Effective Inhibition by Low Dose Aminoglutethimide of Peripheral Aromatization in Postmenopausal Breast Cancer Patients

Aminoglutethimide without glucocorticoid has been shown to be a clinically effective treatment for postmenopausal breast cancer in low dosage (250mgday-1). The mechanism of action of this approach is thought to be the inhibition of peripheral aromatase, the enzyme which converts androstenedione to oestrone. The activity of this enzyme was measured in vivo by injection with 3H-androstenedione and 14C-oestrone and found to be 0.20% + 0.05 in 5 patients on low dose AG therapy. In comparison with previously published data this demonstrates a 92% inhibition of peripheral aromatase activity confirming aromatase inhibition as a viable aim in the endocrine treatment of breast cancer. Aminoglutethimide (AG) is a clinically effective endocrine treatment for advanced postmenopausal breast cancer (Lipton et al. which it has been used almost exclusively in doses of 750-1000mgday-1, in combination with hydro-cortisone (HC). This therapeutic regime was derived with the aim of suppressing adrenal androgen synthesis (Lipton et al., 1974; Wells et al., 1978) which was expected to result from previously reported inhibition by AG of the conversion of cholesterol to pregnenolone by 20,22-desmolase (Cohen, 1967; Dexter et al., 1967). HC was added to the regime to prevent a rise in adrenocorticotrophic hormone, which would result from suppression of cortisol synthesis, and which might overcome the enzyme block (Wells et al., 1978). More recently it has become apparent that AG + HC has little effect on serum levels of adrenal androgens (Samojlik et al., 1980; Harris et al., 1983) and its clinical effectiveness in breast cancer is probably due to inhibition by AG of peripheral (Santen et al., 1978) and perhaps intratumoural aromatization of androgens to oestrogens (Abul-examination of the clinical and endocrine effectiveness of AG at low dosage (250mgday-1) without HC (Harris et al., 1983; Stuart-Harris et al., 1984, 1985). When used in a dose of 1000mg (+ 40mg HG) day-1, AG was found to inhibit peripheral aromatase activity in vivo by at least 95% (Santen et al. 1978). We report here the measurement of this activity in vivo in postmenopausal breast cancer patients undergoing treatment at a lower dosage (250mg day 1). Patients and methods Radioactive injections Five patients treated with AG 125mg twice daily were studied. These patients were part of a clinical study of the effectiveness of this treatment (Stuart-Harris et al., 1984) and their clinical details are given in Table I. Approval from the local ethical committee, informed consent and a DHSS licence were …

Aminoglutethimide (AG) is a clinically effective endocrine treatment for advanced postmenopausal breast cancer (Lipton et al., 1974;Smith et al., 1978;Santen et al., 1981;Harris et al., 1982), in which it has been used almost exclusively in doses of 750-1000mgday-1, in combination with hydrocortisone (HC). This therapeutic regime was derived with the aim of suppressing adrenal androgen synthesis (Lipton et al., 1974;Wells et al., 1978) which was expected to result from previously reported inhibition by AG of the conversion of cholesterol to pregnenolone by 20,22-desmolase (Cohen, 1967;Dexter et al., 1967). HC was added to the regime to prevent a rise in adrenocorticotrophic hormone, which would result from suppression of cortisol synthesis, and which might overcome the enzyme block (Wells et al., 1978).
More recently it has become apparent that AG + HC has little effect on serum levels of adrenal androgens (Samojlik et al., 1980;Harris et al., 1983) and its clinical effectiveness in breast cancer is probably due to inhibition by AG of peripheral  and perhaps intratumoural aromatization of androgens to oestrogens (Abul-HaiJ, 1980;Miller et al., 1982;Tilson-Mallet et al., 1984). The inhibitory potency of AG on aromatase in vitro has been shown to be at least ten-fold greater than on the 20,22 desmolase (Graves & Correspondence: M. Dowsett. Received 18 February 1985;andin revised form 18 March 1985. Salhanick, 1979) and this has led to the examination of the clinical and endocrine effectiveness of AG at low dosage (250mgday-1) without HC (Harris et al., 1983;, 1985. When used in a dose of 1000mg (+ 40mg HG) day-1, AG was found to inhibit peripheral aromatase activity in vivo by at least 95% . We report here the measurement of this activity in vivo in postmenopausal breast cancer patients undergoing treatment at a lower dosage (250mg day 1).

Radioactive injections
Five patients treated with AG 125mg twice daily were studied. These patients were part of a clinical study of the effectiveness of this treatment (Stuart-  and their clinical details are given in Table I. Approval from the local ethical committee, informed consent and a DHSS licence were obtained before commencement of the study. Each patient received by bolus intravenous injection 120 pCi of [7a-3H] androstenedione (A4A, 30 Ci mmol -, New England Nuclear) and 1 pCi of [4-14C] oestrone (E1, 55 mCi mmol-1, Amersham International) in 58 ml isotonic saline between 10.30 and 12.00. One ml of this mixture was retained for estimation of 3H: 14C ratio. All urine passed during the next 72 h was collected and was kept at -20°C until analysis.
©) The Macmillan Press Ltd., 1985 Purification of urinary oestrone The initial stages of purification (i.e. Amberlite chromatography, f-glucuronidase digestion, ethyl acetate extraction and phenolic extraction) were as previously described . After nearly drying the residues from the phenolic extract, they were subjected to thin layer chromatography (TLC) as outlined in Table II. The first system listed was used twice in 4 of the 5 samples; its function was to remove enough extraneous material so that the samples would not smear in subsequent TLCs. The final system was preceded by an acetylation, performed by adding 12 drops of pyridine and 6 drops of acetic anhydride to the sample apd incubating overnight at room temperature.

Results
The 3H: 14C ratio of the injection mixture and of urinary E1 at each stage of purification are shown in Table III together with the calculated p values. The 3H: 14C ratio of urinary E1 was essentially constant between the last 2 chromatographic steps. The mean value for the conversion of A4A to E was 0.20+0.56% (s.d.).
It was not possible to determine pretreatment values in these patients but for comparative purposes values may be drawn from previously published studies (Poortman et al., 1973;MacDonald et al., 1978;Santen et al., 1978) which used the same methodology. These data are shown in Table IV, and are plotted in Figure 1 together with the p values from patients in the current study, and 2 patients treated with 1000mg AG from a previous study . A mean p value of 2.5%+0.8 (s.d.) may be derived from the pooled data. Comparison (unpaired t-test) of these values with those obtained in the 5 patients treated with low dose AG shows that the latter group of values were significantly lower (P<0.001, t=6.77) showing a mean 92% inhibition of conversion of A4A to E1. Comparison of the values in the 5 treated.patients against the 2 obtained previously in patients treated with 1000mg AG daily (p=0.01, 0.08) showed these 2 values to be significantly lower (P<0.05, t=2.64).

Discussion
Aromatase inhibition would appear to be an effective mechanism for the endocrine treatment of postmenopausal breast cancer. This has been previously suggested by demonstration of clinical responses to testololactone (Goldenberg et al., 1973) and aminoglutethimide (Santen et al., 1981) and recently confirmed by the demonstration of   , weight range 45-63 kg; dBreast cancer patients. response to 4-hydroxy-androstenedione, the suicide inhibitor of aromatase (Coombes et al., 1984). AG is a clinically effective agent in postmenopausal breast cancer patients when used without HC at the lower than usual dose of 125mg twice daily . Serum levels of oestrone and oestradiol are significantly suppressed by that dose whilst there are significant increases in the serum levels of androstenedione and testosterone (Harris et al., 1983;, 1985. We have therefore suggested that AG acts through inhibition of peripheral aromatase in this circumstance, and in the current study we have been able to confirm that low dose AG is indeed an effective inhibitor of aromatization in vivo. Comparison of the current data with those from previously published reports is not ideal, but seems acceptable firstly because of the internal consistency of the present data and secondly since the previously published 2 results  Group 3, Table IV) which were obtained from untreated breast cancer patients in the Hershey Laboratory were comparable with those in the other two reports. In addition, there was a close similarity in the techniques used to derive conversion rates between all 3 reports and the current study.
Peripheral aromatization is known to be directly related to patient weight (MacDonald et al., 1978). The mean weight of the 5 patients studied was 66 kg, which is in the upper part of the weight range of the patients in the studies cited (see Legend Table IV). The pretreatment rho values in the 5 patients may therefore have been a little higher than that of the groups used for comparison.
It would appear that the current low dose treatment may be a little less effective in aromatase inhibition than 1000 mg AG although the comparison is on very small numbers and the statistics are of low power. It is probable that the degree of difference is of little clinical significance, but it should be noted that this dose of AG alone also results in a doubling of serum androstenedione levels (Stuart- Harris et al., 1985). The combined effect of less complete aromatase inhibition and higher substrate concentration may make this treatment less effective than conventional dose AG+HC in the suppression of oestrogen synthesis. We are currently comparing the effects of low dose AG with and without HC on oestrogen suppression.