Pharmacology of new aromatase inhibitors

The Breast (1996) 5.202-208 0 1996 Pearson Professional Ltd

ES0 TASK FORCE ARTICLE

Pharmacology of new aromatase inhibitors P. E. Lonning Department of Oncology, Haukeland University Hospital, Bergen, Norway

S UM M A R Y. Recent investigations of novel aromatase inhibitors have addressed several important questions related to the biochemical effects of this class of drugs. While aromatase inhibitors such as aminoglutethimide, formestane (given by the oral and i.m. route), rogletimide and fadrozole administered at different doses have been found to inhibit in vivo aromatization by 60-93%, the novel aromatase inhibitors, letrozole and anastrozole, are shown to inhibit in vivo aromatization by about 98%. Contrary to previous studies on aromatase inhibitors reporting plasma oestrogens sustained at 30-50% of their control levels letrozole and anastrozole suppress plasma oestrone sulphate by approximately 95%. Future studies should explore cross-resistance of aromatase inhibitors and steroidal antioestrogens and between different types of aromatase inhibitors. A major goal is to evaluate alterations in tissue oestrogen concentration as well as growth factor expression in response to oestrogen deprivation with aromatase inhibitors.

INTRODUCTION

stantial number of aromatase inhibitors have been taken into clinical trials.

It has bean known for about 50 years that ablative therapy by adrenalectomy or hypophysectomy can cause tumour regression in postmenopausal women.‘*2 The first generation aromatase inhibitor, an unsuccessful anti-epileptic named aminoglutethimide, was introduced for breast cancer treatment in an attempt to achieve a ‘medical adrenalectomy’ because of its toxic influence on adrenocortical hormone synthesis.3 It was discovered later that, in spite of the fact that aminoglutethimide inhibits several steps in adrenal steroid synthesis,4 androgen synthesis is sustained. On the other hand, treatment with aminoglutethimide was found to produce a substantial fall in plasma oestrogen levels.5 In a classical experiment published in 1978, Santen and co-workers6 were able to show that the conversion of radioactive androstenedione (A) into oestrone (E,) was inhibited by aminoglutethimide in vivo. This introduced the term ‘aromatase inhibition’ as a treatment option for postmenopausal women suffering from advanced breast cancer. Owing to substantial side-effects associated with use of aminoglutethimide and its lack of specificity, many attempts have been made to develop novel, less toxic and more specific aromatase inhibitors. The first selective aromatase inhibitor, formestane (4-hydroxyandrostenedione) was introduced for clinical treatment in 1984.’ Since then, a subAddress correspondence Haukeland University

DIFFERENT INHIBITORS

CLASSES OF AROMATASE

Aromatase inhibitors in clinical use may be divided into two major classes based on their effect on the aromatase enzyme. Steroidal aromatase inhibitors (Fig. 1) are all derivatives of the natural androgen A substituted in different positions. The second are non-steroidal inhibitors with an imidazole or triazole structure (Fig. 2) or a chemical structure resembling aminoglutethimide (Fig. 1).

CURRENT CONTROVERSIES RELATED TO TREATMENT WITH AROMATASE INHIBITORS The major goal in developing novel aromatase inhibitors was to obtain drugs with a better specificity (lack of influence on other steroid synthesizing enzymes) and a less toxic profile than aminoglutethimide. A major concern in relation to treatment with aminoglutethimide (and later also during treatment with second and third generation aromatase inhibitors) is the discrepancy between the degree of aromatase inhibition as measured by in vivo tracer studies and the percentage suppression of plasma oestrogen levels. Thus, while aminoglutethimide as well as the second generation

to: P. E. Lpnning, Department of Oncology, Hospital, N-5021 Bergen, Norway

202

Pharmacology of new aromatase inhibitors CH, O

Table

C%

23 -

0

@

oti

Y k

OH

0

6% Exemestane

P ti

O

9H5\ 0

inhibition

achieved

with different

drugs

Drug

Dose

% aromatase inhibition

Rogletimide Formestane Fadrozole Aminoglutethimide Formestane AG + Form Letrozole Anastrozole

400/800/1600 mg/day 125/250*/250**mg/day 2/4 mg/day 1000 mg/day 250/.500 mgi2 w 500 mglw 100 mg/day, 500 mg/w 0.512.5 mglday 1110 mg/day

50.6l63.5ll3.8 72.3l7O.Ol57.3 82.4192.6 90.6 84.8I91.9I92.5 93.8 98.4/98.9 96.1198.1

All studies performed with use of a sensitive assay based on separation of different urinary oestrogens by HPLC in the Department of Academic Biochemistry, Royal Marsden Hospital, London.t5-*I AG = aminoglutetbimide, Form = formestane, w = week. * 125 mg b.d. ** 250 mg o.d.

Rogletimide

Fig. 1 Aromatase inhibitors in clinical use or trials belonging to the steroidal class (formestane and exemestane) and non-steroidal aromatase inhibitors belonging to the ‘aminoglutethimide’ class (aminoglutetbimide and rogletimide)

_.

N-N

Fadrozole

Letrozole

Anastrozole

Vorozole Fig. 2 Non-steroidal (fadrozole) or triazole

In vivo aromatase

-NH,

Aminoglutethimide

Formestane

1

203

aromatase (vorozole,

inhibitors letrozole,

belonging to the imidazole anastrozole) class.

aromatase inhibitor formestane and the third generation inhibitor fadrozole were all found to inhibit aromatization in vivo by more than 90% when given in optimal doses (Table l), plasma oestrogens were sustained at 3%50% of pretreatment levels.8-‘0 Another controversy relates to the contribution of local oestrogen production (within the cancer cell itself or mesenchymal cells in the tumour cell vicinity) to intratumour oestrogen concentrations. Two studies evaluating the quantitative contribution of circulating oestrogens versus local hormone synthesis to intratumour oestrogen concentration both revealed a substantial variation between individuaIs.“~‘* Steroidal and non-steroidal aromatase inhibitors act differently on the aromatase enzyme in as much as the

steroidal inhibitors bind to the steroid substrate binding site, while the non-steroidal inhibitors bind to the p-450 part of the molecule. This finding has raised the issue whether there may be full cross-resistance between drugs belonging to these two chemical classes. The question is of particular relevance because we have a poor understanding of the mechanisms of acquired resistance to oestrogen deprivation in general. Thus, the following questions need to be addressed in relation to treatment with aromatase inhibitors. 1. How can we explain the finding of sustained oestrogens in patients during treatment with aromatase inhibitors? 2. Is there evidence suggesting a dose response relationship between the degree of plasma oestrogen suppression and the chance of achieving a clinical response to an aromatase inhibitor? 3. Is there evidence suggesting lack of cross-resistance to different aromatase inhibitors in vivo? 4. What are the possible mechanisms of resistance to aromatase inhibitors?

HOW CAN WE EXPLAIN THE FINDING OF SUSTAINED OESTROGENS IN PATIENTS DURING TREATMENT WITH AROMATASE INHIBITORS? In vivo aromatization can be measured by two different methods: either by continuous infusion of [3H]-A and [‘“ClE, with measurement of the isotope ratio in the plasma E, fraction or, alternatively, administering a bolus injection of [3H]-A and [14C]-E, followed by measurement of the isotope ratio in the urinary oestrogens. The second method has the advantage compared with the first that the isotopes in urine may be concentrated to give a much higher radio-activity in the test sample and therefore a better sensitivity of the assay.

204

The Breast

Based on a previous assay designed to separate oestrogen metabolites in urine,13 we have developed a highly sensitive assay to measure in vivo aromatization by isolation of radioactive oestrogen metabolites in urine with HPLC following injection of [3H]-A and [14C]-E,.14A recent formal assessment of sensitivity indicated that inhibition up to 99.1% was detectable.i5 Over recent years this method has been used to study in vivo aromatization with several different aromatase inhibitors such as the first generation inhibitor aminoglutethimide, the second generation inhibitor formestane, the third generation inhibitors fadrozole and rogletimide, and finally the fourth generation inhibitors letrozole and anastrozole.‘5-21 The results obtained with these drugs at different doses are given in Table 1. A direct comparison of the results is justified, in as much as all data were obtained with the same method and the biochemical analysis was conducted in one department (Department of Academic Biochemistry, Royal Marsden Hospital, London, UK). Based on these results, aromatase inhibitors evaluated to date may be separated into three different classes: 1. Drugs causing sub-optimal aromatase inhibition. As may be seen, rogletimide and oral 4-hydroxyandrostenedione cause approximately 50-70% aromatase inhibition. 2. Drugs with a fairly good aromatase inhibition: aminoglutethimide, formestane (given by the intra-muscular route) and fadrozole given in different doses inhibit in vivo aromatization by 82-93%. It is noteworthy that by combining a steroidal and a non-steroidal inhibitor (formestane and aminoglutethimide) it is possible to enhance aromatase inhibition to some extent. 3. Drugs which are highly potent aromatase inhibitors: anastrozole and letrozole, which belong to the triazole class, both cause approximately 98% inhibition of in vivo aromatization. Accordingly, these two drugs are significantly more potent than other aromatase inhibitors. The inhibition achieved with these drugs is significantly better than that achieved even by administering aminoglutethimide and formestane in concert. A major question is whether these highly effective aromatase inhibitors are able to suppress plasma oestrogen levels by the same percentage as they inhibit in vivo aromatization. The difficulty assessing plasma oestrogens in postmenopausal women relates to the detection limits of the assay. Considering plasma Q and E,, mean concentration is about 20 and 70-80 pM in postmenopausal women.22 Radioimmunoassays currently applied have a sensitivity limit for E, and E, of about 2 pM and 7 pM respectively. It may be difficult to detect more than 90% suppression of these plasma oestrogens in the majority of patients. The problem is illustrated by results from recent trials on novel aromatase inhibitors which report many patients obtaining plasma oestrogen levels below the sensitivity limits of the

assays.23,24On the other hand, the oestrogen conjugate oestrone sulphate (E,S) is found in the plasma of postmenopausal women at a concentration of about 400 pM. Plasma E,S is at equilibrium with plasma E, and E,, and as long as drug treatment does not influence the sulphokinase or sulphatase enzymes regulating the conversion of E, to E,S,25plasma E,S may be expected to be suppressed to a similar extent as E, and E,. Recently, we have developed a highly sensitive radioimmunoassay for plasma E,S measurements26 with a sensitivity limit of 2.7 pM. Measuring plasma E,S with this method we found anastrozole, at doses of 1 mg and 10 mg daily, to suppress this oestrogen conjugate by 93.5% and 95.7% respectively.*’ Similar results have been obtained by others in relation to treatment with letrozole.27 Thus, it is clear that treatment with these potent aromatase inhibitors suppresses plasma oestrogen levels by a percentage approaching that evaluated by tracer techniques. These results refute the hypothesis that there may be alternative oestrogen sources in postmenopausal breast cancer patients treated with aromatase inhibitors. Whether previous results are due to technical artifacts cannot be completely addressed at this point. In a recent study we measured plasma levels of E,S, with our new sensitive assay in patients treated with formestane alone or formestane and aminoglutethimide combined. Treatment with formestane suppressed plasma E,S by approximately 70%,28 which is somewhat better than what has been seen in previous studies. In general there have been improved radioimmunoassays in several laboratories over recent years and therefore one should be careful comparing plasma oestrogen levels measured 10 years ago in relation to treatment with first and second generation aromatase inhibitors, to contemporary results with novel drugs. Results from some recent studies reporting percentage suppression of plasma E,S during treatment with aromatase inhibitors are shown in Figure 3.2L~27~28 Aminoglutethimide administered together

Fig. 3 Results from recent studies reporting plasma levels of ElS during treatment with aromatase inhibitors. Values expressed in percentage of control levels.21*27,28 Each bar represent the mean value of an individual study. *Arimidex is trade name for anastrozole.

Pharmacology of new aromatase inhibitors with formestane suppressed plasma E,S by a mean of 93% which is partly due to the fact that aminoglutethimide enhances the plasma clearance rate of E,S by more than loo%.=

IS THERE EVIDENCE SUGGESTING A DOSE RESPONSE RELATIONSHIP BETWEEN THE DEGREE OF PLASMA OESTROGEN SUPPRESSION AND THE CHANCE OF ACHIEVING A CLINICAL RESPONSE TO AN AROMATASE INHIBITOR? Drugs acting on adrenal steroid synthesis and LHRH-agonists suppressing ovarian androgen synthesis in postmenopausal women have all been found to cause minor oestrogen suppression and a small but significant response rate in postmenopausal women. 29While these observations suggest that sub-optimal oestrogen suppression may cause a suboptimal response rate, so far there is little hard evidence from randomized trials to suggest any difference in responserate related to the degree of oestrogen suppression. Randomized studies comparing aminoglutethimide at different doses”e33have all included a limited number of patients and would not be expected to detect a difference in response rate of 5 or 10%. Two recent studies comparing anastrozole, 1 mg or 10 mg daily, to megestrol acetate found the response rate to be similar in all three treatment aims.34 Bearing in mind the number of patients required to detect a difference in response rate of 5 or 10% in a randomized trial (Table 2), we doubt whether it is realistic to document a modest differenCe in response rate by conducting classical ‘phase III trials’. On the other hand, much evidence suggests that patients progressing after oestrogen deprivation (either as de novo or acquired resistance) may achieve a second response to further oestrogen suppression. Thus, four out of 17 patients progressing during treatment with aminoglutethimide 250 mg/day responded to a dose of 1000 mg/day.35 In a pilot study we were able to show that patients progressing on treatment with formestane achieved further oestrogen suppression and in two out of seven patients a clinical response

Table 2 Number of patients needed in each trial arm to validate a difference in response rate between two treatments at a 5% level (2tailed) depending on expected differences in response rate and p-limits demanded45 Low 20 20 25 25

response

rate (%)

Difference 10 10 5 5

(%)

p-limit

Number of patients needed in each arm

0.20 0.10 0.20 0.10

310 410 1300 1700

205

when aminoglutethimide was added.36 Patients previously treated with adrenalectomy or hypophysectomy who have very low plasma oestrogen levels may respond to treatment with aminoglutethimide.37 While many patients treated with aromatase inhibitors have received previous treatment with castration for their breast cancer, in most cases they have received tamoxifen therapy between castration and treatment with the aromatase inhibitor. Only one pilot study3* has evaluated the response to an aromatase inhibitor subsequent to progression on treatment with an LHRH-analogue. In this study, four out of six patients were found to respond to treatment with formestane. Thus, while we lack hard evidence suggesting a dose-response relationship between the degree of oestrogen suppression and the chance of achieving a clinical response, much evidence suggests that step-wise oestrogen suppression may cause subsequent responses in breast cancer patients.

IS THERE EVIDENCE SUGGESTING LACK OF CROSS-RESISTANCE TO DIFFERENT AROMATASE INHIBITORS IN VIVO? As outlined above, patients relapsing after adrenalectomy or hypophysectomy as well as patients progressing on treatment with formestane may all respond to subsequent treatment with aminoglutethimide. On the other hand, it is now clear that patients relapsing on aminoglutethimide may also respond to formestane,28~39and it has been shown that patients relapsing after aminoglutethimide may respond to hypophysectomy.40 Based on current knowledge about plasma oestrogen levels during treatment with the different treatment options it is difficult to explain these observations by enhanced plasma oestrogen suppression. One possibility is that the steroidal and non-steroidal aromatase inhibitors may have a differential effect on intra-tumoral aromatase. Recently, Miller’s group in Edinburgh has shown that the sensitivity of the aromatase enzyme in human tumour specimens to formestane may vary, in as much as the enzyme in some tumours seems to be refractory to the inhibitory effect of the drug.4’ Whether this is due to drug pharmacokinetics (such as ineffective uptake of the drug into the cells) or alterations in the aromatase enzyme itself, is currently not known.

WHAT ARE THE POSSIBLE MECHANISMS OF RESISTANCE TO AROMATASE INHIBITORS? The mechanisms of resistance to oestrogen deprivation are poorly understood. Possible mechanisms may be divided into three groups. First, the tumour may become insensitive to E, as a mitogen. A second possibility is that tumour

206

The Breast

Nr\ 1..El IE* q

!It 4BA PRO

DA

A

BL---J Cancer

cell

Fig. 4 Possible mechanisms of resistance to treatment with aromatase inhibitors. (A) Escape of the local aromatase enzyme to the inhibitory action of the drug (over-expression of normal aromatase enzyme? mutant aromatase enzyme? reduced uptake of the aromatase inhibitor in the cell?) (B) Alterations in oestrogen uptake in the tumour cells. (C) Sensitization of the tomour cells to action of oestrogen hormones either by alterations in the receptor or post-receptor events.

oestrogens escape from deprivation (Fig. 4). Finally, tumour cells may be sensitized to oestrogens and able to respond to E, as a mitogenic stimulus at much lower concentration than it did previously (Fig. 4). It is known from in vitro experiments that breast cancer cells under certain conditions may respond to E, at concentrations as low as l&r4 M.42 Observations that patients relapsing after oestrogen deprivation do sometimes respond to further oestrogen suppression (see above) indicate that such mechanisms may confer tumour growth in vivo. Except for one small study evaluating intra-tumour oestrogens during treatment with formestane,43 little is known about changes in tumour oestrogens in response to treatment with aromatase inhibitors. Neither is it known whether there may be changes occurring over time in relation to development of acquired resistance. It is well known that the tissue concentration of E, is one order of magnitude higher than plasma concentrations in postmenopausal women,44 but we do not know whether this is due to an active transport mechanism or related to specific or non-specific binding of E, to certain elements outside or within the cells. A major target for future research is to address the influence of treatment with aromatase inhibitors on intra-tumoral, hormone concentrations as well as growth factor expression. Several major goals have been reached in relation to treatment with aromatase inhibitors in recent years. We now have highly potent drugs with a favourable toxicity profile. Some of these drugs are able to inhibit in vivo aromatization by about 98% and, perhaps, more importantly, it is now well documented that these drugs also suppress plasma oestrogens by a percentage approaching that for aromatase inhibition. However, several major issues remain to be addressed in relation to the clinical use of these drugs. We need to evaluate whether stepwise oestrogen suppression (e.g. using a less potent aromatase inhibitor followed by a more potent one) is a more suitable treatment option to extend the time period of disease control, compared to maximal oestrogen deprivation as first line treatment. Other possibilities could be to use different aromatase inhibitors in sequence or, alternatively, applying a steroidal and a non-steroidal aromatase inhibitor in concert to achieve maximal suppression of intratumour oestrogens. A third option is to combine a potent aromatase inhibitor with drugs suppressing plasma oestrogen precursors. While previous investigations suggested that drugs like medroxyprogesterone acetate and megestrol acetate suppress plasma androgen and oestrogen levels by about 30%, ongoing investigations in our laboratory suggest these drugs may suppress plasma oestrogens by 70-80%. If this is part of the mechanism of action of progestins in breast cancer, a logical approach would be to use a progestin and a potent aromatase inhibitor in concert following relapse on treatment with an aromatase inhibitor.

Pharmacology of new aromatase inhibitors Acknowledgements The secretarial work of Mrs H. Hjortung and Mrs Y. Homsleth is highly appreciated. The European School of Oncology gratefully acknowledges an educational grant from Zeneca Pharmaceuticals which made the meeting possible.

19.

20.

References 1. Huggins C, Dao TL-Y. Adrenalectomy and oophorectomy in treatment of advanced carcinoma of the breast. JAMA 1953; 151: 1388-1394. 2. Luft R, Olivecrona H, Sjiigren B. Hypofysektomi pa manniska. Nor Med 1952; 14: 351-354. 3. Lonning P E, Dowsed M, Powles T J. Postmenopausal estrogen synthesis and metabolism: alterations caused by aromatase inhibitors used for the treatment of breast cancer. J Steroid Biochem 1990; 5: 355-366. 4. Lonning P E, Kvinnsland S. Mechanisms of action of aminoglutethimide as endocrine therapy of breast cancer. Drugs 1988; 35: 685-710. 5. Samojlik E, Santen R J, Wells S A. Adrenal suppression with aminoglutethimide. II. Differential effects of aminoglutethimide on plasma androstenedione and estrogen levels. J Clin Endocrinol Metab 1977; 45: 48@487. 6. !&ten R J, Santner S, Davis B, Veldhuis J, Samojlik E, Ruby E. Aminoglutethimide inhibits extraglandular estrogen production in postmenopausal women with breast carcinoma. J Clin Endocrinol Metab 1978; 47: 1257-1265. 7. Coombes R C, Goss P, Dowsett M, Gazet J-C, Brodie A. 4hydroxyandrostenedione in treatment of postmenopausal patients with advanced breast cancer. Lancet 1984; ii: 1237-1239. 8. Santen R J, Worgul T J, Lipton A, Harvey H, Boucher A. Aminoglutethimide as treatment of postmenopausal women with advanced breast carcinoma. Ann Intern Med 1982; 96: 94101. 9. Dowsett M, Cunningham D C, Stein R C et al. Dose-related endocrine effects and pharmacokinetics of oral and intramuscular 4-hydroxyandrostenedione in postmenopausal breast cancer patients. Cancer Res 1989; 49: 13061312. 10. Dowsett M, Stein R C, Mehta A, Coombes R C. Potency and selectivity of the non-steroidal aromatase inhibitor CGS 16949A in postmenopausal breast cancer patients. Clin Endocrinol 1990; 32: 623-634. 11. Reed M J, Owen A M, Lai L C et al. In situ oestrone synthesi in normal breast and breast tumour tissue: effect of treatment with 4-hydroxyandrostenedione. Int J Cancer 1989; 44: 233-237. 12. Miller W R. Importance of intratumour aromatase, and its susceptibility to inhibitors. In: Dowsett M, ed. Aromatase inhibition - then, now and tomorrow. London: Partenon Publishing Group, 1994. 13. Lonning P E, Skulstad P, Sunde A, Thorsen T. Separation of urinary metabolites of radiolaballed estrogens in man by HPLC. J Steroid Biochem 1989; 32: 91-97. 14. Jacobs S, Lonning P E, Haynes B, Griggs L, Dowsett M. Measurement of aromatisation by a urine technique suitable for the evaluation of aromatase inhibitors in vivo. J Enzyme Inhibition 1991; 4: 315-325. 15. Dowsett M, Jones A, Johnston S R D, Jacobs S, Trunet P, Smith I E. In vivo measurement of aromatase inhibition by letrozole (CGS 20267) in post menopausal patients with breast cancer. Clin Cancer Res 1995; 1: 1511-1515. 16. MacNeill F A, Jones A L, Jacobs S, Lonning P E, Powles T J, Dowsett M. The influence of aminoglutethimide and its analogue rogletimide on peripheral aromatisation in breast cancer. Br J Cancer 1992; 66: 692497. 17. Jones A L, MacNeill F, Jacobs S, L@ming P E, Dowsett M, Powles T J. The influence of intramuscular 4-hydroxyandrostenedione on peripheral aromatisation in breast cancer patients. Eur J Cancer 1992; 28A: 1712-1716. 18. MacNeill F A, Jacobs S, Dowsett M, Lonning P E, Powles T J. The

2 1.

22.

23.

24.

25.

26.

27.

28.

29. 30.

31.

32.

33.

34.

35.

36.

37.

207

effects of oral 4-hydroxyandrostenedione on peripheral aromatisation in post-menopausal breast cancer patients. Cancer Chemother Pharmacol 1995; 36: 249-254. MacNeill F A, Jacobs S, Lonning P E, Powles T J, Dowsett M. Combined treatment with 4-hydroxyandrostenedione and aminoglutethimide: effects on aromatase inhibition and estrogen suppression. Br J Cancer 1994; 69: 1171-I 175. L@nning P E, Jacobs S, Jones A, Haynes B, Powles T, Dowsett M. The influence of CGS 16949A on peripheral aromatisation in breast cancer patients. Br J Cancer 1991; 63: 789-793. Geisler J, Lonning P E, Dowsett M et al. A randomised, doubleblind, multicentre crossover trial to evaluate in vivo inhibition of aromatase by arimidex (ZD 1033) (1 mg and 10 mg PO OD) in postmenopausal women with breast cancer. The Breast 1995; 4: 227. Lonning P E, Helle S I, Johannessen D C et al. Relations between sex hormones, sex hormone binding globulin, insulin-like growth factor-I and insulin-like growth factor binding protein-l in post-menopausal breast cancer patients. Clin Endocrinol 1995; 42: 23-30. Iveson T J, Smith I E, Ahem J, Smithers D A, Trunet P F, Dowsett M. Phase I study of the oral nonsteroidal aromatase inhibitor CGS 20267 in healthy postmenopausal women. J Clin Endocrinol Metab 1993; 77: 324-331. Johnston S R D, Smith I E, Doody D, Jacobs S, Robertshaw H, Dowsett M. Clinical and endocrine effects of the oral aromatase inhibitor vorozole in postmenopausal patients with advanced breast cancer. Cancer Res 1994; 54: 5875-5881. Lonning P E, Johannessen D C, Thorsen T. Alterations in the production rate and the metabolism of oestrone and oestrone sulphate in breast cancer patients treated with aminoglutethimide. Br J Cancer 1989; 60: 107-l 11. Lonning P E, Ekse D. A sensitive assay for measurement of plasma oestrone sulphate in patients on treatment with aromatase inhibitors. J Steroid Biochem Molec Biol 1995; 55: 409-412. Demers L M. Effects of fadrozole (CGS 16949A) and letrozole (CGS 20267) on the inhibition of aromatase activity in breast cancer patients. Breast Cancer Res Treat 1994; 30: 95-102. Geisler J, Johannessen D C, Anker G, Lonning P E. Treatment with formestane alone and in combination with aminoglutethimide in heavily pretreated cancer patients: clinical and endocrine effects. Eur J Cancer. In press. Lonning P E. New endocrine drugs for treatment of advanced breast cancer. Acta Oncol 1990; 29: 379-386. Bonneterre J, Coppens H, Mauriac L et al. Aminoglutethimide in advanced breast cancer: Clinical results of a French multicenter randomised trial comparing 500 mg and 1 g/day. Eur J Cancer Clin Oncol 1985; 21: 1153-l 158. Bruning P F, Bonfrer J M G, Hart A A M et al. Low dose aminoglutethimide without hydrocortisone for the treatment of advanced postmenopausal breast cancer. Eur J Cancer Clin Oncol 1989; 25: 369-376. Strocchi E, Camaggi C M, Martoni A et al. Aminoglutethimide in advanced breast cancer: Plasma levels and clinical results after low and high dose. Cancer Chemother Pharmacol 1991; 27: 451455. Robustelli GdC. 500 mg versus 1000 mg aminoglutethimide (AG) in advanced breast cancer: 3 years results of an Italian multicentre trial. Eur J Cancer 1991; 27 (Suppl. 2): 69. Jonat W, Buzdar A, Howell A et al. Two randomised trials establishing efficacy and tolerability of arimidex (ZD 1033) in the treatment of postmenopausal women with advanced breast cancer (PABP). Eur J Cancer 1995; 31A (Suppl. 5): 76. Murray R, Pitt P. Low-dose aminoglutethimide without steroid replacement in the treatment of postmenopausal women with advanced breast cancer. Eur J Cancer Clin Oncol 1985; 21: 19-22. Lonning P E, Dowsett M, Jones A et al. Influence of aminoglutethimide on plasma estrogen levels in breast cancer patients on 4-hydroxyandrostenedione treatment. Breast Cancer Res Treat 1992; 23: 57-62. Samojlik E, Santen R J, Worgul T J. Suppression of residual estrogen production with aminoglutethimide in women following

208

38.

39.

40.

41.

The Breast

surgical hypophysectomy or adrenalectomy. Clin Endocrinol 1984; 20: 43-51. Dowsett M, Stein R C, Coombes R C. Aromatisation inhibition alone or in combination with GmRH agonists for the treatment of premenopausal breast cancer patients. J Steroid Biochem Molec Biol 1992; 43: 155-159. Murray R, Pitt P. Aromatase inhibition with 4-OH androstenedione after prior aromatase inhibition with aminoglutethimide in women with advanced breast cancer. Breast Cancer Res Treat 1995; 35: 249-253. Bundred N J, Eremin 0, Stewart H J, Dale B A, Forrest A P M. Beneficial response to pituitary ablation following aminoglutethimide. Br J Surg 1986; 73: 388-389. Sourdaine P, Parker M G, Telford J, Miller W R. Analysis of the

42.

43.

44.

45.

aromatase cytochrome P450 gene in human breast cancers. J Mol Endocrinol 1994; 13: 331-337. Masamura S, Santner S J, Heitjan F, Santen R J. Estrogen deprivation causes estradiol hypersensitivity in human breast cancer cells. J Clin Endocinol Metab 1995; 80: 2918-2925. Reed M J, Aheme G W, Ghilchik M W, Pate1 S, Chakraborty J. Concentrations of oestrone and 4-hydroxyandrostenedione in malignant and normal breast tissue. Int J Cancer 1991; 49: 562-565. Van Landeghem A A J, Poortman J, Nabuurs M, Thijssen J H H. Endogenous concentration and subcellular distribution of estrogens in normal and malignant breast tissue. Cancer Res 1985; 45: 29(X&2904. Simon M. Design and conduct of clinical trials. In: De Vita V T, Hellman S, Rosenberg S, eds. Cancer: Principles and Practice of Oncology. Philadelphia: Lippincott, 1985: 329-350.