(CANCERRESEARCH52, 5933-5939, November1, 1992J Phase I and Endocrine Study of Exemestane (FCE 24304), a New Aromatase Inhibitor, in Postmenopausal Women T. R. Jeffry Evans,' Enrico Di Salle, Giorgio Ornati, Mercedes Lassus, Margherita Strolin Benedetti, Eio Pianezzola, and R. Charles Coombes Department ofMedical OncoIoij@,St. Geoa@ge'sHospital Medical School, Creamer Terrace, London SWI7 ORE, England fT. R. I. E.J; Departments of Oncology IE. D. S., G. 0., M. Li and Pharmacokinetics and Metabolism [M. S. B., E. P.J, Farmitalia Carlo Erba, Via Carlo Imbonati, Milan, Italy; and Department of Medical Oncology, Charing Cross Hospital, FuThoin Palace Roa@ London W6 8RF, England (R. C. C.] ABSTRACT aminoglutethimide and fadrozole (CGS 16949A) (7). Objective tumor regression occurred in approximately 21% of patients Aromatase inhibitors are a useful therapeutic option in the manage treated with 4}IAI@@,2witha low incidence of adverse effects; ment of endocrine-dependent advanced breast cancer. A single-dose 4.5% of patients were withdrawn from treatment because of administration of exemestane (FCE 24304; 6-methylenandrosta-l,4-dl ene-3,17-dione), a new Irreversible aromatase inhibitor, was investi side effects. However, 4HAD undergoes extensive metabolism gated in 29 healthy postmenopausal female volunteers. The compound, in the liver to form the inactive glucuronide (8) and conse given at p.o. doses ofO.5, 5, 12.5, 25, 50, 200, 400, and 800 mg(n = 3—4), quently it is recommended that it is given i.m. rather than p.o. was found to be a well tolerated, potent, long-lasting, and specific in The use of aminoglutethimide as an aromatase inhibitor is re hibitor of estrogen biosynthesis. The minimal dose which produced the stricted by the frequency of its adverse events (9), that of fadro maximum suppression ofplasma estrogens was 25 mg, reducing plasma zole by its interference with aldosterone biosynthesis (10, 11). estrone, estradlol, and estrone sulfate to 35, 28, and 39%ofbasal values, In an attempt to achieve maximal aromatase suppression with respectively. This maximum suppression, observed at 3 days, persisted a p.o. effective, nontoxic compound, a new irreversible aro for at least 5 days after administration of a single dose. However,there matase inhibitor, exemestane (6-methylenandrosta-l,4-diene was no interference on cortisol, aldosterone, 17-hydroxyprogesterone, or 3,17-dione; FCE 24304; Fig. 1), has been developed. Exemes dehydroeplandrostenedione sulfate plasma levels.Peak plasma exemes tane concentrations of 27, 221, 343, and 414 ag/mI were reached within tune was found to cause a time-dependent inactivation of 2 h after administration of 50, 200, 400, and 800 mg, respectively. human placental aromatase (t½,13.9 mm) and to have a K1of Plasma concentrations declined rapidly and fell under the detection 26 flM, thus showing an affinity for the enzyme to be 2.6-fold limit (10 ng/ml) at 4 (50 mg) or 24 h (200 and 400 mg). No clinically higher than the substrate androstenedione (Km 69 nM) (12). significant adverse events which could be attributed to the drug were Exemestane induced 30 and 73% regression ofestablished 7,12- reported. Apart from transient eosinophilia in 3 patients, all biochem dimethylbenz(a)anthracene-induced mammary tumors in rats ical and hematological laboratory parameters were within 1.25-fold of when given daily at 10 and 50 mg/kg s.c., associated with 86 the normal ranges. and 93% decrease in total ovarian aromatase activity (13, 14). Consequently we considered exemestane worthy of further INTRODUCTION study in a human population, and we have studied the endo One-third of human breast tumors are hormone dependent crime and toxicity effects and the pharmacokinetics of an esca (1) and estrogens are the most important hormones involved in lating single dose of exemestane in healthy postmenopausal supporting growth of these hormone-dependent tumors (2, 3). female volunteers. The extraglandular conversion of androstenedione to estrone catalyzed by the aromatase enzyme is considered to be the most MATERIALS AND METHODS important source of circulating estrogens in the postmeno pausal woman (4). Consequently, inhibition ofestrogen biosyn Subjects. Twenty-nine postmenopausal women attending the breast thesis by means ofselective aromatase inhibitors is a potentially clinic at St. George's Hospital, London, were recruited into the study. All gave written informed consent, and the trial was approved by the useful therapeutic option in hormone-sensitive breast cancer ethical committee of St. George's Hospital Medical School. The study (5). The enzyme system is localized in the endoplasmic reticu was conducted in two parts. In both parts female subjects over the age lum of cells in which it is expressed and consists of two com of45 years, who were postmenopausal as defined by greater than I year ponents (6). The first is a form of cytochrome P-450 known as since last menses (FSH > 50 milliunits/ml; LH, 15—40milliunits/ml) or aromatase cytochrome P-450. This hemoprotein is responsible who had had bilateral oophorectomy wereeligible.The mean age of the for binding the C-19 steroid substrate and catalyzing the series subjects recruited was 60 years (range, 48—75 years); the mean time of reactions which leads to the formation of a phenolic A-ring. from menopause was 12.3 years (range, 2—36years).Subjects with a The second component is a flavoprotein, NADPH cytochrome clinically significant history of allergic disorders, with cardiac, hema P-450 reductase, which is an ubiquitous protein in the endo tological, respiratory, endocrine, renal, hepatic, or gastrointestinal dis plasmic reticulum of most cell types and which transfers reduc orders, or who had hypertension were excluded, as well as subjects who had participated in a trial in the previous 3 months or who had taken ing equivalents from NADPH to cytochrome P-450 (6). In the any medication in the previous 2 weeks.All volunteers were assessed by search for potent inhibitors of aromatase, various analogues of history, examination, and appropriate investigations before entering the aromatase substrate androstenedione have been investigat the study. All subjects who had previously had breast cancer were fully ed. One of these, 4-hydroxyandrostenedione, an irreversible ar investigated to exclude any evidence of recurrent disease. omatase inhibitor, has been extensively investigated in clinical In studypartA, 15subjectswereinvestigated;14hadpreviouslyhad trials as have some nonsteroidal aromatase inhibitors such as breast cancer but with no evidence of recurrent malignant disease on Received3/24/9Z accepted8/26/92. The costs ofpublication ofthis article weredefrayedin part by the paymentof 2 The abbreviations used are: 4HAD, 4-hydroxyandrostenedione; DHEAS, pagecharges.Thisarticlemustthereforebe herebymarkedadvertisenaentinaccord dehydroepiandrostenedionesulfate;RIA, radioimmunoassay,FSH, follicle-stimu ance with 18 U.S.C. Section 1734solelyto indicatethis fact. latinghormone;LH, luteinizinghormone;HPLC, highpressureliquidchromatog I To whom requests for reprints should be addressed. raphy. 5933 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1992 American Association for Cancer Research. PHASEI STUDYOF EXEMESTANE column chromatography. An aliquot (0.75 ml) of the aqueous phase (containing estrone sulfate) was retained for enzyme hydrolysis with 2 IU (100 Ml)of arylsulfatase (Helix pomalia; Merck) and 0.75 ml of 1Macetate buffer, pH 5, in a 37C water bath. After 18 h of incubation, the free estrone was extracted twicewith diethyl ether and processed as above prior to Celite column chromatography. To determine total (free and unconjugated) urinary estrone and estradiol, the urinary samples (0.5 ml) were hydrolyzed with 2 IU arylsulfatase and 4 IU @-glucu ronidase (H. pomatia; Merck) and 0.5 ml of 1 Macetate buffer, pH 5, in a 37T water bath and processed as above.In the chromatography stage the redissolved sample was applied to the column and successiveelu tions with increasing concentrations of ethyl acetate in isooctane (0,6, 20, and 40%) were collected. The fractions containing estrone (20% ethyl acetate) and estradiol (40% ethyl acetate) were evaporated under EXEMESTANE (FCE 24304) N2 and the dried samples were used for radioimmunoassay. The final recovery rates for the procedures, monitored with [3Hjestrone, [3HJ Fig. I. Structure of exemestane. estradiol, I3Hlestrone sulfate, and [3HJestrone glucuronate are shown in Table 1. Estrone and estradiol assays were performed using the commercially entering the study, and 1 had had benign breast disease. A cohort of 3 available radioimmunoassay kits [3H]estrone (BioMerieux) and [125I]@ subjects was studied at each of 5 dose levels: 25, 50, 200, 400, and 800 estradiol (Baxter). The standard curve of the estradiol kit (supplied in . mg in open uncontrolled conditions. Doses were escalated from an serum) was substituted with a standard curve in buffer. Each purified initial 25 mg to 800 mg or until the first occurrence ofan adverse event dry sample was redissolved in the appropriate incubation buffer and an or a laboratory abnormality related to the study drug. At each dose level aliquot of this suspension was subjected to the specific RIA procedure. assessment, subjects were observed for 2 weeks (first 2 subjects) or >2 The sensitivities of the assays were approximately 2.5 and 0.5 pg/tube days (third subject) before escalation to the next dose level.All subjects for estrone and estradiol, respectively. The intraassay coefficients of were admitted to the hospital the day before drug administration for variation (n = 8) were for estrone 12.2 and 2.7% at 5 and 40 pg/tube, clinical evaluation and a 24-h (day 0) basal urine collection commenced. respectively,and the coefficients for estradiol were 7.6 and 4.8% at 0.5 . On the day ofadministration, clinical evaluation was repeated and basal and 4 pg/tube, respectively. The hormone concentrations were cor blood levels were obtained. A single dose of exemestane was adminis rected for the recovery of the labeled hormones and expressed in terms tered within 15—30mmafter a high lipid breakfast and blood samples of free hormones.