Dose-Related Endocrine Effects and Pharmacokinetics of Oral and Intramuscular 4-Hydroxyandrostenedione in Postmenopausal Breast Cancer Patients1

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(CANCER RESEARCH 49. 1306-1312. March 1, 1989] Dose-related Endocrine Effects and Pharmacokinetics of Oral and Intramuscular 4-Hydroxyandrostenedione in Postmenopausal Breast Cancer Patients1

M. Dowsett,2 D. C. Cunningham, R. C. Stein, S. Evans, L. Dehennin, A. Hedley, and R. C. Coombes Department of Biochemical Endocrinology, Royal Marsden Hospital, Fulham Road, London SW3 6JJ (M. D., S. E.J, and Department of Medical Oncology, St. George's Hospital, London SW17 OQT[D. C. C., R. C. S., A. H., R. C. C.J, United Kingdom, and Fondation de Recherche en Hormonologie, 66-77 Boulevard Pasteur, 94260 Fresnes, France (L. D,]

ABSTRACT sion, whereas a single SOO-mginjection led to maximal suppres sion for over 14 days in 6 of 7 patients. Repeated weekly 4-Hydroxyandrostenedione (CGP32349; 4-OHA) is a clinically effec injections of 500 mg i.m. resulted in persistent estrogen tive treatment for advanced postmenopausal breast cancer by both the parenteral and p.o. routes, as a result of its inhibition of Â¡immutasi-and suppression but at this and higher doses sterile abscesses and lumps at the injection site were noted in some patients (13). It consequent suppression of plasma estrogen levels. Thirty patients were randomized to treatment with 250 mg 4-OHA orally once, twice, and 4 was therefore important to determine to what degree the dose times daily for 2 weeks and 29 of these plus a further 11 patients were of 4-OHA could be reduced without loss of efficacy. then randomized to treatment with 250 or 500 mg i.m. every 2 weeks to Suppression of estradiol levels has been used to determine determine the optimal dose for each route according to the suppression the optimum dose by the i.m. route (11). This approach is of serum estradici levels. There was no significant difference between the utilized in the current study to compare the effectiveness of 250 3 oral doses in their suppression of estradiol levels indicating that the and 500 mg 4-OHA i.m. every 2 weeks, and of 250 mg 4-OHA maximum required p.o. dose of 4-OHA is probably 250 mg daily. p.o. once, twice, or four times a day. Pharmacokinetic profiles Suppression by the parenteral dose of 250 mg every 2 weeks was were obtained for both routes of administration. marginally suboptimal but clinical considerations of response and toler- We previously reported that estrone levels, as measured by ability indicate this as the optimal dose for i.m. injection. 4-OHA had no effect on serum levels of androstenedione, testosterone, or 5a-dihydrotes- radioimmunoassay after chromatography, were not signifi cantly affected by i.m. 4-OHA treatment (13). To clarify this tosterone when given by either route but p.o. treatment with 4 doses of 250 mg daily reduced sex hormone-binding globulin levels by a mean of unexpected result, samples from parenterally treated patients 34%. Serum levels of estrone as measured by gas chromatography-mass were measured in this study by the definitive technique of spectrometry were suppressed to approximately 40% of baseline by GCMS. 4-OHA has no effect on LH, FSH, or SHBG levels parenteral treatment. The half-life of 4-OHA p.o. was approximately 3 when given i.m. at a dose of at least 500 mg weekly (13), but h, whereas the apparent half-life of injected drug was between 5 and 10 no report has been made of the effects of p.o. 4-OHA on these days after a more rapid clearance during the first 4 days after injection. parameters or of the effects on androgen levels of 4-OHA administration by either route. Both of these effects have been INTRODUCTION documented in this study. Inhibition of aromatase, the enzyme complex which converts androgens to estrogens, was first established as an effective MATERIALS AND METHODS measure in the endocrine treatment of advanced breast cancer in postmenopausal women by the use of aminoglutethimide (1- Patients. All patients were either postmenopausal (at least 2 years of amenorrhea) or surgically ovariectomized women and all had histolog- 3). There are a number of disadvantages to the use of this drug. ically or cytologically proven progressive metastatic breast cancer. No These include its inhibition of some other steroid hydroxylases patient had received endocrine or cytotoxic chemotherapy within 4 (4, 5), which necessitates its combination with a glucocorticoid weeks of starting treatment. Informed consent was obtained from all for maximum effectiveness (6) and possibly therapeutic safety patients, and the study was approved by the Ethics Committee at St. (7), and its side effects such as subclinical hypothyroidism in George's Hospital. Patients were free to withdraw at any time. Disease some patients (8) and the frequent occurrence of a skin rash response was assessed according to standard Union Internationale (9). This has led to efforts on the part of many research groups Contre le Cancer criteria (15). Drug. 4-OHA was provided by Ciba-Geigy Pharmaceuticals as a to derive more specific and more potent aromatase inhibitors, sterile microcrystalline formulation (CGP 32349) in ampuls and was which lack significant clinical side effects. stored at 4Â°C.The powder was suspended in physiological saline (125 We have previously reported that 4-OHA3 (Ciba-Geigy, CGP mg/ml) immediately prior to i.m. injection and in water or saline (50 32349) is such an inhibitor and is the first selective inhibitor to mg/ml) for p.o. administration. be studied clinically (10, 11). The compound has been charac Oral Study. Thirty patients were randomized from random number terized as a suicide inhibitor in vivo (12) and has been shown tables to receive 250 mg 4-OHA p.o. once, twice, or four times daily to be clinically effective by both the i.m. and p.o. routes (10, for 2 weeks. Four patients were withdrawn from the study because of 13, 14). Serum estradiol levels are consistently and markedly rapidly progressive disease and they were replaced by four others who suppressed by both p.o. and parenteral 4-OHA (11). For the were assigned in a random manner to the appropriate groups. The p.o. route daily administration is required to maintain suppres- mean ages were 60.2 Â±10.4 (SD), 63.4 Â±10.4, and 68.4 Â±8.4 years and the mean weights were 59.3 Â±6.3, 68.6 Â±20.3, and 65.9 Â±11.9 Received 6/29/88; revised 11/10/88; accepted 11/14/88. kg for the once, twice, and four times daily treatment groups, respec The costs of publication of this article were defrayed in part by the payment tively, and were not statistically significantly different between the of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. groups. No treatment was given for at least 7 days after the last p.o. 1Some of these data were the subject of a presentation at "Hormones and dose of 4-OHA. At least one clotted blood sample was drawn from Cancer, 3" Hamburg, September 1987. each patient on the day before starting treatment and a further pretreat 2To whom requests for reprints should be addressed. ment sample was obtained immediately prior to the first dose of 4- ' The abbreviations used are: 4-OHA, 4-hydroxyandrostenedione; GCMS, gas OHA. On-treatment blood samples were collected 5, 10, 15, 20, 30, chromatography-mass spectrometry; 1.11. luteinizing hormone; FSH, follicle- stimulating hormone; SHBG, sex hormone-binding globulin; 5Â«DHT, Sa-dihy- 45, 60, and 90 min; 3, 4, 6, 8, 10, 12, and 24 h; and 2, 4, 7, and 14 drotestosterone; 4-OHT, 4-hydroxytestosterone; 4-OHA-G, 4-OHA-glucuronide. days after starting treatment. A further sample was taken 7 days after 1306

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1989 American Association for Cancer Research. 4-HYDROXYANDROSTENEDIONE IN BREAST CANCER finishing p.o. treatment. The on-treatment blood samples at 6, 12, and ether/dichloromethane (4/1) to remove the unconjugated 4-OHA and 24 h were taken immediately prior to administration of 4-OHA. Sam was then treated with 0.5 mg bovine liver tÃ-glucuronidase(type B-3; ples were unobtainable at one or more time points in some patients, as Sigma, Poole, United Kingdom) in 500 n\ acetate buffer, pH 4.5, for indicated in the figures. 24 h at 37Â°C.Experiments were conducted which established that these Parenteral Study. Forty patients were randomized into groups and hydrolytic conditions achieved >90% of 4-OHA-G. After hydrolysis received either 250 mg (n = 22) or 500 mg (n - 18) i.m. injections of [3H]4-OHA (donated by Dr. R. Wade, Ciba-Geigy, Horsham, United 4-OHA every 2 weeks. The first 29 patients were the same patients that Kingdom) was added (about 1000 cpm) as a recovery control and the completed the p.o. study which is detailed above. The mean ages were mixture was extracted with 2 doses of 3 ml diethyl ether/dichlorometh 60.2 Â±12.3 (SD) and 66.2 Â±9.3 years and the mean weights were 66.2 ane. Thereafter the technique was identical to that for unconjugated 4- Â±14.5and 68.6 Â±16.7kg, for the 250- and 500-mg groups, respectively, OHA. and were not statistically significantly different between the groups. Statistical Tests. Within- and between-dose comparisons were made The time between finishing the oral study and beginning the parenteral applying analysis of variance to the logarithms of the original values. study was between 7 and 14 days for all except one patient who had a Two-sided tests of significance were applied, with a significance level 35-day gap. Clotted blood samples were drawn on the day before and of 5%. Whenever possible a repeated-measures analysis of variance was on the day of the first injection immediately before the injection was applied, with the Greenhouse-Geisser correction for within-dose com given. Samples were then taken at 1,2, 4, 7, 10, and 14 days after the parisons; otherwise, within- and between-dose comparisons were tested first injection and at weekly intervals thereafter. The samples which for significance, in the event of a statistically significant F test applying were taken on the same day as the drug was given were drawn imme the Bonferroni correction. diately prior to injection. In a number of patients samples were unavail able at some time points. RESULTS Hormone Assays. The assays for estradiol (11), LH, FSH (16), and SHBG (17) were performed according to methodologies which have Oral Treatment. The effect of p.o. 4-OHA on serum estradiol been described in detail previously. Androstenedione, testosterone, and levels is shown in Table 1 for all three doses throughout the 2 SttDHT were analyzed by radioimmunoassays after ether extraction and thin layer Chromatographie purification to avoid cross-reaction weeks of treatment. The effect of doses on estradiol levels on days 7 and 14 of treatment are compared in Fig. 1. The mean with the drug or its putative metabolites. The solvent system used was benzene/ethyl acetate/methanol, 80/20/2, and the support was silica levels were significantly reduced within 3 h of starting treat gel on plastic-backed plates. Recovery was monitored and corrected for ment. After 4 days the mean levels were less than 50% of with tracer quantities of the appropriate tritiated steroids. The radio- pretreatment levels in all three groups. During the next 10 days immunoassay system for testosterone was the St. Thomas's Hospital of treatment there was little variability in the degree of suppres (London, United Kingdom) ether extraction, iodinated tracer kit, which sion, but within 7 days of stopping p.o. therapy the mean uses an antibody raised against a 3-linked conjugate. The androstene- estradiol levels returned to within 16% of pretreatment values dione antibody was raised to an androstenedione-19-carboxymethyl- in all groups. The mean pretreatment estradiol level for the bovine serum albumin conjugate and was used with an iodinated ligand 500-mg group was over 10 pmol/liter higher than those for the (both from Radioassay Systems Laboratories, Carson, CA). The anti other two groups. Although this was not statistically significant body to 5Â«DHTwas raised to a 3-linked conjugate (Chelsea Hospital it is likely that this may have some effect on the levels during for Women) and was used with a tritiated tracer. After chromatography there was no significant interference in any of the androgen assays treatment. It is therefore difficult to perform meaningful com when serum was spiked with 300 ng/ml 4-OHA or with 30 ng/ml 4- parisons with this group. However, comparisons between the OHT. In this study 4-OHA levels were measured in over 700 samples. lowest and highest dose groups showed no significant difference In two samples values were >200 ng/ml, but no values >300 ng/ml in suppression at any point during treatment. were found. There is little information on the metabolism of 4-OHA The concentrations of 4-OHA in serum were measured for in humans, but in rhesus monkeys circulating levels of 4-OHT were the first 24 h after the first dose of 4-OHA. 4-OHA was detected approximately 15% of those of 4-OHA (18). These data therefore in 9 of 10 patients at 5 min postdosing. For most subjects the indicate that the assays are not subject to significant interference by subsequent serum profiles were irregular, with more than one circulating levels of 4-OHA or its potential metabolite, 4-OHT. In all peak or trough in the profile. Maximum serum concentrations assays the within- and between-assays coefficients of variation were less ranged from 23 to 235 ng/ml and were reached 0.5 to 4 h than 9 and 15%, respectively. Estrone levels were measured by GCMS basically as described pre postdosing. The mean levels are shown in Fig. 2. Where the viously (19). Some minor modifications were made: extraction of 2 ml elimination phase could be determined the decline in plasma plasma with dichloromethane and liquid chromatography was per concentrations was adequately described by a monoexponential formed on Sephadex LH-20 columns (120 x 4 mm) with hexane/ decay with a half-life of about 3 h. Seven of the patients who ethanol/acetic acid (80/20/1 ) as an eluent. The first 4 ml were discarded had a pharmacokinetic profile had either overt liver mÃ©tastases and estrone was eluted in the next 4 ml. After evaporation to dryness, or abnormal liver function. The mean level of 4-OHA at 1.5 h estrone was derivatized to the trimethylsilyl ether. after the first dose was 65.4 Â±63.5 (SD) ng/ml in these patients Accuracy was checked by standard additions to a plasma pool (10- compared with 55.6 Â±40.6 ng/ml in the rest of the group (P, 200 pg/ml range). Linear regression of added (x Â±SD) on found (y) not significant). estrone gave the equation Serum levels of 4-OHA-G were measured in the same 24 y= 1.03(Â±0.09)X+ 12.4(Â±1.3) patients 90 min after administration of the first dose of 4-OHA. The mean level was 2.40 Â±1.07 Mg/ml and at the same time The interassay coefficient of variation was 10.4% at the 30-pg/ml level. point the mean level of 4-OHA was 55.2 Â±43.6 ng/ml. The Samples from the same patient were analyzed in the same batch for mean ratio of conjugated to free 4-OHA (determined after all assays. conversion to molar equivalents) was 36.3 Â±19.8 (range, 5- 4-Hydroxyandrostenedione Assay. Levels of 4-OHA were measured with a cross-reacting androstenedione antibody after prior organic 96). The coefficients of variation of these three measurements at 90 min were thus 79% (4-OHA), 44% (4-OHA-G), and 55% extraction and Chromatographie purification, as previously described (4-OHA-G/4-OHA). There was no statistically significant dose and validated (II), with the minor modification that extraction was performed with 2 doses of 3 ml diethyl ether/dichloromethane (4/1). relationship between the free and conjugated forms when ex 4-OHA-G levels were measured after enzymic hydrolysis of the amined by linear regression analysis (r = 0.38, 0.05 < P < conjugate. Serum (50 jil) was extracted with 2 doses of 3 ml diethyl 0.10). 1307

Table 1 Effect ofp.o. 4-OHA treatment on serum estradiÂ»!levels(mean Â±SEM) in postmenopausal women treatmentUnitspmol/liter on Daily dose of 4-OHA(mg)250Time days post- h19.4 day14.4 days14.6 days12.2 days11. days11.6 treatment22.3 + 3.8 Â±2.7 Â±2.4 Â±2.9 Â±2.0 + 3.1 Â±1.5 1Â±1.943.7 Â±1.9 Â±3.5 % of pretreatment 100.03h20.485.9 Â±6.96h18.672.2 Â±4.91271.1 Â±6.61 56.0 Â±4.32 56.0 Â±12.34 48.0 Â±6.47 Â±4.414 47.0 Â±6.67 89.8 Â±9.4 levelPre26.3

500 pmol/liter 37.6 Â±7.8 29.7 Â±6.4 26.1 Â±7.6 28.4 Â±8.2 21.0 Â±6.3 11.8 + 3.8 10.8 + 2.0 9.6 Â±2.0 14.0 Â±3.0 27.3 Â±8.7 % of pretreatment 100.0 84.8 + 7.4 72.1 Â±9.9 78.3 Â±14.7 69.8 Â±15.0 41.4 Â±7.9 35.7 Â±6.6 28.6 Â±4.9 40.9 Â±5.1 95.5 + 19.4 level

1000 pmol/liter 27.2 Â±5.5 22.3 Â±5.6 20.5 Â±3.1 16.8 + 2.6 12.4 Â±2.4 12.0 Â±2.2 8.7 Â±1.6 11.0 Â±1.9 8.3+1.1 22.5 + 4.8 % of pretreatment 100.0 86.8 Â±5.5 85.3 Â±8.9 73.0 Â±8.8 57.2 Â±11.0 59.8 Â±13.4 35.5 Â±8.7 50.4+10.7 41.5 Â±8.2 84.5 Â±14.2 level

PRE The effect of p.o. 4-OHA treatment in LH, FSH, SHBG, testosterone, SaDHT, and androstenedione levels is shown in 40Estradiol Table 2. There were no significant differences between the groups for any of the analytes before treatment. LH, testoster 30 one, SaDHT, and androstenedione levels were unaffected by pmol/L2010n=iK)\10iIODI4D7rh. p.o. 4-OHA treatment. For FSH there was a minor significant increase in levels (by a mean 16% above pretreatment levels, P < 0.05) in patients treated with 250 mg twice daily. SHBG 1.uriT - T . T.Â» r l levels were significantly suppressed at both the lowest and r|_loj_9_jjoj1 9 IOâ€¢Ã•-1J highest doses, by means of 9.5% (P < 0.05) and 34.0% (P < xl x2 xl x2 x4 xl x2 x4 0.01), respectively. DAILY DOSE 4-OHA (x250mg) Parenteral Treatment. The mean serum levels of estradiol before and during treatment with i.m. 4-OHA are shown for 100% each dose in Fig. 3. Levels were significantly suppressed to of8Â°pretreatment60 approximately 60% of baseline levels for both doses after 24 h. On day 7 the mean (Â±SEM) levels were 9.2 Â±1.5 pmol/liter (26.5 Â±3.0% of baseline) and 8.7 Â±0.9 pmol/liter (42.4 Â± 7.2% of baseline) for the lower and higher doses, respectively. 4020'D7DI4â€¢nÂ«ilIOi"9ilIOt|9T1K>Â» Thereafter, levels were maintained below 50% of baseline throughout the next 9 weeks in both groups. There was no statistically significant difference in estradiol levels between the two doses at any point during treatment. However, the suppres sion was more variable between time points with 250 mg than xl x2 x4 xl x2 x4 DAILY DOSE 4-OHA (x250mg) with 500 mg. For the lower dose the estradiol levels were Fig. 1. Effect of 2 weeks of p.o. 4-OHA on mean serum estradici levels in 30 significantly higher on day 14 than on day 7 and on day 28 postmenopausal breast cancer patients, liars, SEM. Top, estradici levels expressed than on day 21. This phenomenon of recovery just prior to the as pmol/liter; bottom, estradici levels expressed as percentage of pretreatment next injection also appeared to occur prior to the fifth and sixth levels. D, day. injections but this was not statistically significant. It can be seen from Fig. 4, in which individual patient values are plotted during the first 4 weeks of treatment, that the recovery of estradiol levels in the 250-mg group was most apparent in those patients with pretreatment values >35 pmol/ 60r liter. However, of the 6 patients treated with 250 mg 4-OHA every 2 weeks who showed an objective clinical response to therapy, 3 had pretreatment estradiol levels >35 pmol/liter. Only 1 of these 3 responders had on-treatment estradiol levels which were maintained below 20 pmol/liter. There were 5 patients in the 500-mg group with pretreatment estradiol levels >35 pmol/liter, but there was little evidence of a recovery phenomenon even within that subgroup. A mathematical estimate of the quantitative importance of the recovery phenomenon in the patients in the 250-mg group with pretreatment estradiol values >35 pmol/liter is made in Fig. 5. The day 7 and day 21 estradiol values are taken as minimal on-treatment levels after the first and second injections Time after of 4-OHA and the lines connecting these points with the day 14 and day Fig. 2. Mean serum levels of 4-OHA after a single 250-mg p.o. dose. Bars, SEM. The numbers of observations made at each time point are indicated under 28 values, respectively, are taken to represent the intermediate bars. estrogen levels. The nonshaded areas then indicate the degree 1308

Table 2 Effect ofp.o. 4-OHA treatment on mean (Â±SEM)serum level ofLH, FSH, SHBG, testosterone (testo), SaDHT, and androstenedione (\4A) in postmenopausal women The numbers in parentheses indicate the number of observations. Pre- and on-treatment samples were analyzed for the same patients in all cases. 250 mg in 1 dose 250 mg twice daily 250 mg 4 times daily Hormone Units Pre On Pre On Pre On LHFSHSHBGTestoSaDHTA4AIU/literIU/liternmol/liternmol/liternmol/liternmol/liter27.8Â±3.3(10)36.8 Â±4.136.1 Â±6.5(9)37.9 Â±6.342.7 10.5(10)40.6 Â±8.239.4 Â±3.0(10)81.5 Â±3.574.5+ + 7.6(9)60.0 Â±8.2Â°55.8 Â±9.5(10)77.0+ Â±9.246.4 Â±12.2(10)1.08 12.1Â°1.18 Â±9.5(9)1.15 Â±8.71.24 12.4(10)0.89 +5.5*1.40 (8)0.40Â±0.30 +0.280.41 +0.30(6)0.33 Â±0.250.31 Â±0.20(5)0.40 Â±0.240.42 Â±0.06(10)1.73 Â±0.101.76 Â±0.07(7)1.62 Â±0.062.08 Â±0.07(8)1.94 Â±0.072.99 + 0.28(10)27.9 + 0.3133.1 Â±0.38 (7)35.7 + 0.2240.2+ Â±0.43 (8)35.0 Â±0.59

(126) 250 500 60

IIII 60

21 28 35 42 49 20 DAYS ON TREATMENT Fig. 3. Effect of i.m. 4-OHA every 2 weeks on mean serum estradiol levels in 10 40 postmenopausal breast cancer patients. Bars, SEM. O, 250 mg; â€¢¿.500mg. Arrows, time of injection.

PRE I PRE I 3 4 of suppression during the second and fourth weeks as being 40 84.5 and 83.7% maximal. An equivalent estimate for all patients in the 250-mg group gives values of 85.5 and 87.6% maximal 30 suppression, respectively. The subgroup with the higher pre treatment estradiol levels is thus little different from the whole group in this respect. Twenty-nine of the patients that received p.o. therapy were 10 also included in the parenteral study. A comparison of the estradiol suppression achieved by the two routes is made in Fig. 6. It can be seen that there was little difference in any of the six PRE I Â» 3 4 PRE I 2 3 4 subgroups after the change from p.o. to i.m. treatment. The WEEKS ON TREATMENT subgroups are too small to allow meaningful statistical analysis, Fig. 4. Individual serum estradiol levels in patients during the first 4 weeks of but an overall comparison of p.o. versus parenteral treatment treatment with 250 mg (left) or 500 mg (right) 4-OHA i.m. every 2 weeks. Top showed no statistically significant difference (P = 0.18). and bottom, levels in patients with pretreatment serum estradiol levels >35 pmol/ After the first injection serum levels of 4-OHA were consist liter and <35 pmol/liter, respectively. ently lower in the 250-mg group than in the 500-mg group [approximately one-half (Fig. 7)]. Peak levels occurred between Nine patients had either overt liver mÃ©tastasesor abnormal 1 and 2 days after the first injection, but by day 4 levels had liver function. The mean level of 4-OHA in the five treated fallen to less than one-half of the peak values. Thereafter the with 250 mg was 2.9 Â±0.9 (SD) ng/ml after 7 days and in the fall in serum 4-OHA levels was slower and approximately log- four treated with 500 mg was 6.2 Â±6.6 ng/ml. The comparable linear with an apparent half-life of between 5 and 10 days. After values of the other patients in the two groups were 2.3 Â±0.9 the second and subsequent injections serum drug levels were and 5.6 Â±2.5 ng/ml, respectively. There was no significant measured at weekly intervals and were found to show an in difference in the levels according to the presence or absence of verted V pattern with lowest levels just prior to the next liver disease. injection (Fig. 8). For the 250-mg group the mean levels (Â± The data were examined to determine whether there was any SEM) at the midpoint between injections became progressively correlation on days 1, 7, 14, 21, and 28 between 4-OHA higher (day 7, 2.4 Â±0.2 ng/ml; day 63, 4.5 Â±0.7 ng/ml). The concentrations and the suppression of estradiol levels, when the 10 patients who completed 10 weeks of treatment on 250 mg latter were expressed as absolute concentrations or as percent had similar serum 4-OHA levels during the first month of ages of pretreatment. No significant relationship was found for therapy as the whole group. There was therefore no evidence either dose on any of these days. that a patient selection might be responsible for the progres Serum levels of estrone were measured by GCMS in 9 pa sively higher serum levels. tients before and during treatment with either 250 mg (n = 5) 1309

MEAN 60 PRETREATkCNT 2pmol/L 500 mg ---250 mg

40 845% 837%

20 MEAN'MINIMAL' ON-TREATMENT 155% LEVEL I 2 4 IO 14 DAYS ON TREATMENT IO 14 21 28 Fig. 7. Mean serum levels of 4-OHA after a single i.m. injection of 250 mg DAYS ON TREATMENT ( ) or 500 mg ( ) 4-OHA. Bars, SEM. The numbers of observations made Fig. 5. Estimation of mean quantitative importance of recovery phenomenon at eact time point are indicated alongside the bars. in patients treated with 250 mg 4-OHA i.m. every 2 weeks and pretreatment estradio! levels >35 pmol/liter. The points at days 7 and 21 are taken as being the mean minimal on-treatment estradici level after the first and second injection. LI degree by which suppression is submaximal during the second week after injection, quantified as a percentage of the oblongs enclosed by the dashed lines.

250mg, i.m 500mg, im Ã¬ 4

nÂ«4 250mg x I. p.p. 19 17

3456789 IO WEEKS ON TREATMENT Fig. 8. Mean serum levels of 4-OHA 7 and 14 days after repeated 2 weekly i.m. injections with 250 or 500 mg 4-OHA. Bars, SEM. The numbers of obser Pre p.0 i.m. Pre p.c. im 40 vations made at eact time point are indicated alongside the bars.

n-5 250mg x2,po 125

IOO- Estrone pmol/L 75 Pre po. Â¡m Pre p.a.Im

30' 30 50 ~nnÂ«4 20 20 25Omgx4,poT 25-

IO 10nÂ»5.T1~

PRE ON Pre p.0.im Pre po Lm. Fig. 9. Effect of 4-OHA i.m. every 2 weeks on serum estrone as measured by Fig. 6. Comparison of the effect of p.o. and parenteral 4-OHA treatment on GCMS. â€¢¿,250mg; O, 500 mg. / test, P = 0.001. mean serum estradiol levels. Bars, SEM. Top, middle, and bottom, patients who received 250 mg in l dose, 2 doses or 4 doses p.o. daily, respectively. Left and Table 3 Effect of i.m. 4-OHA treatment on mean (Â±SEM)serum levels of right, those who transferred to 250 or 500 mg i.m. every 2 weeks, respectively. testosterone, androstenedione (\4A), and SaDHT (nmol/liter) On-treatment levels were measured on day 21 of therapy. The numbers in or 500 mg 4-OHA every 2 weeks. On-treatment levels were parentheses indicate the number of observations. made after at least 21 days of therapy (range, 21-36 days). 250 mg 500mg Levels were suppressed in all patients, the mean fall being from Hormone Pre On Pre On 82.3 Â±10.3 [SEM] to 33.0 Â±9.2 pmol/liter, i.e., to a mean Testosterone 0.78 Â±0.09 (10) 0.75 Â±0.07 1.35 Â±0.29 (9) 1.09 Â±0.17 40.3 Â±7.0% of baseline [P = 0.001 (Fig. 9)]. The serum SaDHT 0.38 Â±0.06 (10) 0.38 Â±0.04 0.41 Â±0.05 (9) 0.41+0.07 estradiol levels in these same patients were suppressed to a A4A 1.67 Â±0.26 (10) 1.97 Â±0.40 1.87 Â±0.35 (9) 1.59 Â±0.27 mean 29.4% of baseline at the same time points. There was no statistically significant difference in any of the androgen levels as a result of the low toxicity of the medical regimens now between samples taken prior to treatment and those taken on day 21 of i.m. therapy with either dose of 4-OHA (Table 3). available. Aromatase inhibition with aminoglutethimide achieves a similar profile in terms of clinical efficacy to the antiestrogen tamoxifen (20, 21), which has become the bench DISCUSSION mark for new endocrine treatments, but aminoglutethimide has Endocrine treatment is the favored first line therapy for breast significant side effects (8, 9) and requires concurrent glucocor- cancer in tumors identified as estrogen receptor positive, largely ticoid administration for maximal effectiveness (6). Our earlier 1310

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1989 American Association for Cancer Research. 4-HVDROXYANDROSTENEDIONE IN BREAST CANCER reports (10, 11, 13, 14) indicate that 4-OHA is a potent, specific by p.o. but not parenteral treatment is probably due to the high aromatase inhibitor with few significant clinical side effects levels of drug presented by p.o. treatment to the liver, the organ when given by either the p.o. or i.m. route. In this report we of SHBG synthesis. have continued to use the suppression of serum estradiol levels The levels of androstenedione, testosterone, and SaDHT as a marker of the effectiveness of aromatase inhibition with 4- were not significantly affected by either p.o. or parenteral OHA and have conducted pharmacokinetic analyses as well as therapy. It therefore seems unlikely that the effect of high doses clarifying a number of endocrine questions which we had not of the drug on 170-hydroxysteroid dehydrogenase and 5a- approached previously. reductase which have been noted in vitro (23, 25) will be of any We have shown in an earlier report that in 6 patients receiving importance to the pharmacological activity of 4-OHA at ther 500 mg 4-OHA p.o. for 7 days, serum estradiol levels were apeutic doses in postmenopausal breast cancer patients. suppressed to 44.5 Â±7.9% of baseline which was not signifi Comparison of 250 mg with 500 mg 4-OHA given i.m. every cantly different from that after 500 mg i.m. (11). Although 2 weeks indicates that the lower dose may be slightly suboptimal there was no significant difference between the suppression in terms of estradiol suppression since there is an increase in achieved by 250- and 500-mg p.o. daily, in that report the trend mean estradiol levels prior to the next injection which was was in favor of a greater effect at the higher dose (to 58.3 Â± statistically significant during the first month of treatment but 6.1% of baseline with 250 mg; 50.8 Â±7.9% with 500 mg). In also apparent after the fourth and fifth injections. It was also the current study a comparison of 250 mg given once, twice, or clear, however, that this recovery phenomenon was most ap four times daily was made but interpretation of the data was parent in those patients with the higher pretreatment levels of complicated by the higher pretreatment estradiol levels in the estradiol (>35 pmol/liter). It may therefore have been consid twice daily group. Nonetheless, the suppression with 250 mg/ ered that these patients might benefit from higher dose treat day was very similar to that achieved with 1000 mg/day. This ment but the observations that 3 of the 6 responders to treat lower dose therefore appears to be an acceptable dose for ment with 250 mg every 2 weeks were in the group with therapeutic study. To confirm that this is the minimal dose pretreatment levels >35 pmol/liter and that only 1 of these 3 which achieves maximal estradiol suppression, we are conduct responders had estradiol levels which were suppressed consist ing a further pharmacoendocrine study to compare 62.5, 125, ently below 20 pmol/liter throw doubt on the significance of 250, and 500 mg 4-OHA p.o. daily. the recovery of estradiol levels. The measurement of serum 4-OHA levels has demonstrated The estimates of the quantitative importance of recovery in that the drug is rapidly absorbed and reaches mean peak levels the groups with pretreatment levels of estradiol above and below at approximately 90 min after administration. There was very 35 pmol/liter may provide an explanation for the apparent lack marked variability in levels of 4-OHA between patients. Thus, of importance of the suboptimal estradiol levels. Firstly, the a 10-fold difference existed between the highest and lowest peak estimates indicate that the suppression is suboptimal by only levels. It is likely that there are many determinants of this about 16% during the second weeks after injection and over the variability. Although the heaviest patient (109 kg) had the 2-week injection period this will amount to about 8%. Indeed, lowest peak values, weight was not closely related to 4-OHA this may be a high estimate since the recovery may follow a levels, and the degrees of absorption and metabolism during concave exponential function rather than a straight line. Sec first pass through the liver also probably affect circulating levels. ondly, the proportional recovery is similar between the group It has been demonstrated that 4-OHA-G is the major metab with higher estradiol levels and the group as a whole. In a Phase olite of 4-OHA (22) and we have shown here that the circulating II study of 250 mg i.m. every 2 weeks the response rate was levels of the conjugate are between 5- and 100-fold higher than identical (33%) to that in a previous study of higher dose (500 free-drug 90 min after dosing. The variability of conjugate levels mg weekly) treatment. The incidence of pain and sterile abscess was markedly less that that for the free drug. This may relate formation at the injection side was, however, substantially to variability in absorption and in first pass liver metabolism of reduced at the lower dose.4 In the light of the clinical efficacy the drug. of the lower dose of 4-OHA, the lower incidence of side effects The half-life of about 3 h for the parent compound leads to and the marginal nature of the submaximal suppression of levels which are largely undetectable (<0.8 ng/ml) 24 h after a estradiol, 250 mg i.m. every 2 weeks would appear to be the 250-mg p.o. dose. This may, however, be relatively unimportant optimal parenteral dose. in the light of the irreversible nature of the inhibition by this The long half-life of 4-OHA after i.m. administration indi drug of aromatase (12). The data in Table 1 indicate that there cates that a depot of drug is formed. The release of the com is continued progressive estradiol suppression during the first pound from the depot appears to be more rapid during the first 24 h of treatment with a single dose of 250 mg daily despite few days than after 4 days; the levels fall by about 50% between this rapid clearance of 4-OHA from the blood. days 2 and 4 but thereafter the apparent half-life is approxi We have found previously that parenteral treatment does not mately 5-10 days. The much lower levels of drug during the affect serum LH, FSH, or SHBG levels (13). In this study oral few days prior to the next injection undoubtedly relate to the treatment had no effect on LH and the minor, statistically recovery of estradiol levels. We previously published prelimi significant increase in FSH levels at the dose of 250 mg twice nary data from 6 patients indicating that the circulating levels daily seems likely to have been a chance observation since the of 4-OHA were directly related to the maintenance of estradiol highest dose had no effect. However, there was a very marked suppression-recovery to above 50% of pretreatment levels did suppressive effect on SHBG with the highest p.o. dose. This is not occur until levels had fallen below 3 ng/ml (11). In this probably a reflection of the minor androgenic activity of the current study there were 6 patients receiving 250 mg every 2 compound which has been noted in vitro (23) and in rats in vivo weeks who showed this degree of recovery during the first 2 (24) but had not been found previously in our human studies. weeks of treatment, and their serum levels of 4-OHA at the The importance of this is marginal since the lower dosages 4 R. C. Stein, M. DowseÂ«,A. Hedley, J. Davenport, and R. C. Coombes. which are likely to be used therapeutically had only a minor Treatment of advanced breast cancer in postmenopausal women with 4-hydrox- effect on SHBG levels. The occurrence of this change in SHBG yandrostenedione. submitted for publication. 1311

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1989 American Association for Cancer Research. 4-HYDROXYANDROSTENEDIONE IN BREAST CANCER time of recovery ranged between 0.6 and 2.3 ng/ml. This Proc. Soc. Exp. Biol. Med., 127: 1086-1090, 1969. 5. Touitou, Y.. Bogdan, A., Legrand, J. C., and Desgrez, P. Aminoglutethimide supports our original observation although there was no statis and glutethimide: effects on 18-hydroxycorticosterone biosynthesis by human tical correlation between serum levels of 4-OHA and estradiol. and sheep adrenals in vitro. Acta Endocrinol., XII:517-526, 1975. After the first injection the ratio of serum 4-OHA levels 6. Dowsett, M., Harris, A. L., Stuart-Harris, R., Hill, M., Cantwell, B. K. J., Smith, I. E., and Jeffcoate, S. L. A comparison of the effects of low dose between groups receiving 250 and 500 mg every 2 weeks was aminoglutethimide with and without hydrocortisone in postmenopausal approximately 1/2. The finding that the ratio increased as breast cancer patients. Br. J. Cancer, 52: 525-529, 1985. 7. Murray, R. M. L., and Pitt, P. Treatment of advanced metastatic breast treatment proceeded was unexpected and remains unexplained. cancer, carcinoma of the prostate, and endometrial cancer with aminoglute It was not due to a selection process whereby those patients thimide. ///: G. A. Nagel and R. J. Santen (eels.). Aminoglutethimide as an who had the highest 4-OHA levels remained on treatment Aromatase Inhibitor in the Treatment of Cancer, pp. 109-122. Berne, Switzerland: Hans Huber, 1984. longer. With the long half-life of the injected drug the apparent 8. Santen, R. J., Wells, S. A., Cohen, N., Deniers. L. M., Mistin, R. I., and accumulation with the 250-mg group is more in line with Foltz, E. L. Compensatory increase in TSH secretion without effect on expectations than the more static levels of the 500-mg group. prolactin secretion in patients treated with aminoglutethimide. J. Clin. En docrinol. Metab., 45:739-746, 1977. This difference in pattern between the two doses may relate to 9. Stuart-Harris, R. C., and Smith, I. E. Aminoglutethimide in the treatment population variability or it might reflect greater induction of of advanced breast cancer. Cancer Treat. Rev., //: 189-204, 1984. 10. Coombes, R. C.. Goss, P., Dowsett, M., Gazet, J. C., and Brodie, A. M. H. metabolizing enzymes at the higher dose. 4-Hydroxyandrostenedione in treatment of postmenopausal patients with Our earlier finding that estrone levels as measured by radio- advanced breast cancer. Lancet, 2:1237-1239, 1984. immunoassay did not suppress with 4-OHA treatment was open 11. Dowsett, M., Goss, P. E., Powles, T. J., Brodie, A. M. H., Jeffcoate, S. L., and Coombes, R. C. Use of the aromatase inhibitor 4-hydroxyandrostene- to a number of explanations (13). The current demonstration dione in postmenopausal breast cancer: optimization of therapeutic dose and that levels measured by GCMS are consistently suppressed on route. Cancer Res., 47: 1957-1961, 1987. 12. Brodie, A. M. H., GarreÂ«,W. M., Hendrickson, J. R., Tsai-Morris, C. H., parenteral therapy to a degree similar to that found with estra Marcotte, P. A., and Robinson, C. H. Inactivation of aromatase in vitro by diol indicated that the earlier result was due to a cross-reacting 4-hydroxyandrostenedione and 4-acetoxyandrostenedione and sustained ef unidentified metabolite of 4-OHA which cochromatographs on fects in vivo. Steroids, 38:693-702, 1981. Lipidex 5000 and that inhibition of aromatase by 4-OHA 13. Goss, P. E., Powles, T. J., Dowsett, M., Hutchinson, G., Brodie, A. M. H., Gazet, J. C., and Coombes, R. C. Treatment of advanced postmenopausal affects estradiol and estrone levels equally. breast cancer with an aromatase inhibitor, 4-hydroxyandrostenedione: Phase In conclusion, these data indicate that 4-OHA achieves max II report. Cancer Res., 46: 4823-4826, 1986. 14. Cunningham, D., Powles, T. J., Dowsett, M., Hutchinson, G., Brodie, A. M. imally effective estradiol suppression at a p.o. dose of 250 mg H., Ford, H. T., Gazet, J. C., and Coombes, R. C. Oral 4-hydroxyandroste daily and at a parenteral dose of 500 mg every 2 weeks. Further nedione, a new endocrine treatment for disseminated breast cancer. Cancer Chemother. Pharmacol., 20: 253-255, 1987. study of lower p.o. doses is indicated. The slightly submaximal 15. Hayward, J. L., Rubens, R. D., Carbone, P. P., Heuson, J-C, Kumablea, S., suppression achieved by 250 mg i.m. every 2 weeks is probably and Segaloff, A. Assessment of response to therapy in advanced breast cancer. not clinically significant and the lower incidence of local side Br. J. Cancer, 35: 292-298, 1977. 16. Ferguson, K., Hayes, M., and Jeffcoate, S. L. A standardized multicentre effects and the demonstrable clinical efficacy of this lower dose procedure for plasma gonadotrophin radioimmunoassay. Ann. Clin. leads to its selection as the optimal parenteral dose. The only Biochem.,/9: 358-361, 1982. endocrine side effect encountered is the androgenic effect on 17. Dowsett, M., Attree, S. L., Virdee, S. S., and Jeffcoate, S. L. Oestrogen related changes in sex hormone binding globulin levels during normal and SHBG levels when the drug is given p.o. This effect is small at gonadotrophin-stimulated menstrual cycles. Clin. Endocrinol., 23:303-312, doses which will be used therapeutically and no clinical andro 1985. 18. Brodie, A. M. H., Romanoff, L. P., and Williams, K. I. H. Metabolism of genic symptoms such as hirsutism were observed at any dose. the aromatase inhibitor 4-hydroxyandrostenedione by male rhesus monkeys. Apart from pharmacokinetics which are not ideal for the p.o. J. Steroid Biochem., 14:693-696, 1982. route of administration, 4-OHA continues to fulfil the criteria 19. Reiffsteck, A., Dehennin, L., and Scholler, R. Estrogens in seminal plasma of human and animal species: identification and quantitative estimation by of an ideal aromatase inhibitor. gas chromatography-mass spectrometry associated with stable isotope dilu tion. J. Steroid Biochem., 17: 567-572, 1982. 20. Smith, I. E., Harris, A. L., Morgan, M., Gazet J-C., and MacKinna, J. A. ACKNOWLEDGMENTS Tamoxifen iw.vw.vaminoglutethimide versus combined tamoxifen and ami noglutethimide in the treatment of advanced breast carcinoma. Cancer Res. We are grateful to Ciba-Geigy Pharmaceuticals for their help in this (Suppl.), 42: 3430s-3433s, 1982. project, to Joanne Nicholls and Anshumala Mehta for their technical 21. Lipton, A., Harvey, H. A., Santen, R. J., Boucher, A., White, D., Bernath, assistance, and to Shani Jayawardene to typing the manuscript. Dr. A., Dixon, R., Richards, G., and Shafik, A. Randomized trial of aminoglu tethimide versus tamoxifen in metastatic breast cancer. Cancer Res. (Suppl.), Peter Lloyd is thanked for his help with pharmacokinetic analyses. 42: 3434s-3436s, 1982. 22. Goss, P. E., Jarman, M., Wilkinson, J. R., and Coombes, R. C. Metabolism of the aromatase inhibitor 4-hydroxyandrostenedione in vivo. Identification REFERENCES of the glucuronide as a major urinary metabolite in patients and biliary metabolite in the rat. J. Steroid Biochem., 24:619-622, 1986. 1. Santen, R. J., Santner, S., Davis, B., Veldhuis, J., Samojlik, E., and Ruby, 23. Brodie, A. M. H., Brodie, H. J., Romanoff, L., Williams, J. G., Williams, K. E. Aminoglutethimide inhibits extraglandular estrogen production in post- I. H., and Wu, J. T. Inhibition of estrogen biosynthesis and regression of menopausal women with breast carcinoma. J. Clin. Endocrino!. Metab., 47: mammary tumours by aromatase inhibitors. Hormones Cancer Adv. Exp. 1257-1265, 1978. Med. Biol., 138: 179-190, 1982. 2. Harris, A. L.. Dowsett, M., Smith, I. E., and Jeffcoate, S. L. Endocrine 24. Brodie, A. M. H., Schwarzel, W. C., Shaikh, A. A., and Brodie, H. J. The elTects of low dose aminoglutethimide alone in advanced postmenopausal effect of an aromatase inhibitor 4-hydroxy-4-androstene-3,17-dione, on es breast cancer. Br. J. Cancer, 47: 621-627, 1983. trogen-dependent processes in reproduction and breast cancer. Endocrinol 3. Stuart-Harris, R., Dowsett, M., D'Souza, A., Donaldson, A., Harris, A. L., ogy, 100: 1684-1695, 1977. JelTcoate, S. L., and Smith, I. E. Endocrine effects of low dose aminoglute 25. Motta, M., Zoppi, S., Brodie, A. M. H., and Martin, L. Effect of 1,4,6- thimide as an aromatase inhibitor in the treatment of breast cancer. Clin. androstenedione-3,17-dione (ATD), 4-hydroxy-4-androstene-3,17-dione (4- Endocrinol., 22: 219-226, 1985. OH-A) and 4-acetoxy-4-androstene-3,17-dione (4-Ac-A) on thÃ©5rÃ©duction 4. Cohen, M. P. Aminoglutethimide inhibition of adrenal desmelase activity. of androgens in thÃ©ratprostate. J. Steroid Biochem., 25: 593-600, 1986.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1989 American Association for Cancer Research. Dose-related Endocrine Effects and Pharmacokinetics of Oral and Intramuscular 4-Hydroxyandrostenedione in Postmenopausal Breast Cancer Patients

M. Dowsett, D. C. Cunningham, R. C. Stein, et al.

Cancer Res 1989;49:1306-1312.

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