie, The steroidal antiestrogen ICI 182,780 is an inhibitor of cellular aromatase activity, J. Steroid Biochem. Molec. Biol. 67 (4) (1998) 293 – 304. A.S. Bhatnagar, A. Ha¨ usler, K. Schieweck, C. Batzl-Hartmann, M. Lang, P. Trunet, Estrogen depletion in advanced breast cancer: why, how and where are we going?, in: R.D. Rubens (Ed.), Advanced Breast Cancer: reassessing hormonal therapy, Parthenon Publishing Group, London, 1996, pp. 21 – 30. A.S. Bhatnagar, R.M. Bowman, K. Schieweck, C. Batzl-Hartmann, M. Lang, P.F. Trunet, Letrozole: from test tube to patients, in: H.T. Mouridsen (Ed.), New options for the therapy of advanced breast cancer, Parthenon Publishing Group, London, 1997, pp. 9 – 14. A.S. Bhatnagar, W.R. Miller, Pharmacology of inhibitors of estrogen biosynthesis, in: M. Oettel, E. Schillinger (Eds.), Handbook of Experimental Pharmacology, vol. 135/II, Estrogens and Antiestrogens II, Pharmacology and clinical application of estrogens and antiestrogens, Springer Verlag, Berlin, 1999, pp. 223 – 230. J. Geisler, J. Anker, M. Dowsett, P.E. Lønning, Letrozole suppresses plasma estrogen levels in postmenopausal breast cancer patients more completely than anastrozole, Proc. Am. Soc. Clin. Oncol. 19 (2000) 102a. M. Dowsett, J. Geisler, B.P. Haynes, G. Anker, E. Lønning, Letrozole achieves more complete inhibition of whole body aromatisation than anastrozole, Eur. J. Cancer, 36 (Suppl. 5) (2000) S88 (Abs. 221).
Intracellular aromatase and its relevance to the pharmacological efficacy of aromatase inhibitors A.S. Bhatnagar a, A.M.H. Brodie b, B.J. Long b, D.B. Evans a,*, W.R. Miller c b
a No6artis Pharma AG, CH-4002, Basel, Switzerland Department of Pharmacology and Experimental Therapeutics, School of Medicine, Uni6ersity of Maryland, Baltimore, MD 21201, USA c Breast Research Unit, Western General Hospital, Edinburgh EH4 2XU, Scotland, UK
Abstract An important feature of the pharmacological profile of aromatase inhibitors is the ability of the various inhibitors to inhibit intracellular aromatase. It is now well documented that a large proportion of breast tumors express their own aromatase. This intratumoral aromatase produces estrogen in situ and therefore may contribute significantly to the amount of estrogen to which the cell is exposed. Thus it is not only important that aromatase inhibitors potently inhibit the peripheral production of estrogen and eliminate the external supply of estrogen to the tumor cell, but that they in addition potently inhibit intratumoral aromatase and prevent the tumor cell from making its own estrogen within the cell. To study the inhibition of intracellular aromatase we have compared the aromatase-inhibiting potency of the non-steroidal aromatase inhibitors, letrozole, anastrozole and fadrozole in a variety of model cellular endocrine and tumor systems which contain aromatase. We have used hamsters ovarian tissue fragments, adipose tissue fibroblasts from normal human breast, the MCF-7Ca human breast cancer cell line transfected with the human aromatase gene and the JEG-3 human choriocarcinoma cell line. Although letrozole and anastrozole are approximately equipotent in a cell-free aromatase system (human placental microsomes), letrozole is consistently 10 – 30 times more potent than anastrozole in inhibiting intracellular aromatase in intact rodent cells, normal human adipose fibroblasts and human cancer cell lines. Whether these differences between letrozole and anastrozole are seen in the clinical setting will have to await the results of clinical trials which are currently in progress. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Fibroblasts; Genes; Letrozole
1. Introduction Estrogen deprivation is effective therapy for the treatment of hormone dependent breast cancer [1,2]. Since the final step in estrogen biosynthesis is mediated by the enzyme aromatase, aromatase inhibitors offer the best opportunity of potently and selectively inhibiting estrogen biosynthesis. Two such non-steroidal aromatase inhibitors (AIs) which are available commercially today for the treatment of advanced
Proceedings of the 14th International Symposium of the Journal of Steroid Biochemistry and Molecular Biology ‘‘Recent Advances in Steroid Biochemistry and Molecular Biology’’ (Quebec, Canada 24 – 27 June 2000). * Corresponding author. Tel.: + 41-61-6965605; fax: + 41-616963835. E-mail address: [email protected] (D.B. Evans).
breast cancer are letrozole (Femara®) and anastrozole (Arimidex®) [3]. In pharmacologically profiling non-steroidal AIs, we have used both endocrine and anti-tumor effects in assessing efficacy [4]. We have used suppression of uterine weight as a key endocrine parameter and defined the consequences of ovariectomy as the maximal effects which could be obtained. To assess anti-tumor efficacy, we have used carcinogen-induced (DMBA) mammary carcinoma models in rodents. We have reported previously that letrozole when administered once-daily orally, is a highly effective and selective aromatase inhibitor [4]. Further, it has also been shown that treatment with letrozole can achieve total estrogen deprivation in normal adult cycling female rats by mimicking the endocrine sequelae of ovariectomy [5]. We have also reported previously the presence of the aromatase enzyme in human breast cancer tissue and
0960-0760/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0960-0760(01)00050-4
A.S. Bhatnagar et al. / Journal of Steroid Biochemistry & Molecular Biology 76 (2001) 199–202
200
that the enzymatic characteristics of the aromatase found in breast cancer tissue are generally similar to those of aromatase found in other tissues like placenta or ovary [6–9]. In postmenopausal women, intracellular aromatase is present not only in peripheral adipose tissue but also in the breast tumor itself, thus providing the breast tumor cell with two different sources of estrogen (Fig. 1). The first being from the peripheral conversion of androgens to estrogens in adipose tissue which are then transported by the circulation to the tumor cell and the second being the in situ production of estrogens within the tumor cell. Several clinical studies have been performed in the past demonstrating the endocrine effects of aromatase inhibitors on the levels of serum estrogen which are derived from peripheral conversion in adipose tissue in postmenopausal women [10,11]. Here we report on studies performed in vitro where we have taken various sources of aromatase, both cellular and non-cellular, and have compared the inhibition of this aromatase by the two non-steroidal third generation aromatase inhibitors, letrozole and anastrozole and the second generation non-steroidal aromatase inhibitor, fadrozole. With the intent of studying the ability of aromatase inhibitors to inhibit intratumoral
aromatase and as a consequence the in situ production of estrogens, the cellular sources of aromatase that were used included human breast and choriocarcinoma cancer cell lines.
2. Experimental Letrozole, anastrozole and fadrozole used in these studies were all synthesized in the laboratories of Novartis Pharma AG, Basel, Switzerland. Preparations of aromatase were obtained from the following sources, human placental microsomes, hamster ovarian tissue, human adipose fibroblasts obtained by normal female breast tissue obtained at reduction mammoplasty, MCF-7Ca cells which are transfected with the aromatase gene and the human choriocarcinoma cell line JEG-3. The experimental conditions under which aromatase inhibition was measured in human placental microsomes [12], hamster ovarian tissue [13], human adipose fibroblasts [9], MCF-7Ca cells transfected with the aromatase gene and the human choriocarcinoma cell line JEG 3 [14], have been described previously.
Fig. 1. Peripheral and intratumoral production of estrogen in postmenopausal women with breast cancer. Table 1 IC50 (concentration for 50% inhibition) for the inhibition of aromatase Inhibitor
Letrozole Anastrozole Fadrozole
IC50 (nM) Human placental aromatase
Hamster ovarian tissue
Human breast fibroblasts
MCF-7Ca cancer cells
JEG-3 cancer cells
11 23 5
20 600 30
0.8 14 1
0.07 0.82 0.05
0.07 0.99 0.07
A.S. Bhatnagar et al. / Journal of Steroid Biochemistry & Molecular Biology 76 (2001) 199–202
201
Fig. 2. Relative potencies with which letrozole, anastrozole and fadrozole inhibit aromatase from non-cellular and intracellular sources.
3. Results The results obtained from the incubation studies with the three non-steroidal inhibitors in the non-cellular and cellular preparations are shown in Table 1 and Fig. 2. Table 1 lists the IC50s (the concentration of inhibitor at which 50% inhibition of enzyme activity was seen) for inhibition of estrogen production by each of the inhibitors in each of the systems. Using the IC50s from Table 1, the results are plotted as relative potency in Fig. 2, with the potency of anastrozole being set arbitrarily as one. From the results, it is apparent that in a non-cellular preparation of aromatase such as microsomal aromatase prepared from human placenta, all three inhibitors inhibit aromatase equally well. However, in all of the cellular systems used in this study, letrozole and fadrozole are at least 10 fold more effective at inhibiting aromatase compared with anastrozole. This increased potency of letrozole and fadrozole over anastrozole is seen both in non-tumor as well as tumor cells and in cells of either human or rodent origin. Thus both letrozole and fadrozole more effectively inhibit the in situ production of estrogens through the aromatase enzyme than does anastrozole.
4. Discussion In the past we have reported on the differences between letrozole, anastrozole and fadrozole seen in a large variety of in vitro and in vivo preclinical experimental studies [15,16]. Letrozole was demonstrated as being the most effective of the three aromatase inhibitors with an endocrine efficacy equivalent to that of ovariectomy in the rat. Surprisingly, neither fadrozole
nor anastrozole were effective in reducing uterine weight in normal cyclic female rats. One of the explanations offered to explain this finding was the relatively short half lives of both aromatase inhibitors compared to that of letrozole [17]. The results described here offer an additional explanation for the previously reported difference between anastrozole and letrozole. Recently, results from a clinical study in postmenopausal women with breast cancer were reported. In this study, the authors measured effects on circulating estrogens [18] and also determined the effects on in vivo aromatization [19] using the commercially available aromatase inhibitors Femara® (letrozole) and Arimidex® (anastrozole). The authors showed that Femara® was able to reduce circulating levels of serum estrone and estrone sulfate significantly better than Arimidex®. Furthermore, Femara® was able to inhibit in vivo aromatisation significantly more effectively than Arimidex®. One could speculate that these results may be a consequence of the differences in the in situ inhibition of aromatase by the two aromatase inhibitors reported in this publication.
References [1] G.T. Beatson, On the treatment of inoperable cases of carcinoma of the mamma: suggestions for a new method of treatment with illustrative cases, Lancet 2 (1896) 104 – 107. [2] C.K. Osborne, Aromatase inhibitors in relation to other forms of endocrine therapy for breast cancer, Endocrine-Related Cancer 6 (1999) 271 – 276. [3] I.E. Smith, Clinical overview of aromatase inhibitors, in: W.R. Miller, R.J. Santen (Eds.), Aromatase Inhibition and Breast Cancer, Marcel Dekker, New York, 2001, pp. 51 – 62. [4] A.S. Bhatnagar, C. Batzl, A. Ha¨ usler, K. Schieweck, M. Lang, P.F. Trunet, Pharmacology of non-steroidal aromatase inhibitors, in: J.R. Pasqualini, B.S. Katzenellenbogen (Eds.), Hor-
202
[5]
[6]
[7] [8] [9]
[10]
[11]
[12]
[13]
A.S. Bhatnagar et al. / Journal of Steroid Biochemistry & Molecular Biology 76 (2001) 199–202 mone-Dependent Cancer, Marcel Dekker, New York, 1996, pp. 155 – 168. A.S. Bhatnagar, C. Batzl, A. Ha¨ usler, V. Nogue´ s, The role of estrogen in the feedback regulation of follicle-stimulating hormone secretion in the female rat, J. Steroid Biochem. Molec. Biol. 47 (1 – 6) (1993) 161 –166. W.R. Miller, Steroid metabolism in breast cancer, in: B.A. Stoll (Ed.), Breast Cancer: Treatment and Progress, Blackwell Scientific Publications, Oxford, 1986, pp. 156 – 172. W.R. Miller, J. O’Neill, The importance of local synthesis of estrogen within the breast, Steroids 50 (1987) 537 – 540. W.R. Miller, Uptake and synthesis of steroid hormones by the breast, Endocrine-Related Cancer 4 (1997) 307 – 311. W.R. Miller, Biology of aromatase inhibitors: pharmacology/endocrinology within the breast, Endocrine-Related Cancer 6 (1999) 187 – 195. J. Geisler, N. King, M. Dowsett, L. Ouestad, S. Lundgren, P. Walton, P.O. Kormeset, P.E. Lønning, Influence of anastrozole (Arimidex®) a non-steroidal aromatase inhibitor, on in vivo aromatisation and plasma estrogen levels in postmenopausal women with breast cancer, Br. J. Cancer 74 (1996) 1286 – 1291. M. Dowsett, A. Jones, S. Johnston, S. Jacobs, P. Trunet, I.E. Smith, In vivo measurements of aromatase inhibition by letrozole (CGS 20267) in potmenopausal patients with breast cancer, Clin. Cancer Res. 1 (1995) 1511 –1515. A.S. Bhatnagar, A. Ha¨ usler, K. Schieweck, Inhibition of aromatase in vitro and in vivo by aromatase inhibitors, J. Enzym. Inhib. 4 (1990) 1021 –1027. A. Ha¨ usler, L. Schenkel, C. Kra¨ henbu¨ hl, G. Monnet, A.S. Bhatnagar, An in vitro method to determine the selective inhibi-
.
[14]
[15]
[16]
[17]
[18]
[19]
tion of estrogen biosynthesis by aromatase inhibitors, J. Steroid Biochem. 33 (1989) 125 – 131. B.J. Long, S.L. Tilghman, W. Yue, A. Thiantanawat, D.N. Grigoryev, A.M.H. Brodie, The steroidal antiestrogen ICI 182,780 is an inhibitor of cellular aromatase activity, J. Steroid Biochem. Molec. Biol. 67 (4) (1998) 293 – 304. A.S. Bhatnagar, A. Ha¨ usler, K. Schieweck, C. Batzl-Hartmann, M. Lang, P. Trunet, Estrogen depletion in advanced breast cancer: why, how and where are we going?, in: R.D. Rubens (Ed.), Advanced Breast Cancer: reassessing hormonal therapy, Parthenon Publishing Group, London, 1996, pp. 21 – 30. A.S. Bhatnagar, R.M. Bowman, K. Schieweck, C. Batzl-Hartmann, M. Lang, P.F. Trunet, Letrozole: from test tube to patients, in: H.T. Mouridsen (Ed.), New options for the therapy of advanced breast cancer, Parthenon Publishing Group, London, 1997, pp. 9 – 14. A.S. Bhatnagar, W.R. Miller, Pharmacology of inhibitors of estrogen biosynthesis, in: M. Oettel, E. Schillinger (Eds.), Handbook of Experimental Pharmacology, vol. 135/II, Estrogens and Antiestrogens II, Pharmacology and clinical application of estrogens and antiestrogens, Springer Verlag, Berlin, 1999, pp. 223 – 230. J. Geisler, J. Anker, M. Dowsett, P.E. Lønning, Letrozole suppresses plasma estrogen levels in postmenopausal breast cancer patients more completely than anastrozole, Proc. Am. Soc. Clin. Oncol. 19 (2000) 102a. M. Dowsett, J. Geisler, B.P. Haynes, G. Anker, E. Lønning, Letrozole achieves more complete inhibition of whole body aromatisation than anastrozole, Eur. J. Cancer, 36 (Suppl. 5) (2000) S88 (Abs. 221).