Development of Antiestrogens and Their Use in Breast Cancer: Eighth Cain Memorial Award Lecture1

Home , Dienestrol, Estradiol benzoate, Estrogenic substances, Ethamoxytriphetol, Hexestrol, Nafoxidine

[CANCER RESEARCH 50, 4177-4189. July 15, 1990]

Special Lecture Development of Antiestrogens and Their Use in Breast Cancer: Eighth Cain Memorial Award Lecture1

Leonard J. Lerner and V. Craig Jordan Department of Pharmacology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 [L. J. L.J, and Departments of Human Oncology and Pharmacology, University of Wisconsin Clinical Cancer Center, Madison, Wisconsin 53792 [V. C. J.J

Abstract not involved in the pathology. However, it is reasonable to expect a complex tissue such as the mammary gland, which This paper describes the laboratory discovery and clinical testing of the first nonsteroidal antiestrogen, MER-25 (ethantoxytriphetol). The requires a number of hormones and probably growth factors for its normal growth and function, to have the involvement of compound blocks estrogen action in all species tested and has only slight but transient estrogenic effects. No other antisteroidal actions are noted. more than one hormone in its pathology. MER-25 is antiestrogenic in primates and was investigated in the clinics Certainly there are a number of possible ways of interfering in a wide range of gynecological conditions, including breast and endo- with estrogen interaction with its target tissue other than by metrial cancer. Unfortunately toxic side effects (hallucinations, etc.) removing the steroid-secreting glands. Reduction of pituitary precluded further investigation. A derivative of triphenylethylene, clom- gonadotropin secretion by sex steroids or by blocking the bio- iphene, has some partial agonist (estrogen-like) actions in laboratory conversion of androgen to estrogen are ways of decreasing the animals and following clinical evaluation is now an established agent for endogenous pool of estrogens. Biologically available estrogen the induction of ovulation in subfertile women. Although clomiphene is can also be reduced by increased catabolism or altering the active in advanced breast cancer, it was not developed further. In the late plasma level of estrogen-binding protein. Another way to inter 1960s a related compound, tamoxifen, was evaluated to treat a number of estrogen-responsive disorders but was successfully introduced in the fere with estrogen activity is to block it at the level of its 1970s for the treatment of advanced breast cancer. Although there was receptor in estrogen target tissues. only modest initial interest in the palliative use of tamoxifen, an enormous increase in basic and applied studies with antiestrogens resulted in a definition of the target site-specific and tumoristatic actions of tamoxifen. Development of Nonsteroidal Estrogens Close cooperation between laboratory and clinical evaluation has guided The most common of the wide spectrum of biological activi the subsequent development of tamoxifen which is now available to treat ties associated with estrogens, regardless of chemical classifi all stages of breast cancer. Long-term adjuvant tamoxifen therapy, a cation or source, is the uterotrophic response and the cornifi- concept developed in the laboratory, is currently the treatment strategy of choice. The considerable success of tamoxifen has focused attention cation of vaginal epithelium. These activities have been incor on new antiestrogens with different pharmacological properties for other porated into the bioassays that have defined every natural and potential clinical applications. synthetic compound designated as an estrogen. Development of a sensitive bioassay in rodents by Allen and Doisy (4) enabled characterization and classification according to potency of the Introduction natural steroidal estrogens and nonsteroidal plant estrogens. Estrogen is involved in a large number of physiological phe This formed the basis to identify the structural requirements nomena throughout the body. The mechanism by which this for activity, as well as potency. hormone produces its effects is complex and may not be uni Cook et al. (5) found that the phenanthrene nucleus was not versal for all of the activities reported to be estrogenic. Estrogen necessary for estrogenic activity. The laboratory of Dodds and is also involved in some pathologies. Here too, its role has not his collaborators therefore synthesized a large number of di- been completely defined. The removal of estrogen or interfer phenolic and triphenolic compounds resulting in the develop ence with its action is helpful in elucidating the role of estrogen ment of potent, clinically important estrogenic substances such and the mechanism of its activity in both physiological and as diethylstilbestrol (6), hexestrol, and dienestrol (7). At this pathological processes. same time in the mid-1930s, Robson and Schonberg reported Perhaps the earliest association of reproductive tissue and that triphenylethylene had estrogenic activity (8) and that oral behavioral responses to a hormonal secreting organ was the potency and prolongation of activity could be achieved by cessation of estrus in domesticated animals following ovariec- bromination (9). This finding led, in the 1940s, to the synthesis tomy and the diminution of aggressive behavior in male animals of a number of triphenylethylene derivatives including more and humans after castration. Indeed, almost 100 years ago there potent halogenated compounds by the William S. Merrell Co. was recognition that the ovary was involved in mammary car in the United States (10, 11) and by Imperial Chemical Indus cinoma and that removal of that endocrine gland could be tries in the United Kingdom. beneficial to the patient (1). While all of the compounds called estrogens increase uterine Ablation of other endocrine organs including adrenalectomy weight and stratify and cornify the vaginal epithelium, the (2) and hypophysectomy (3) were also found to be beneficial in spectrum of other biological activities associated with estradiol some breast cancers. Surgical removal of these glands or the may or may not be present in all of these estrogens. Moreover, ovaries, however, did more than decrease or eliminate endoge the quantitative aspect of one of the activities may not be related nous estrogen from the body. A number of hormones and to other activities of the same compound. nonhormonal substances were removed, some of which were Knowledge of structure-activity relationships allows for the rational design of chemical agents with greater specificity for a Received 4/5/90. 1Presented at the 80th Annual Meeting of the American Association for particular pharmacological action. Cancer Research, May 24, 1989, San Francisco, CA. Some of the physiological interactions of the steroidal hor- 4177

mones were known in the first half of the 20th century. These interactions could be additive, augmentative, synergistic, or antagonistic depending upon the particular steroids, dosage of each, order and time course of administration, as well as the end point and species of animal under study. A small dose of an estrogen administered to an immature rabbit prior to treat

ment with progesterone allows for maximal growth and devel ETHAMOXYTRIPHETOL (MER 25) opment of the uterus. It is now known that this effect is a result of increased estrogen-induced uterine hyperemia and induction of proges terone receptors. Large doses of the estrogen, however, can reduce the subsequent effect of the progestational agent in the rabbit.

Estrogen Antagonism Treatment of an immature or ovariectomized rodent with a progestogen, androgen or corticoid can antagonize the uterotrophic effect of estrogens (12-16). Antagonism, however, is closely linked to the relative and absolute dose of the agonist and the antagonist. The first estrogens that demonstrated antagonism of the N1TROMIPHENE (CN-55, 945) NAFOXIDINE(U-11,100A) stimulation of another estrogen were estriol (17) and 16-epies- triol (18). These weak estrogenic steroids partially inhibited the uterotrophic effect of estradiol in the rat. This antiestrogenic activity was manifested only over a narrow range of doses (19), and at lower doses the two estrogens induced additive uterine growth responses (20). Moreover, for the end point of vaginal cornification in rats and mice, estriol was always additive with TAMOXIFEN (ICI 46, 474) estradiol (21). Fig. 1. Structures of MER-25 and antiestrogens derived from triphenyleth The nonsteroidal synthetic estrogens and their structurally ylene. related weak or inactive relatives were an attractive source in the search for modulators or inhibitors of estrogenic activity. given MER-25 with or without estradiol benzoate were identical Several weakly estrogenic stilbene and triphenylethylene com pounds demonstrated partial antagonism of estrone- or estra- to those of animals given injections of the olive oil alone. In diol-induced uterotrophic activity in mice in my laboratory (L. addition, uterine vascularity and intraluminal fluid, which also increases with estrogen treatment, were absent in the MER-25- J. L.). At a presentation of data (22) on the antiestrogenic treated groups. Moreover, body weight remained normal. The activity of one of the diphenolic compounds Dr. Roy Hertz study was repeated several times and the complete blockade of from the NIH remarked that the findings were interesting but the estrogen-induced uterotrophic response was confirmed. a clinically useful antiestrogen would have to be much more potent. The inhibition of estradiol action in the mouse uterus by MER-25 was dose related and simultaneous administration of graded doses of estradiol was able to prevent the antiestrogenic Discovery and Development of Ethamoxytriphetol (MER-25) effects of MER-25 in a dose-related manner (Fig. 2). This study In 1954, a compound related to triphenylethylene and chlo- demonstrated that MER-25 is a competitive antagonist of es rotrianisene was synthesized at Merrell to study its possible trogen action. cardiovascular effects, but it was found to be inactive. The The nature of the competitive mechanism of this and deriv structural similarities ofthat compound to those of the triphen- ative antiestrogens was elucidated when Jensen et al. (23, 24) ylethylenes that were being studied for antiestrogenic activity found that they prevented binding of labeled estradiol to the rat in my laboratory (L. J. L.) prompted us to test its endocrine uterus. Numerous studies in vivo and in vitro have confirmed activity. that the nonsteroidal antiestrogens act at the level of the estro That compound, l-/?-2-(diethylamino)ethoxyphenyl-2-(/>- gen receptor. However, the exact nature of the mechanism(s) is methoxyphenyl)-l-phenylethanol was later given the generic still incompletely understood. Moreover, other activities have name of ethamoxytriphetol and the reference designation of been documented for this class of pharmacological agents. MER-25 (Fig. 1). The initial dose of MER-25 was selected on These include inhibition of estrogen-induced protein synthesis, the basis of experience that showed that even the most active thymidine incorporation, DNA synthesis, cell cycle blockage at of the compounds tested up to that time were of low potency the GÃ¬phase, inhibition of calmodulin, and non-estrogen-re and only partially inhibited the uterotrophic effect of estrone lated events. or estradiol. Not all of these activities, however, have been found univer Weanling mice were given s.c. injections of MER-25 in olive sally with all antiestrogens and results from studies in vivo do oil for 3 days at a daily dose of 1.7 mg (170 mg/kg). One-half not necessarily support findings in vitro. Some of these effects of the animals were also given daily injections of estradiol may be a result of the relative estrogenic to antiestrogenic benzoate (0.1 ^g/animal). The results of this experiment were activities of the compound studied or to the biological half-life surprising since the uterine weights of the mice that had been of each activity. A comparison of an estrogenic antiestrogen 4178

50 (11F) Estradiol 0.3 Mg (11F) ~. 40 33 (16F,3P,4N) O) (1N,3P,7F) (9N,1P,1F) Fig. 2. Uterotrophic and antiestrogenic ac l (1N.7P.2F) tivities of MER-25 in immature mice. Groups of 30 animals were treated for 3 days with (A) estradici ^ benzoate (0.3 ng) and MER-25 (O) or MER-25 /> 4N.4P.3F) alone (A) or (B) different doses of estradici benzoate with (d) or without (â€¢)5mg MER-25. 20 All doses are 3-day total dosages. Data in paren theses, number of animals exhibiting full (/"), partial (P), or no (AOuterine intraluminal fluid. 'Control rControl (25N) (1P.33N) (38N) nI 0.2 1.0 5.0 0.03 0.3 30.0 3000

TOTAL DOSE MER-25 (mg) TOTAL DOSE ESTRADIOL

(partial agonist) with a nonestrogenic antiestrogen (pure antag MER-25 was also a complete inhibitor of the effect of endog onist) for each of the activities may help resolve this problem. enous estrogen or administered estrogen at the level of the The antiuterotrophic effect of MER-25 was also demon vagina. Cornification of vaginal epithelium by estrogen was strated in immature, ovariectomized, and adult rats (25-30). In blocked and MER-25 did not induce any cornification when the immature rat, a MER-25:estradiol benzoate ratio of 400:1 administered to immature or ovariectomized mice or rats, dem reduced the Uterotrophic effect of the estrogen by approximately onstrating a lack of inherent estrogenicity. 45% and complete suppression of the estrogen activity was obtained with a ratio of 10,000:1 (Fig. 3). Uterine as well as Further Studies with MER-25 oviductal growth stimulated by estradiol was inhibited in a dose-related fashion as were a number of biochemical parame There was some evidence that this compound did posses very ters elevated by estrogen. These included nucleic acids, phos- weak and short-lived estrogen activity at other end points. At pholipids, glucose-6-phosphate dehydrogenase and other en relatively low doses, it induced a weak Uterotrophic effect which, zyme activities, nitrogen, and water. many times, was only borderline in significance. When a single These initial studies prompted my colleagues and I (L. J. L.) dose of this agent was administered to immature mice, a slight to investigate the pharmacological spectrum of activities of increase in uterine weight was noted 48 h after treatment but MER-25 in different species in various stages of maturity. Our was not seen on subsequent days (26). Those doses that were studies evaluated the antihormonal and antifertility properties weakly Uterotrophic also elevated uterine alkaline phosphatase of MER-25 in acute and long-term studies (15, 26). Each study (27). brought new information but also raised new questions. MER- In immature rats, lower doses induced minimal increases in 25 was an antiuterotrophic agent regardless of the route of uterine weight (28). Low doses of MER-25 induced a short administration or the estrogen used as the agonist, but it had lived increase in glucose-6-phosphate dehydrogenase, glucose little or no activity as an antagonist of androgen or progester isomerase, and lipids (30). In the immature rabbit pretreated one-induced Uterotrophic activity. with estrogen to allow for progesterone-induced endometrial development, MER-25 effectively inhibited the estrogen activity (presumed induction of progesterone receptors), thereby blunt 140 ing the effect of the progesterone (26). MER-25, however, when administered instead of estradiol, permitted a weak uterine growth response to subsequent progesterone treatment. Other indications of weak estrogenic activity were the acceleration of vaginal opening in prepuberal rats and mucification of the adult rat vagina (26, 27). Offspring of rats treated with MER-25 during the last third of gestation demonstrated early opening of their vaginas (27). MER-25 has only weak antigonadotropin activity, but it effectively blocks estrogen- or castration-induced pituitary hy pertrophy and gonadotroph degranulation (26). It was found to be devoid of any other hormonal activity. Mouse uteri stimulated by testosterone were unaffected by simultaneous treatment with this estrogen antagonist (26, 27). High doses of MER-25, however, partially inhibited androgen- Total Dose of MER-25 - mg stimulated uterine weight increase and biochemical changes in Fig. 3. Uterotrophic and antiestrogenic activities of MER-25 in ovariecto the immature rat (29). Immature castrated male rats given mized immature rats. Groups of animals were treated for 4 days with a total dose injections of MER-25 and testosterone propionate demon of 2.0 fig estradiol benzoate ( ), MER-25 <&\,estradiol benzoate plus MER- 25 (â€¢)orinjection vehicle (sesame oil) alone ( ). Animals were killed on day strated no antagonism of the androgen-induced changes in the 5 and blotted uteri were weighed wet. seminal vesicles although there was a partial inhibition of the 4179

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1990 American Association for Cancer Research. ANTIESTROGENS AND BREAST CANCER augmented weight and nucleic acids of the ventral prostate (32). one such agent was developed for postcoital antifertility utility Antiestrogenic activity was also found in the chick. MER-25 (40), but it was never marketed. inhibited estrogen-induced oviductal growth and elevated MER-25 was also shown to augment pregnancy maintenance plasma phospholipids (26). MER-25 did not alter these same by progesterone in Ovariectomized rats similar to that obtained estrogen target parameters at any dose used, indicating a lack with estrone (27). However, in rats fed a protein-free diet where of estrogenicity in this species. Tamoxifen and other triphen- estrone helps maintain pregnancy, the administration of MER- ylethylene antiestrogens (33, 34) were later also found to be 25 negated the supportive effect of the estrogen (27). devoid of estrogen activity in the chick oviduct. This is confirm The activities of MER-25 were expressed relative to the atory evidence for a species-related hormonal profile for this hormonal and developmental state of the animal. In adult chemical class of compounds. cycling rats, this compound decreased the weights and function MER-25, if it were to have clinical potential, had to have of the ovaries, pituitary, and uterus. It also antagonized the antiestrogenic activity in a primate. Ovariectomized Macaca effect of estrogen on the latter two organs (26). Administration mulatta monkeys, maintained on estradici, were periodically of MER-25 to pubertal rats, however, produced different re given varying p.o. doses of MER-25. The antagonist induced a sults. Ovarian weights, nucleic acids, and enzyme activities were sharp reduction in the cornified vaginal cells of these estrogen- elevated after 4 days of treatment (41). The increases in the ized animals and at higher doses the decrease in cornified cells ovaries were similar to that obtained by treatment with human was to the castration level. Accompanying this was a lessening chorionic gonadotropin (41). Estradici treatment at the same of the sex skin swelling and reddening and eventually estrogen time prevented the MER-25-induced augmentation of ovarian withdrawal bleeding (26). Other studies in intact cycling mon weight and biochemical activities. keys demonstrated that endogenous estrogen was inhibited by The increase in ovarian activity following MER-25 treatment antagonism or via reduced gonadotropin secretion since the of the pubertal rat may parallel the ovarian stimulation in menstrual cycles were lengthened and the percentage of corni women after administration of antiestrogens including MER- fied cells in the vaginal smear decreased. 25 and clomiphene citrate. These studies paralleled those in intact rats where a daily p.o. dose of 1 mg lengthened the estrous cycle and 5 mg/day stopped cycling (26). The rats resumed normal cycling of 2-6 days Utility of Antiestrogens for Ovulation Induction following the last dose. In the 1950s Dr. James H. Leathern used the human cho This prototype antiestrogen was further investigated in order rionic gonadotropin-treated hypothyroid rat as a model for to determine its pharmacological properties especially in rela polycystic ovaries. His work stimulated us to study the effect tion to those biological phenomena where endogenous estrogen of MER-25 in this model. The antiestrogen blocked the uterine or gonadotropins had been shown to be involved. hypertrophy induced by the hyperactive ovary and reduced ovarian weight; however, cystic follicles were still evident. These Effect of Antiestrogens on Reproduction studies were repeated and extended by Leathern and his grad uate students. MER-25 prevented the induction of cystic ovaries The reproductive system and all of its accessory tissues re and alteration in ovarian ascorbic acid concentration (42). Fur quire a changing balance of hormones for their development thermore, pretreatment with MER-25 prior to induction of the and function. Changes in the absolute and relative quantities of hypothyroid state followed by gonadotropin administration was these hormones may lead to functional disorders and pathology. also effective in blocking cystic development (43). Treatments that increase or decrease the available pool of the A preliminary study in nymphomaniac cows bearing cystic particular hormone or alter its activity may correct the problem. ovaries was performed by W. Hansel and R. M. Melampy using Similarly a physiological process such as ovulation can be a low use of MER-25 that I (L. J. L.) had suggested. These interrupted by addition of hormones. cows promptly resumed normal behavior; however, no meas It was interesting to explore the actions of MER-25, an urement of ovarian follicular size or ovulation was recorded. antiestrogen having weak gonadotropin-inhibitory and very These findings in animals encouraged us to initiate explora weak selective estrogenic activities, on reproduction. The com tory studies on selected patients to determine the effects of pound was administered to female rats at various times prior MER-25 on menstrual or ovarian disorders. Kistner and Smith to or after mating (26, 27). None of the pretreated animals (44) reported that patients treated for endometrial hyperplasia mated, since none of the animals exhibited estrus behavior or and carcinoma with MER-25 demonstrated higher urinary ex showed vaginal cornification. Treatments that were initiated cretion of estrogen and follicle-stimulating hormone and had after mating on day 1 of pregnancy probably inhibited nidation temperature changes indicative of ovulation. These investiga or altered ova transport since no fetuses or products of concep tors also induced ovulation with this compound in patients with tion were found. Administration of the compound on days 4-7 Stein-Leventhal syndrome (45). At this same time Tyler et al. postmating prevented blastocyst implantation. When treatment (46) reported that 6 of 18 patients with severe anovulatory was delayed until the 13th day of pregnancy, gestation contin problems ovulated within 7 days of starting MER-25 therapy. ued and live pups were delivered, but parturition was delayed This antiestrogen, however, induced undesirable and unex and was difficult with several of the young dying probably due pected central nervous system symptoms leading to cessation to the effects of estrogen deprivation on uterine contraction. of its use. Fortunately my laboratory (L. J. L.) had been devel The postcoital antifertility effect of MER-25 was confirmed oping, at the same time, another related but more potent by other investigators (35, 36) and similar results were obtained antiestrogen that had some qualitative differences from MER- for other triphenylethylene antiestrogens including clomiphene 25. That compound, MRL-41 or clomiphene citrate (47) (orig and MRL-37 in my laboratories (L. J. L.) and by other inves inally called chloramiphene) (Fig. 1), was, in comparison to tigators (37, 38). None of the antiestrogenic agents were suffi MER-25, weakly estrogenic (25). Clomiphene was provided to ciently efficacious in primates, although ovum implantation in R. W. Kistner, E. T. Tyler, and R. B. Greenblatt to study its the monkey uterus was inhibited by several (39) and at least ability to induce ovulation. Shortly thereafter Greenblatt et al. 4180

(48) reported that this compound successfully induced ovulation tion, 4) fertility in the female, 5) dysmenorrhea, 6) all kinds of in women and similar successes were reported by the other menstrual disturbances including menorrhagia or prolonged investigators. Ovulation was induced with doses that were menstrual periods, 7) menopause, 8) endometriosis, 9) acne, smaller than those used for MER-25 and no central nervous 10) cancerâ€”a) to test for or substitute for adrenalectomy in system disturbances were found. cancer, b) as a substitute for ovariectomy, c) mammary carci These studies ushered in a new mode for treating anovulatory noma, d) prostatic cancerâ€”alone or in combination with an and infertile patients having functional ovaries which has had estrogen, in an attempt to maintain the effects of estrogen on widespread use in in vitro fertilization technology. the prostate and avoid side reactions of estrogens, 11) benign prostatic hypertrophy, 12) postpartum breast engorgement, and 13) precipitate the menopause when desirable." Effect of Antiestrogens on Lipids While that list was compiled largely on the basis of laboratory Estrogens are known to regulate the synthesis and metabo data on MER-25 presented by L. J. L., it is interesting that the lism of lipids. This produces a change in the relative concentra anticancer indications were not derived from animal tumor tion of various lipid fractions in the plasma and disposition in models since such models were not available. The stimulation tissues. Indeed endogenous estrogen is a normal protective for such evaluation in human cancers came largely from the regulator for the cardiovascular system and the decreased syn availability of a unique compound that could block the effect of thesis of these steroids at menopause is associated with an estrogen completely and also have weak gonadotropin inhibi increase in atherosclerosis and coronary disease. Men are more tion activity. prone to these disorders than are premenopausal women. A Several well known competent clinical investigators were chemical compound that demonstrates an estrogen-like activity suggested for each of the areas. The two who were asked to on lipids without affecting the reproductive system would there investigate MER-25 in breast disorders including cancers were fore be desirable. Another possible partitioning of the biological Dr. Roy Hertz at the NIH and Dr. Robert Kistner at Harvard spectrum of activities of estrogen might result from the admin University. The dosages suggested were derived from the labo istration of an estrogen such as estradiol and of a "nonestro- ratory studies in animals including those performed in the genic" antiestrogen. monkey. Dr. Kistner also investigated other diseases of the An experiment in 1-week-old chicks showed that estradiol breast, uterus, and ovaries. benzoate could increase oviductal weight by 1500% while dou Both investigators confirmed antiestrogenic activity in their bling the plasma phospholipid concentration. MER-25 admin patients. Kistner and Smith (44) reported that patients with istration did not alter either end point even at doses 300 times chronic cystic mastitis not only had pain relief but also had larger than that of the estrogen. When both substances were reduced glandular proliferation and breast size. He also found administered to these birds the estrogenic effect on the plasma that two of four patients with breast carcinomas experienced phospholipids as well as on the oviduct were blocked (26). pain relief and a reduction of calcium excretion while on MER- Even though MER-25 failed to show an estrogen-like effect 25 therapy. Hertz also treated several patients with metastatic on the chick plasma phospholipids, this antiestrogen and a breast cancer and obtained similar favorable results. These number of other compounds having different spectra of estro studies demonstrated the utility of an antiestrogen in breast genic and/or antiestrogenic activities were investigated in the cancer. rat. MER-25, clomiphene citrate, and a related compound, Unfortunately the investigation of MER-25 as a therapeutic triparanol, were hypocholesterolemic (41) with the latter com agent had to be discontinued because Drs. Hertz, Kistner, and pound being 5 times more potent than the other two. Studies Tyler reported that their patients were experiencing hallucina in vitro with rat liver homogenates demonstrated that MER-25 tions, psychotic episodes, nightmares, disturbed sleep, or other and clomiphene inhibited the conversion of mevalonate to indications of undesirable central nervous system activity. cholesterol whereas triparanol produced its major blocking Clomiphene which was the next most advanced in preclinical effect after mevalonic acid (41, 49). investigation or another of the structurally related antiestrogens The nonestrogenic weak antiestrogen 2,2'"-(l-methyl-4,4- could have been evaluated for anticancer activity in the early diphenylbutylidene)bis(/>-phenyleneoxy)bistriethylamine oxa- 1950s. However, the clinical evaluation of these compounds for late lowered plasma cholesterol in rats fed normal and hyper- those indications was never undertaken due to the concentration lipemic diets by blocking cholesterol biosynthesis between mev of effort on the use of clomiphene in induction of ovulation in alonate and lanosterol (49). It also reduced serum free fatty anovulatory infertile women. In fact clomiphene citrate was acids and triglycÃ©rides. later shown to have efficacy in breast cancer in a few patients It may be interesting to investigate the relationship of cho in 1964(50). lesterol biosynthesis inhibition and anticancer potential of the Resumption of investigation of similar antiestrogens for antiestrogens in light of present knowledge concerning the breast cancer had to wait for more than a decade. Chemists and catalogue of factors required for cellular growth, development, biologists in the United Kingdom reexplored the triphenylethy- regulation, and multiplication. lenes for antiestrogenic and antifertility activity and eventually for antitumor activity. The clinical efficacy of one of these compounds, tamoxifen, established antiestrogens as the stand Demonstration of the Efficacy of an Antiestrogen in Breast ard endocrine treatment in breast cancer. Cancer Laboratory studies with MER-25, clomiphene, and many Development of Tamoxifen other related compounds suggested a variety of potential clinical utilities based on the particular pharmacological spectrum of Tamoxifen (ICI 46,474; Nolvadex) (Fig. 1) is a nonsteroidal each compound. In mid-1956 a list of some of these utilities antiestrogen with antifertility properties in laboratory animals was suggested for evaluation with MER-25. These included, (51-54). The compound exhibits an interesting species-specific "1) precocious development, 2) anti-fertility, 3) habitual abor pharmacology (55); it is a pure antiestrogen in chicks (33) and 4181

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1990 American Association for Cancer Research. ANTIESTROGENS AND BREAST CANCER an antiestrogen with partial estrogenic activity in rats (52) but, past half-decade interest has focused on an evaluation of the in short-term tests, it is classified as an estrogen in mice (51, antitumor action of tamoxifen in athymic mice, hetero- 52, 56). However, it appears that the prolonged administration transplanted with breast cancer cell lines (91-95). of tamoxifen to ovariectomized mice causes the uterus and Carcinogen-induced Models. Mammary tumors can be in vagina to become refractory to the stimulatory effects of estra duced in 50-day-old Sprague-Dawley rats by a single feeding dici (37, 57, 58). (20 mg in 2 ml peanut oil) of DMBA. Tamoxifen will inhibit Tamoxifen is a competitive inhibitor of estradici binding to the initiation (85, 87) and growth (61, 88, 96-98) of DMBA2- estrogen receptors (59, 60) and the compound inhibits the induced tumors. The model system could be likened to the binding of estrogen to estrogen target tissues in vivo (57, 61- treatment of advanced breast cancer but the tumors do not 64). metastasize and therefore a direct analogy with breast cancer is Antiestrogens can inhibit estrogen-regulated events in cells impossible. This fact is important inasmuch as the fashion for in culture; e.g., tamoxifen slows the growth of MCF-7 breast the treatment of breast cancer was changing in the mid-1970s. cancer cells [in phenol red containing media (65, 66)]; however, A strategy was devised to use adjuvant hormonochemotherapy the addition of increasing concentrations of estradiol will re to destroy micrometastases following mastectomy, a treatment verse the effects of tamoxifen and the cells will continue to aimed at curing a majority of patients. Unfortunately no animal grow ("estrogen rescue") (67). Nevertheless, high concentra model system was, or is, available to duplicate the clinical tions of antiestrogens will cause estrogen irreversible actions situation to test the value of this treatment strategy in the (68, 69). An "antiestrogen binding site" has been described (70) laboratory. Nevertheless the DMBA- and yV-methylnitrosourea- but its relevance to the cytocidal actions of tamoxifen is not induced rat mammary carcinoma models were adapted to eval clear. It is clear, however, that the high affinity of tamoxifen to uate whether antiestrogens could "cure" animals with a micro a variety of proteins causes ubiquitous binding of the drug in scope tumor burden. vivo and probably accounts for its long biological half-life When DMBA is administered to Sprague-Dawley rats, the Current research is focused on the action of growth factors initiated mammary cells are promoted by circulating hormones to regulate the breast cancer cell cycle. Estrogens decrease cell to effect transformation. Palpable tumors appear 100-150 days cycle time and tamoxifen causes a reversible blockade at the G, after DMBA. There is therefore a period when microscopic phase (71, 72). Antiestrogens inhibit estrogen-stimulated in disease can be treated to establish whether it is possible to cure creases in transforming growth factor a, a stimulatory factor the animals with antiestrogens. If different doses are adminis (73), and there is a complementary rise in transforming growth tered for 4 weeks starting at different times after DMBA (99, factor ÃŸ,aninhibitory growth factor (74). Although the actual 100), a delay occurs in the appearance of tumors. In contrast regulatory mechanisms that orchestrate the inhibitory and stim continuous therapy causes the majority of animals to remain ulatory actions of growth factors are unclear, this is an area of tumor free (101-103). Nevertheless of tamoxifen therapy is intense investigation. stopped, tumor growth recurs (104). The knowledge that tamoxifen is an inhibitor of estrogen A similar situation occurs with the carcinogen jV-methylni- action focused attempts to develop an appropriate clinical ap trosourea; a short course of tamoxifen delays the appearance plication. Tamoxifen was initially evaluated in a number of of tumors but the animals remain tumor free during a contin endocrine-responsive conditions (75-80) and the drug subse uous treatment regimen (105, 106). quently became available in some countries for the induction of Athymic Mouse Models. Breast cancer cell lines can be het- ovulation in subfertile women. However, the discovery that the erotransplanted into athymic immunodeficient mice. Estradiol presence of the estrogen receptor may predict the hormone encourages the growth of hormone-responsive lines (e.g., MCF- responsivness of advanced breast cancer (81) naturally led to 7) but hormone nonresponsive lines (e.g., MDA-MB-231) will the evaluation of tamoxifen to control tumor growth [tamoxifen grow with or without estrogen treatment. Tamoxifen and its inhibits the binding of estrogen to estrogen receptors derived metabolites inhibit estrogen-stimulated growth of estrogen re from breast tumors (82)]. Much of the credit for steering the ceptor-positive tumors but in general do not inhibit the growth use of tamoxifen toward the treatment of advanced breast of receptor-negative tumors (91, 92). However, this model has cancer must go to the late Dr. Arthur L. Walpole of ICI, been used successfully to determine whether tamoxifen pro Pharmaceuticals Division, Macclesfield, United Kingdom. Ear duces a tumoristatic or tumoricidal effect on implanted hor lier in his career Dr. Walpole had been particularly interested mone-responsive breast cancer cells. If animals are treated for in carcinogenesis and cancer therapy but in the 1960s his 1, 2, or 6 months with sustained release preparations of tamox attention was directed, as head of the fertility control program, ifen the tumor cells are not specifically destroyed. When the to the development of antifertility agents (83). Much of the animals are treated with estradiol to identify surviving tumor early preclinical antitumor work was subsequently conducted cells, tumors regrow in every case (92, 93). in my (V. C. J.) laboratory funded through the good offices of Overall the experimental evidence points to the fact that Arthur Walpole at ICI. tamoxifen is a tumoristatic rather than a tumoricidal agent. Long-term adjuvant tamoxifen treatment, or treatment until relapse, is predicted to be the best therapeutic strategy to control Antitumor Actions of Tamoxifen in Laboratory Models the recurrence of breast cancer following mastectomy. This The first systematic laboratory study of the antitumor action application of tamoxifen can be called chemosuppression. of tamoxifen was conducted at the Worcester Foundation for Experimental Biology in Shrewsbury, MA (82, 84-88). One of Long-Term Adjuvant Tamoxifen Therapy the results of this research (89, 90) was to encourage the clinical evaluation of tamoxifen in the United States by the Eastern Despite the developing laboratory data that demonstrated Cooperative Oncology Group. that tamoxifen is a tumoristatic agent (99-102), the vast ma- Most of the early research on tamoxifen used carcinogen- induced rat mammary tumor models (85-90) but during the 2The abbreviation used is: DMBA, dimethylbenzanthracene. 4182

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1990 American Association for Cancer Research. ANTIESTROGENS AND BREAST CANCER jority of clinical trials initially evaluated 1 or 2 years of adjuvant menopausal women receiving tamoxifen alone and women with tamoxifen therapy. There were several sound reasons for this: node-negative disease who might be treated for decades. This (a) a short course of tamoxifen might produce a tumoricidal expanded and extended use of tamoxifen requires an ongoing effect before the onset of resistance since it was known that evaluation of the pharmacology and potential toxicological patients with advanced disease respond to tamoxifen for only consequences of indefinite therapy. Obviously it is important 1-2 years; (b) the adjuvant use of tamoxifen might encourage to establish the safety of tamoxifen to reassure both physicians the early outgrowth of receptor-negative disease (the rationale and patients and to avoid unwarranted restrictions on this was that it would be wiser to save the drug until recurrence so valuable chemosuppressive therapy. that physicians would have something available to treat the patient); and (c) the long-term side effects of tamoxifen were unknown (thousands of women with advanced disease had been Clinical Pharmacology treated, but only until relapse, 1-2 years later). Although tamoxifen was initially used conservatively the The clinical pharmacology of tamoxifen was initially inves tigated in patients with advanced breast cancer (115-118). The recent overview of all randomized clinical trials (107) has demonstrated the survival advantage of postmenopausal, node- drug is readily absorbed following p.o. administration and accumulates to steady-state levels by the end of the first month positive women who received 1 or 2 years of adjuvant tamoxifen therapy. Swayed by the encouraging clinical trial results and of treatment (116). The serum levels of tamoxifen at steady state are extremely variable (50-300 ng/ml) but blood levels the laboratory findings (101, 107) clinical trials are currently focused on an evaluation of 5, 10, or in some cases indefinite are not related to response (119). The principal metabolite of adjuvant tamoxifen therapy in pre- and postmenopausal pa tamoxifen is ,/V-desmethyltamoxifen which produces blood lev tients with node-positive and node-negative disease. els approximately twice as high as those of tamoxifen by the In 1977, on the basis of encouraging laboratory data (101), end of the first month of treatment. The serum half-lives of tamoxifen and W-desmethyltamoxifen are extremely long (7 Dr. Douglass C. Tormey organized a pilot nonrandomized adjuvant clinical study to identify any unusual side effects and 14 days, respectively) (120), probably because the com produced by long-term adjuvant tamoxifen therapy (5 years) pounds are highly protein bound. and to evaluate the potential efficacy of the treatment strategy Tamoxifen is extensively metabolized in patients (121, 122) (108). The results demonstrated the safety of tamoxifen and (Fig. 4) but to date no significant quantities of estrogenic provided promising preliminary data to establish the Eastern metabolites have been noted that would cause concern during long-term adjuvant tamoxifen therapy. ,/V-Desmethyltamoxifen Cooperative Oncology Group protocols EST 4181 and EST 5181 to evaluate chemotherapy and different lengths of tamox is further metabolized to metabolite Y which has a glycol side ifen therapy in randomized clinical trials (109). chain (123, 124). Tamoxifen is also converted to 4-hydroxyta- The National Surgical Adjuvant Breast and Bowel Project moxifen but this is observed only at low concentrations in has chosen a similar course of action. Based upon their suc serum (118). However, this metabolite has an extremely high cessful evaluation of 2 years of adjuvant tamoxifen and chemo binding affinity for the estrogen receptor (the same as that of therapy (110) they conducted a registration study of chemo estradici) (125); therefore it probably plays a significant role in supporting the antitumor action of tamoxifen. Recently 4- therapy and tamoxifen for 2 years followed by an additional hydroxy-yV-desmethyltamoxifen has been identified in patients year of tamoxifen for those patients who did not have recur rence. The results are encouraging and support the view that (126). This metabolite has been shown in the laboratory to be long-term tamoxifen therapy benefits the patient (111). Several clinical trials have also evaluated the efficacy of long- term adjuvant tamoxifen therapy alone. A French study (112) has compared 3 years of adjuvant tamoxifen therapy versus no treatment. Tamoxifen produced a survival advantage especially in receptor-positive patients. Similarly a large clinical trial in Scotland (113) has evaluated whether it is an advantage to treat patients for 5 years with adjuvant tamoxifen. This clinical study is particularly interesting because it addresses the question of whether to use tamoxifen as an adjuvant therapy or to use the drug only when the patient develops advanced disease. In the Scottish study, patients in the control arm received tamoxifen at first recurrence. The results demonstrate that long-term adjuvant therapy with tamoxifen provides an effective control of disease recurrence and a survival advantage for the patient. There appears to be no need to delay the drug until recurrence N-DESMETHYLTAMOXIFEN 4-HYDROXY N-DESMETHYL because early drug resistance does not develop. TAMOXIFEN The National Surgical Adjuvant Breast and Bowel Project is currently conducting a trial of long-term adjuvant tamoxifen therapy (10 years) in pre- and postmenopausal patients with node-negative estrogen receptor-positive disease. The prelimi nary results are again encouraging (114) inasmuch as tamoxifen-treated women have an extended disease-free survival. Overall the clinical trials community has demonstrated some advantages for long-term adjuvant tamoxifen therapy. To date N.N. DIDESMETHYLTAMOXIFEN METABOLITE Y the trial population has been diverse with both pre- and post Fig. 4. Metabolites of tamoxifen identified in patients. 4183

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1990 American Association for Cancer Research. ANTIESTROGENS AND BREAST CANCER formed from either 4-hydroxytamoxifen or jV-desmethyltarnox- the growth of the breast tumor is controlled by tamoxifen. One ifen(127). large clinical trial (139) has shown that a similar phenomenon The blood levels of tamoxifen and its metabolites are stable can occur during long-term adjuvant tamoxifen therapy; Le, for up to 10 years of adjuvant therapy and no metabolic toler second breast tumors are reduced but there is an increase in ance seems to occur. This fact and the low incidence of reported endometrial carcinoma. To date these data have not been con side effects (121) makes tamoxifen an ideal long-term chemo- firmed by any other major clinical trials organization and it is suppressive agent for the adjuvant treatment of breast cancer. important to state that adjuvant tamoxifen therapy should not be denied to a patient on the basis of this potential side effect. It should be obvious that tamoxifen is of proven benefit (107) Potential Side Effects of Long-Term Adjuvant Tamoxifen Ther for the treatment of breast cancer, a disease that is invariably apy fatal, and endometrial carcinoma, should it occur, has a good prognosis. Routine gynecological examination must be per Tamoxifen therapy is being extended beyond a decade. Indeed node-negative patients with only a moderate risk of recurrence formed to monitor the health and well-being of the patients during adjuvant tamoxifen therapy. may in fact receive the drug indefinitely. This raises important questions about the potential long-term side effects of nonste- Finally, since more and more premenopausal patients are receiving adjuvant tamoxifen therapy, it should be stressed that roidal antiestrogens (Table 1). they are at risk for pregnancy and require counseling about Estrogen is important in prevention of the development of barrier contraceptives. Short-term tamoxifen treatment causes osteoporosis in women; therefore the long-term administration of an "antiestrogen" would seem to imply that women may an increase in ovarian estrogen synthesis (140) and premeno pausal patients on long-term adjuvant tamoxifen regimens (with become predisposed to osteoporosis. Similarly estrogen pro or without chemotherapy) continue to menstruate and have tects women from coronary heart disease by altering the circu lating levels of cholesterol. Again it would seem that the long- elevated circulating ovarian steroid hormone levels (141, 142). term administration of an "antiestrogen" might not be benefi It is currently not known what the long-term effects of tamox cial. However, tamoxifen exhibits some estrogen-like effects ifen on the ovary will be; this is a topic for future clinical investigation. that appear to have target site specificity (55). Tamoxifen is an Clearly the elevated levels of estrogen, a known tumor mito- antiestrogen in the rat uterus (52) but displays estrogen-like gen, might be able to impair the antitumor action of the effects in bone (128, 129). There are only few clinical data (130, antiestrogen. Tamoxifen is, however, an effective antitumor 131) to support these beneficial laboratory findings but tamox agent in premenopausal women with advanced disease (121). ifen is clearly not detrimental to bone in patients. The beneficial This clinical situation has been investigated in the athymic effects of nonsteroidal antiestrogens on serum cholesterol has animal model, and similar high circulating levels of estradici been noted previously in animals (41) and tamoxifen has been (500-800 pg/ml) do not reverse the antitumor action of tamox shown to lower serum cholesterol (mainly, low density lipopro- ifen (50 ng/ml).3 However, compliance issues with tamoxifen tein) in patients (132, 133). will be very important in premenopausal patients during long- Obviously the beneficial estrogen-like effects of tamoxifen in term adjuvant tamoxifen. Strategies to lower circulating estro some target sites may prove to be a disadvantage in others. gen like oophorectomy or the administration of luteinizing Estrogens can cause an increase in thromboembolic disorders hormone-releasing hormone agonists during adjuvant tamoxi and estrogen replacement therapy has been associated with an fen therapy should be evaluated in future clinical trials. increase in endometrial carcinoma. Tamoxifen does not, how ever, cause a significant increase in thromboembolic disorders when administered alone but there is a small decrease in anti- New Antiestrogens thrombin III during long-term adjuvant tamoxifen therapy (132, 134). The decrease (generally not more than 30%) is, A study of the structure-activity relationships of tamoxifen however, not great enough to cause clinical concern. Neverthe has provided an important insight into the mechanism of action less patients with a history of thromboembolic disease probably of both estrogens and antiestrogens. Early work demonstrated should not be treated with tamoxifen. that antiestrogens had a weak interaction with the estrogen There is very little information about the effects of tamoxifen receptor (59, 143) and it was widely believed that this property on the human uterus although tamoxifen does have some was essential to inhibit hormone action (144); i.e., an estrogen efficacy for treating endometrial carcinoma (135). Nevertheless would bind tightly to the receptor and the resulting complex tamoxifen has been implicated (anecdotally) in the development could initiate estrogen-dependent events whereas an antiestro of endometrial carcinoma (136). Realistically, however, there gen would bind to the receptor to block the access of estrogen is little possibility that tamoxifen could control the growth of but the complex would "fall apart" before it could itself initiate all endometrial tumors as only about 1 of 3 are hormone estrogen action. The discovery of the pharmacological proper responsive. Interestingly enough there is one provocative labo ties of monohydroxytamoxifen (4-hydroxytamoxifen) (125, ratory observation (137) that tamoxifen can encourage the 145) changed this view of antiestrogen action. 4-Hydroxyta- growth of a human endometrial carcinoma in athymic mice. moxifen has an affinity for the estrogen receptor similar to that Indeed this effect appears to be tumor specific (138). If athymic of estradici but it is a potent inhibitor of estrogen action. mice are bitransplanted with the endometrial tumor EnCalOl Subsequent confirmation of this work (146, 147) and the avail and the breast tumor MCF-7 and treated with estrogen and ability of radiolabeled material for studies in vitro and in vivo tamoxifen, the endometrial tumor is stimulated to grow whereas (147-150) has established the drug as a standard laboratory biochemical. Table 1 Potential side effects of long-term adjuvant tamoxifen therapy During the 1980s a number of assay systems in vitro were Antiestrogenic actions Osteoporosis, coronary heart disease used to describe the structure-function relationships of anties- Estrogenic actions Clotting, uterine stimulation Reproductive Ovarian, teratogenesis 3Y. lino and V. C. Jordan, unpublished observation. 4184

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1990 American Association for Cancer Research. ANTIESTROGENS AND BREAST CANCER trogens (151-157). These model systems are important as they provide information about the direct effects of ligands on the cell without metabolic transformation. In the future this knowl edge will undoubtedly dovetail with current research on the O molecular biology of the estrogen receptor to understand the

complex conformation changes that occur in the receptor pro 111 tein to program cell replication. K As a result of the successful use of tamoxifen to treat breast DC cancer, several new antiestrogens are now being evaluated in O the laboratory and the clinic. The structures of several of the D new compounds are shown in Fig. 5. The literature on new antiestrogens is increasing dramatically and interested readers should consult recent reviews (122, 158). One notable advance is the investigation of nonestrogenic antiestrogens for potential clinical applications. The discovery WEEKS process appears to have gone full circle (MER-25 has few Fig. 6. Effect of the nonestrogenic antiestrogen ICI 164,384 on tamoxifen- estrogenic properties) to find compounds without the estrogen- stimulated growth of endometrial tumor EnCa 101 in the athymic mouse. Animals were treated with a sustained release preparation of tamoxifen (2-cm Silastic like properties of the triphenylethylenes. At present only one capsule) (D), a control capsule (O), or ICI 164,384, 1 mg/0.1 ml peanut oil s.c. 3 compound (ICI 164,384) is available for evaluation and it has times weekly (â€¢)ortamoxifen plus ICI 164,384 (â€¢). demonstrated activity as an inhibitor of estradiol action both in vivo and in vitro (159-162). Interestingly the steroid also has Current and Future Uses of Antiestrogens the ability to inhibit tamoxifen-stimulated tumor growth in the laboratory (Ref. 94; Fig. 6). Various pure antiestrogens are In the 30 years since the first publication of the pharmaco currently being evaluated in the laboratory and some are logical properties of MER-25, the clinical application of anti- undergoing toxicology testing. When (or if) any of these new estrogens has proved to be a remarkable success. Tamoxifen is compounds become available for clinical application, they may now the antihormonal treatment of choice for advanced breast provide some additional clues about estrogen-regulated breast cancer (pre- and postmenopausal). As valuable as this applica cancer growth. One question has been whether the antitumor tion is as a palliative treatment, the remarkable advances have activity of tamoxifen is related to its estrogenic or antiestrogenic undoubtedly been made in the application of antiestrogens to properties (or neither?) This could soon be resolved. It is treat all stages of breast cancer. Tamoxifen is the antihormonal possible that pure antiestrogens could produce longer responses treatment of choice for the adjuvant therapy of postmenopausal in advanced disease or be useful as a second line therapy after node-positive women. Two years of treatment is known to long-term adjuvant tamoxifen treatment. This presupposes that produce a survival advantage, and longer treatment schedules a significant proportion of patients who eventually fail tamox are being evaluated to exploit the known tumoristatic qualities ifen therapy do so because of the estrogen-like properties of of tamoxifen. We already know that long-term adjuvant tamox tamoxifen. Unfortunately the potential of pure antiestrogens to ifen therapy (5 years) is beneficial compared to saving tamoxifen produce osteoporosis or early atherosclerosis may preclude until first recurrence, so 10-year and indefinite schedules are their use as a front line long-term adjuvant therapy in node- being evaluated. negative disease. The proven efficacy and low incidence of side effects have encouraged the evaluation of adjuvant tamoxifen therapy in pre- and postmenopausal women with node-negative disease. At present the long-term toxicological concerns appear to be negligible compared to the known course of breast cancer without treatment. Hundreds of thousands of women could be TRIOXIFENE being treated indefinitely with tamoxifen by the turn of the century. Indeed it has even been suggested that antiestrogens could be used to prevent breast cancer. However, the timing and cause of the carcinogenic insult are unknown; therefore the use of antiestrogens as true preventives seems impractical. A better term, also applicable to adjuvant therapy, is chemosuppressive; i.e., the drug can be used to suppress subclinical disease and prevent the appearance of a primary tumor (163). In some adjuvant trials tamoxifen inhibits the development of second primary breast tumors (114, 139, 164) and an early clinical evaluation of the toxicology of tamoxifen in normal women has been reported (165) as a first step in a prevention study. Nevertheless there is a real concern about being able to target TOREMIFENE DROLOXIFENE the right population. We cannot predict who will develop breast cancer; we can only guess at the probability. Furthermore "high risk" women are, in fact, only a minority of those who will develop breast cancer so any success must be balanced against '"ICH j),0C N(CMjljCH, ICI 164, 384 CH, as yet unknown accumulative toxicities. Fig. 5. Structures of new antiestrogens that are being evaluated in the labo Is this the end of the possible applications for antiestrogens? ratory and the clinics. Certainly not. We have obtained valuable clinical information 4185

acetate, desoxycorticosterone acetate and testosterone on estradiol-17 in about this group of drugs that can be applied in other disease duced uterine growth. Endocrinology, 59: 165-169, 1956. states. Research does not travel in straight lines and observa 15. Edgren, R. A., and Calhoun, D. W. Estrogen antagonisms: inhibition of tions in one field of science often become major discoveries in estrone-induced uterine growth by testosterone propionate, progesterone and 17-ethyl-19-nortestosterone. Proc. Soc. Exp. Biol. Med., 94: 537-539, another. Important clues have been garnered about the effects 1957. of tamoxifen on bone and lipids so it is possible that derivatives 16. Lerner, L. J., Holthaus, F. J., Jr., and Thompson, C. R. A myotrophic could find targeted applications to retard osteoporosis or ath agent and gonadotrophin inhibitor, 19-nortestosterone-17-benzoate. Endo crinology, 64: 1010-1016, 1959. erosclerosis. The ubiquitous application of novel compounds to 17. Hisaw, F. L., Velardo, J. T., and Goolsby, C. M. Interaction of estrogens prevent diseases associated with the progressive changes after on uterine growth. J. Clin. Endocrino!., 14: 1134-1143, 1954. 18. Velardo, J. T., and Sturgis, S. H. Interaction of 16-epi-estriol and estradiol- menopause may, as a side effect, significantly retard the devel 17 on uterine growth. Proc. Soc. Exp. Biol. Med., 90: 609-610, 1955. opment of breast cancer. The target population would be post- 19. Muggins, C., and Jensen, E. V. The depression of estrone-induced uterine menopausal women in general, thereby avoiding the require growth by phenolic estrogens with oxygenated functions at position 6 or 16: the impeded estrogens. J. Exp. Med., 102: 335-346, 1955. ment to select a high risk group to prevent breast cancer. 20. Edgren, R. A., and Calhoun, D. W. Oestrogen antagonisms: the effects of oestriol and 16-epioestriol on oestrone-induced uterine growth in spayed rats. J. Endocrinol., 20: 325-330, 1960. Acknowledgments 21. Edgren, R. A., and Calhoun, D. W. Interaction of estrogens on the vaginal smear of spayed rats. Am. J. Physiol., 189: 355-357, 1957. This paper is not intended to be an encyclopedic review article but is 22. Lerner, L. J. Uterotrophic and antiestrogenic activity of a non-steroidal offered only as a personal recounting of research conducted in our compound in mice. In: Conference on Comparison of Biological Properties of Steroids and Hormones, Detroit 1954, Committee on Research, Council respective laboratories during a period that has spanned almost 40 on Pharmacy and Chemistry, American Medical Association, pp. 115-117. years. We sincerely acknowledge the support of colleagues, students, 23. Jensen, E. V., Jacobson, H. I., Flesher, J. W., Saha, N. N., Gupta, G. N., and assistants in our respective laboratories. We also acknowledge the Smith, S., Colucci, V., Shiplacoff, D., Neumann, H. G., DeSombre, E. R., important additional contributions made to the understanding of an and Junglut, P. W. Estrogen receptors in target tissues. In: T. Nakao, G. Pincus, and J. Tait (eds.), Steroid Dynamics, pp. 133-157. New York: tiestrogen action by James H. Clark, Kathryn B. Horwitz, Benita and Academic Press, 1966. John Katzenellenbogen, Marc E. Lippman, C. Kent Osborne, Robert 24. Jensen, E. V., Numata, M., Smith, S., Suuki, T., Brecker, P. I., and I. Nicholson, Henri Rochefort, Robert L. Sutherland, and Alan E. DeSombre, E. R. Estrogen-receptor interaction in target tissues. Dev. Biol., Wakeling. The influence of our teachers the late James H. Leatham Suppl. 3, 151-171, 1969. 25. Lerner, L. J. Estrogen antagonistic activity of l-(/7-2-diethylaminoethoxy- and the late James B. Allison (L. J. L.) and Edward R. Clark (V. C. J.) phenyl)-l-phenyl-2-/>-anisylethanol (MER-25). Fed. Proc., 17: 388, 1958. was important for the direction and development of our investigative 26. Lerner, L. J., Holthaus, F. J., Jr., and Thompson, C. R. A non-steroidal approach. Dr. Lerner is ever grateful for the encouragement he received estrogen antagonist l-(/)-2-diethylaminoethoxyphenyl)-l-phenyl-2-^-meth- as a developing scientist from Roy O. Creep, Roy Hertz, the late Albert oxyphenyl ethanol. Endocrinology, 63: 295-318, 1958. 27. Lerner, L. J. Hormone antagonists: inhibitor of specific activities of estrogen Segaloff, and the late Ralph Dorfman. Dr. Jordan wishes to acknowl and androgen. Recent Prog. Horm. Res., 20: 435-490, 1964. edge William L. McGuire, Jack Gorski, Elwood V. Jensen, the late 28. Lerner, L. J. Steroid hormones and uterine growth. Ann. NY Acad. Sci., Arthur L. Walpole, and colleagues at ICI for their invaluable help and 75:460-462, 1959. support and Paul P. Carbone, Richard R. Love, Bernard Fisher, Sydney 29. Lerner, L. J., Hilf, R., Turkheimer. A. R., Michel, I., and Engel, S. L. Effects of hormone antagonists on morphological and biochemical changes E. Salmon, and Douglass C. Tormey for the clinical application of induced by hormonal steroids in the rat uterus. Endocrinology, 78: 111- many of the concepts and results described here. The EnCalOl tumor 124, 1966. was kindly provided by Dr. P. G. Satyaswaroop, Department of Ob 30. Lerner, L. J., Harris, D. N., Hilf, R., Bianchi, A., and Raskin, B. K. stetrics and Gynecology, Hershey School of Medicine, Hershey, PA, Responses of the rat uterus and oviduct to sex steroids as influenced by time for the experiment in Fig. 6 performed by Dr. Marco M. Gottardis and and antagonists. In: Proceedings of the Second International Congress on Hormonal Steroids, Milan, 1966. Excerpta Medica International Congress Michelle Ricchio. Series No. 132, Amsterdam, pp. 628-638. Amsterdam: Excerpta Medica, 1967. 31. Harris, D. N., Lerner, L. J., and Hilf, R. The effects of progesterone and References ethamoxytriphetol on estradiol-induced changes in the immature rat uterus. Trans. NY Acad. Sci., 30: 774-782, 1968. 1. Beatson, G. T. On the treatment of inoperable cases of carcinoma of the 32. Lerner, L. J., Hilf, R., and Harris, D. N. The effect of antagonists on mamma: suggestions for a new method of treatment with illustrative cases. androgen- and estrogen-induced changes in the male accessory sex organs Lancet, 2: 104-107, 162-167, 1896. of the rat. Trans. NY Acad. Sci., 30: 783-793, 1968. 33. Sutherland, R. L., Mester, J., and Baulieu, E. E. Tamoxifen is a potent 2. Dao, T. L., and Muggins, C. Bilateral adrenalectomy in the treatment of "pure" antioestrogen in the chick oviduct. Nature (Lond.), 267: 434-435, cancer of the breast. A. M. A. Arch. Surg., 71: 645-657, 1955. 3. Pearson, O. H., and Ray, B. S. Results of treatment of metastatic mammary 1977. carcinoma. Cancer (Phila.), 12: 85-92, 1959. 34. Sutherland, R. L. Estrogen antagonists in chick oviduct: antagonist activity 4. Allen, E., and Doisy, E. A. The induction of a sexual mature condition in of eight synthetic triphenylethylene derivatives and their interactions with immature females by injection of the ovarian follicular hormone. Am. J. cytoplasmic and nuclear estrogen receptors. Endocrinology, 109: 2061- Physiol., 69: 577-588, 1924. 2068, 1981. 5. Cook, J. W., Dodds, E. C., and Hewett, C. L. A synthetic oestrus-exciting 35. Segal, S. J., and Nelson, W. O. An orally active compound with antifertility compound. Nature (Lond.), 131: 56-57, 1933. effects in rats. Proc. Soc. Exp. Biol. Med., 98: 431-436, 1958. 6. Dodds, E. C., Golberg, L., Lawson, W., and Robinson, R. Oestrogenic 36. Chang, M. C. Degeneration of ova in the rat and rabbit following oral activity of certain synthetic compounds. Nature (Lond.), 141: 247-248, administration of l-(p-2-diethylaminoethoxyphenyl)-l-phenyl-2-/j-anisylethanol. Endocrinology, 65: 339-342, 1959. 1938. 7. Dodds, E. C., Golberg, L., Lawson, W., and Robinson, R. Oestrogenic 37. Emmens, C. W. Compounds exhibiting prolonged antiestrogenic and anti- activity of alkalated stilboestrols. Nature (Lond.), Â¡42:34, 1938. fertility activity in mice and rats. J. Reprod. FÃ©rtil.,26: 175-182, 1971. 8. Robson, J. M., and Schonberg, A. Oestrous reactions, including mating, 38. Clark, E. R., and Jordan, V. C. Oestrogenic, anti-oestrogenic and fertility produced by triphenylethylene. Nature (Lond.), 140: 196, 1937. properties of a series of compounds related to ethamoxytriphetol (MER- 9. Robson, J. M., and Schonberg, A. A new synthetic estrogen with prolonged 25). Br. J. Pharmacol., 57:487-493, 1976. action when given orally. Nature (Lond.), 150: 22-23, 1942. 39. Morris, J. M., VanWagenen, G., McCann, J., and Jacobs, D. Compounds 10. Thompson, C. R., and Werner, H. W. Studies on estrogen tri-p-anisylchlo- interfering with ovum implantation and development. II. Synthetic estrogens roethylene. Proc. Soc. Exp. Biol. Med., 77: 494-497, 1951. and antiestrogens. FÃ©rtil.Steril., 18: 18-34, 1967. 11. Shelton, R. S., VanCampen, M. G., Jr., Meisner, D. F., Parmeter, S. M., 40. Kamboj, V. P., Setty, B. S., Chandra, H., Roy, S. K., and Kar, A. B. Andrews, E. R., Allen, R. E., and Wyckoff, K. K. Synthetic estrogens. Biological profile of centchromanâ€”a new post-coital contraceptive. Ind. J. Halotriphenyl ethylene derivatives. J. Am. Chem. Soc., 75: 5491-5493, Exp. Biol., /5: 1144-1146, 1977. 1953. 41. Lerner, L. J. The first non-steroidal antioestrogen-MER-25. In: R. L. 12. Robson, J. M. Quantitative data on the inhibition of oestrus by testosterone, Sutherland and V. C. Jordan (eds.), Non-steroidal antioestrogens: Molecular progesterone and certain other compounds. J. Physiol., 92: 371-382, 1938. Pharmacology and Antitumor Activity, pp. 1-16. Sydney, Australia: Aca 13. Szego, C. M., and Roberts, S. Pituitary-adrenal antagonism to estrogenic demic Press, 1981. stimulation of the uterus of the ovariectomized rat. Am. J. Physiol., 152: 42. Adams, W. C., France, E. S., and Leathern, J. H. The influence of an 131-140, 1948. estrogen antagonist on reproductive physiology in the female rat. Proc. Pa. 14. Velardo, J. T., Hisaw, F. L., and Bever, A. T. Inhibitory action of cortisone Acad. Sci., 34: 155-158, 1960. 4186

43. Leathern, J. H., and Adams, W. C. Prevention of ovarian cyst formation by Expression of transforming growth factor a and its messenger ribonucleic ethamoxytriphetol (MER-25). Proc. Soe. Exp. Biol. Med., 113: 240-242, acid in human breast cancer: its regulation by estrogen and its possible 1963. functional significance. Mol. Endocrino!., 2: 543-555, 1988. 44. Kistner, R. W., and Smith, O. W. Observations on the use of a non-steroidal 74. Knabbe, C., Lippman, M. E., Wakefield, L. M., Flanders, K. C., Kasid, A., estrogen antagonist: Mer-25. Surg. Forum, 10: 725-729, 1960. Derynck, R., and Dickson, R. B. Evidence that transforming growth factor 45. Kistner, R. W., and Smith, O. W. Observations on the use of a nonsteroidal ÃŸis a hormonally regulated negative growth factor in human breast cancer estrogen antagonist: Mer-25. II. Effects in endometrial hyperplasia and cells. Cell, 48: 417-428, 1987. Stein-Leventhal syndrome. FÃ©rtil.Steril., 12: 121-141, 1961. 75. Cole, M. P., Jones, C. T. A., and Todd, I. D. H. A new antioestrogenic 46. Tyler, E. T., Olson, H. J., and Gotlib, M. H. The induction of ovulation agent in late breast cancer: an early clinical appraisal of ICI 46,474, Br. J. with an anti-estrogen. Int. J. FÃ©rtil.,5:429-436, 1960. Cancer, 25: 270-275, 1971. 47. Holtkamp, D. E., Greslin, J. G., Root, C. A., and Lerner, L. J. Gonadotro- 76. Ward, H. W. C. Antioestrogen therapy for breast cancer: a trial of tamoxifen phin inhibiting and anti-fecudity effects of chloramiphene. Proc. Soc. Exp. at two dose levels. Br. Med. J., /: 13-14, 1973. Biol. Med., 705: 197-201, 1960. 77. O'Halloran, M. J., and Maddock, P. G. ICI 46,474 in breast cancer. J. Ir. 48. Greenblatt, R. B., Barfield, W. E., Jungck, E. C., and Ray, A. W. Induction Med. Assoc., 67: 38-39, 1974. of ovulation with MRL-41. Preliminary report. JAMA, 178:101-104,1961. 78. Klopper, A., and Hall, M. New synthetic agent for the induction of ovula 49. Lerner, L. J., Harris, D. N., Yiacas, E., Hilf, R., and Michel, I. Cholesterol tion. Preliminary trial in women. Br. Med. J., /: 152-154, 1971. synthesis inhibitor showing reduction of lipids and hormone antagonism. 79. El-Sheikha, Z., Klopper, A., and Beck, J. S. Treatment of menometrorrhagia Am. J. Clin. Nutr., 23: 1241-1250, 1970. with antioestrogen. Clin. Endocrinol., /: 275-282, 1972. 50. Herbst, A. L., Griffiths, C. T., and Kistner, R. W. Clomiphene citrate (NSC- 80. Williamson, J. G., and Ellis, J. D. The induction of ovulation by tamoxifen. 35770) in disseminated mammary carcinoma. Cancer Chemother. Rep., 43: J. Obstet. Gynaecol. Br. Commonw., 80: 844-847, 1973. 39-41, 1964. 81. Jensen, E. V., Block, G. E., Smith, S., Kyser, K., and DeSombre, E. R. 51. Harper, M. J. K., and Walpole, A. L. Contrasting endocrine activities of m Estrogen receptors and breast cancer response to adrenalectomy. Nati. and trans isomers in a series of substituted triphenylethylenes. Nature Cancer Inst. Monogr., 34: 55-70, 1971. (Lond.), 2/2:87, 1966. 82. Jordan, V. C., and Koerner, S. Tamoxifen (ICI 46,474) and the human 52. Harper, M. J. K., and Walpole, A. L. A new derivative of triphenylethylene: carcinoma 8S estrogen receptor. Eur. J. Cancer, 11: 205-206, 1975. effect on implantation and mode of action in rats. J. Reprod. FÃ©rtil.,13: 83. Jordan, V. C. The development of tamoxifen for breast cancer therapy: a 101-119, 1967. tribute to the late Arthur L. Walpole. Breast Cancer Res. Treat., 11: 197- 53. Harper, M. J. K., and Walpole, A. L. Mode of action of ICI 46,474 in 209, 1988. preventing implantation in rats. J. Endocrinol., 37: 83-92, 1967. 84. Investigational Drug Brochure for ICI 46,474. Clinical Research Depart 54. Labhsetwar, A. P. Role of estrogens in ovulation. A study using the estrogen ment, ICI Americas Inc., Wilmington, DE, March 1974. antagonist ICI 46,474. Endocrinology, 87: 542-551, 1970. 85. Jordan, V. C. Antitumour activity of the antioestrogen ICI 46,474 (tamox 55. Jordan, V. C., and Robinson, S. P. Species-specific pharmacology of anti- ifen) in the dimethylbenzanthracene (DMBA)-induced rat mammary carci estrogens: role of metabolism. Fed. Proc., 46: 1870-1874, 1987. noma model. J. Steroid. Biochem., 5: 354, 1974. 56. Terenius, L. Structure-activity relationships of anti-oestrogens with regard 86. Jordan, V. C., Koerner, S., and Robison, C. Inhibition of oestrogen-stimu to interaction with 17ÃŸ-oestradiolin the mouse uterus and vagina. Acta lated prolactin release by antioestrogens. J. Endocrinol., 65:151-152,1975. Endocrinol. Copenh., 66: 431-447, 1971. 87. Jordan, V. C. Effect of tamoxifen (ICI 46,474) on initiation and growth of 57. Jordan, V. C. Prolonged antioestrogenic activity of ICI 46,474 in the DMBA-induced rat mammary carcinomata. Eur. J. Cancer, 12: 419-424, ovariectomized mouse. J. Reprod. FÃ©rtil.,52:251-258, 1975. 1976. 58. Jordan, V. C., Lababidi, M. K., and Mirecki, D. M. Inhibition of mouse 88. Jordan, V. C., and Koerner, S. Tamoxifen as an antitumour agent: role of mammary tumorigenesis by the antiestrogen tamoxifen. In: Proceedings of oestradiol and prolactin. J. Endocrinol., 68: 305-310, 1976. the British Pharmacological Society, King College, London, Abstract 22, 89. Jordan, V. C. The antiestrogen tamoxifen (ICI 46,474) as an antitumor December 1988. agent. Proceedings of the Eastern Cooperative Oncology Group, Miami, 59. Skidmore, J. R., Walpole, A. L., and Woodburn, J. Effect of some triphen FL, February 11-12, 1974. ylethylenes on oestradiol binding in vitro to macromolecules from uterus 90. Jordan, V. C. Tamoxifen: mechanism of antitumor activity in animals and and anterior pituitary. J. Endocrinol., 52: 289-298, 1972. man. Proceedings of the Eastern Cooperative Oncology Group, Jaspar, 60. Jordan, V. C., and Prestwich, G. Binding of [3H] tamoxifen in rat uterine Alberta, Canada, June 22-25, 1974. cytosols. A comparison of swinging bucket and vertical tube rotor sucrose 91. Osborne, C. K., Hobbs, K., and Clark, G. M. Effect of estrogens and density gradient analysis. Mol. Cell. Endocrinol., 8: 179-188, 1977. antiestrogens on growth of human breast cancer cells in athymic mice. 61. Jordan, V. C., and Dowse, L. J. Tamoxifen as an antitumour agent: effect Cancer Res., 45: 584-590, 1985. on oestrogen binding. J. Endocrinol., 68: 297-303, 1976. 92. Gottardis, M. M., Robinson, S. P., and Jordan, V. C. Estradiol-stimulated 62. Jordan, V. C., Dix, C. J., Rowsby, L., and Prestwich, G. Studies on the growth of MCF-7 tumors implanted in athymic mice: a model to study the mechanisms of action of the non-steroidal antioestrogen (ICI 46,474) in the tumoristatic action of tamoxifen. J. Steroid. Biochem., 30: 311-314, 1988. rat. Mol. Cell. Endocrinol., 7: 177-192, 1977. 93. Gottardis, M. M., and Jordan, V. C. Development of tamoxifen-stimulated 63. Jordan, V. C., Rowsby, L., Dix, C. J., and Prestwich, G. Dose-related effect growth of MCF-7 tumors in athymic mice after long-term antiestrogen of non-steroidal antioestrogens and non-steroidal oestrogens on the meas administration. Cancer Res., 48: 5183-5187, 1988. urement of cytoplasmic oestrogen receptors in the rat and mouse uterus. J. 94. Gottardis, M. M., Jiang, S. Y., Jeng, M. H., and Jordan, V. C. Inhibition Endocrinol., 78: 71-81, 1978. of tamoxifen-stimulated growth of an MCF-7 tumor variant in athymic 64. Jordan, V. C., and Naylor, K. E. Binding of [3H]oestradiol in the immature mice by novel steroidal antiestrogens. Cancer Res., 49:4090-4093, 1989. rat uterus during the sequential administration of antioestrogens. Br. J. 95. Jordan, V. C., Gottardis, M. M., Robinson, S. P., and Friedl, A. Immune- Pharmacol., 65: 167-173, 1979. deficient animals to study "hormone-dependent" breast and endometrial 65. Berthois, Y., Katzenellenbogen, J. A., and Katzenellenbogen, B. S. Phenol cancer. J. Steroid. Biochem., 34: 169-176, 1989. red in tissue culture media is a weak estrogen: implications concerning the 96. Jordan, V. C. The antitumour affect of tamoxifen in the dimethylbenzan- study of estrogen-responsive cells in culture. Proc. Nati. Acad. Sci, USA, thracene-induced mammary carcinoma model. Proceedings of a Symposium Â«5:2496-2500, 1986. on the Hormonal Control of Breast Cancer, Alderley Park, pp. 11-17. 66. Bindal, R. D., Carlson, K. E., Katzenellenbogen, B. S., and Katzenellenbo Macclesfield, United Kingdom: ICI Pharmaceuticals Division PLC, 1975. gen, J. A. Lipophilic impurities, not phenosulfophthalein, account for the 97. Nicholson, R. I., and Golder, M. P. Effect of synthetic antioestrogens on estrogenic activity in commercial preparations of phenol red. J. Steroid the growth and biochemistry of rat mammary tumours. Eur. J. Cancer, //: Biochem., 31: 287-293, 1988. 571-579, 1975. 67. Lippman, M. E., and BolÃ¡n,G. Oestrogen-responsive human breast cancer 98. Jordan, V. C., and Jaspan, T. Tamoxifen as an antitumour agent: oestrogen in long-term tissue culture. Nature (Lond.), 256: 592-595, 1975. binding as a predictive test for tumour response. J. Endocrinol., 68: 453- 68. Reddel, R. R., Murphy, L. C., and Sutherland, R. L. Effects of biologically 460, 1976. active metabolites of tamoxifen on the proliferation kinetics of MCF-7 99. Jordan, V. C., Dix, C. J., and Allen, K. E. The effectiveness of long-term human breast cancer cells in vitro. Cancer Res., 43: 4618-4614, 1983. treatment in a laboratory model for adjuvant hormone therapy of breast 69. Taylor, C. M., Blanchard, B., and Zava, D. T. Estrogen receptor mediated cancer. In: S. E. Salmon and S. E. Jones (eds.), Adjuvant Therapy for Cancer and cytotoxic effects of the antiestrogens tamoxifen and 4-hydroxytamoxi- II, pp. 19-26. New York: GruÃ±eand Stratton, 1979. fen. Cancer Res., 44: 1409-1414,1984. 100. Jordan, V. C., and Allen, K. E. Evaluation of the antitumor activity of the 70. Sutherland, R. L., Murphy, L. C., Foo, M. S., Green, M. D., Wybourne, A. nonsteroidal antioestrogen monohydroxytamoxifen in the DMBA-induced M., and Krozowski, Z. S. High affinity antioestrogen binding site distinct rat mammary carcinoma model. Eur. J. Cancer, 16: 239-251, 1980. from the oestrogen receptor. Nature (Lond.), 288: 273-275, 1980. 101. Jordan, V. C. Use of the DMBA-induced rat mammary carcinoma system 71. Sutherland, R. L., Green, M. D., Hall, R. E., Reddel, R. R., and Taylor, I. for the evaluation of tamoxifen treatment as a potential adjuvant therapy. W. Tamoxifen induces accumulation of MCF-7 human mammary carci Rev. Endocrinol. Rei. Cancer, October Suppl., 49-55, 1978. noma cells in the Go/G] phase of the cell cycle. Eur. J. Cancer Clin. OncoL, 102. Jordan, V. C., Allen, K. E., and Dix, C. J. The pharmacology of tamoxifen 19: 615-621, 1983. in laboratory animals. Cancer Treat. Rep., 64: 745-759, 1980. 72. Osborne, C. K., Boldt, D. H., Clark, G. M., and Trent, J. M. Effect of 103. Robinson, S. P., and Jordan, V. C. Reversal of the antitumor effect of tamoxifen on human breast cancer cell cycle kinetics: accumulation of cells tamoxifen by progesterone in the 7,12-dimethylbenzanthracene-induced rat in early G, phase. Cancer Res., 43: 3583-3586, 1983. mammary carcinoma model. Cancer Res., 47: 5386-5390, 1987. 73. Bates, S. E., Davidson, N. E., Vilverius, E. M., FrÃ©ter,C.E., Dickson, R. 104. Robinson, S. P., Mauel, D. A., and Jordan, V. C. Antitumor actions of B., Tarn, J. P., Kudlow, J. E., Lippman, M. E., and Salomon, D. S. toremifene in the 7,12-dimethylbenzanthracene (DMBA)-induced rat mam- 4187

mary tumor model. Eur. J. Cancer Clin. Oncol., 24: 1817-1821, 1988. 131. TÃ¼rken,S., Siris, E., Seidin, E., Seidin, D., Plaster, E., Hyman, G., and 105. Wilson, A. J., Tehrani, F., and Baum, M. Adjuvant tamoxifen therapy for Lindsay, R. Effects of tamoxifen on spinal bone density in women with early breast cancer: an experimental study with reference to oestrogen and breast cancer. J. Nati. Cancer Inst., */: 1086-1088, 1989. progesterone receptors. Br. J. Surg., 69: 121-125, 1982. 132. Bertelli, G., Pronzoto, P., and Amoroso, D. Adjuvant tamoxifen in primary 106. Gottardis, M. M, and Jordan, V. C. The antitumor actions of keoxifene breast cancer: influence on plasma lipids and antithrombin III. Breast Cancer and tamoxifen in the A'-nitrosomethylurea-induced rat mammary carcinoma Res. Treat., 12: 307-310, 1988. model. Cancer Res., 47: 4020-4024, 1987. 133. Love, R. R., Newcomb, P. A., Wiebe, D. A., Surawicz, T. S., Jordan, V. C., 107. Early Breast Cancer Trialists' Collaborative Group. Effect of adjuvant Carbone, P. P., and DeMets, D. C. Lipids and lipoprotein effects of tamoxifen and of cytotoxic therapy on mortality in early breast cancer. N. tamoxifen therapy in postmenopausal women with node negative breast Engl. J. Med., 319: 1681-1692, 1988. cancer. Breast Cancer Res. Treat., 13: Abstract 204, 1989. 108. Tormey, D. C., and Jordan, V. C. Long-term tamoxifen adjuvant therapy 134. Jordan, V. C., Fritz, N. F., and Tormey, D. C. Long-term adjuvant therapy in node positive breast cancer: a metabolic and pilot clinical study. Breast with tamoxifen effects on sex hormone binding globulin and antithrombin Cancer Res. Treat., 4: 297-302, 1984. III. Cancer Res., 47: 4517-4519, 1987. 109. Falkson, H. C., Gray, R., and Wolberg, W. M. Adjuvant therapy of post- 135. Swenerton, K. D. Treatment of advanced endometrial adenocarcinoma with menopausa! women with breast cancerâ€”an ECOG phase III study. Abstract tamoxifen. Cancer Treat. Rep., 64: 805-811, 1980. 67. Proc. ASCO San Francisco May, 1989. 136. Killackey, M. A., Hakes, T. B., and Pierce, V. K. Endometrial adenocarci 110. Fisher, B., Redmond, C., Brown, A., Fisher, E. R., Wolmark, N., Bowman, noma in breast cancer patients receiving tamoxifen. Cancer Treat. Rep., 69: D., Plotkin, D., Wolter, J., Bornstein, R., Legault-Poisson, S., Saffer, E. 237-238, 1985. A., and Other NSABP Investigators. Adjuvant chemotherapy with and 137. Satyaswaroop, P. G., Zaino, R. J., and Mortel, R. Estrogen-like effects of without tamoxifen in the treatment of primary breast cancer: 5 year results tamoxifen on human endometrial carcinoma transplanted into nude mice. from the National Surgical Adjuvant Breast and Bowel Project Trial. J. Cancer Res., 44: 4006-4010, 1984. Clin. Oncol., 4: 459-471, 1986. 138. Gottardis, M. M., Robinson, S. P., Satyaswaroop, P. G., and Jordan, V. C. 111. Fisher, B., Brown, A., Wolmark, N., Redmond, C, Wickerham, L., Wittliff, Contrasting actions of tamoxifen on endometrial and breast tumor growth J., Dimitrov, N., Legault-Poisson, S., Schipper, H., Prager, D., and Other in the athymic mouse. Cancer Res., 48: 812-815, 1988. NSABP Investigators. Prolonging tamoxifen therapy for primary breast 139. Fornander, T., Rutquist, L. E., Cedermark, B., Glas, U., Mattson, A., cancer. Ann. Intern. Med., Â¡06:649-654, 1987. Siljversward, J. D., Skoog, L., Somell, A., Theve, T., Wilking, N., Asker- 112. Delozier, T., Julien, J-P., Juret, P., Veyret, C., Couette, J-E., Grai, Y., gren, J., and Hjolmar, M. L. Adjuvant tamoxifen in early breast cancer: Olliver, J-.M.. and de Ranier, E. Adjuvant tamoxifen in postmenopausal occurrence of new primary cancers. Lancet, 1: 117-120, 1989. breast cancer: preliminary results of a randomized trial. Breast Cancer Res. 140. Groom, G. V., and Griffiths, K. Effect of the antioestrogen tamoxifen on Treat., 7: 105-110, 1986. plasma levels of luteinizing hormone, follicle-stimulating hormone, prolac- 113. Breast Cancer Trials Committee, Scottish Cancer Trials Office (MRC). tin, oestradiol and progesterone in normal premenopausal women. J. En Adjuvant tamoxifen in the management of operable breast cancer: the docrinol., 70:421-428, 1976. Scottish Trial. Lancet, 2: 171-175, 1987. 141. Jordan, V. C., Fritz, N. F., and Tormey, D. C. Endocrine effects of adjuvant 114. Fisher, B., Constantino, J., Redmond, C., and Other Members of the chemotherapy and long-term tamoxifen administration on node positive NSABP. A randomized clinical trial evaluating tamoxifen in the treatment patients with breast cancer. Cancer Res., 47: 624-630, 1987. of patients with node-negative breast cancer who have estrogen receptor- 142. Ravdin, P. M., Fritz, N. F., Tormey, D. C., and Jordan, V. C. Endocrine positive tumors. N. Engl. J. Med., 320: 479-484, 1989. status of premenopausal node positive breast cancer patients following 115. Fromson, J. M., Pearson, S., and Bramali. S. The metabolism of tamoxifen adjuvant chemotherapy and long-term tamoxifen. Cancer Res., 48: 1026- (ICI 46,474) Part II in female patients. Xenobiotica, 3: 711-713, 1973. 1029, 1988. 116. Fabian, C., Sternson, L., and Barrett, M. Clinical pharmacology of tamox- 143. Korenman, S. G. Relation between estrogen inhibiting activity and binding ifen in patients with breast cancer: comparison of traditional and loading to cytosol of rabbit and human uterus. Endocrinology, 87:1119-1123,1970. dose schedules. Cancer Treat. Rep., 64: 765-773, 1980. 144. Bouton, M. M., and Raynaud, J. P. The relevance of interaction kinetics in 117. Adam, H. K., Gay, M. A., and Moore, R. H. Measurement of tamoxifen in determining biological response to estrogens. Endocrinology, 705:509-575, serum by thin layer densitometry. J. Endocrinol., 84: 35-42, 1982. 1979. 118. Daniel, C. P., Gaskell, S. J., Bishop, H., and Nicholson, R. I. Determination 145. Jordan, V. C., Dix, C. J., Naylor, K. E., Prestwich, G., and Rowsby, L. of tamoxifen and a hydroxylated metabolite in plasma from patients with Nonsteroidal antiestrogens: their biological effects and potential mecha advanced breast cancer using gas chromatography-mass spectrometry. J. nisms of action. J. Toxicol. Environ. Health, 4: 364-390, 1978. Endocrinol., 83: 401 -408, 1979. 146. Binan. N., Catelli, M. H., Geynet, Puri, V., Hahnel, R., Mester, J., and 119. Bratherton, D. G., Brown, C. H., Buchanan, R., Hall, V., Kinglsey Pillers, Baulieu, E. E. Monohydroxytamoxifen: an antiestrogen with high affinity E. M., Wheeler, T. K., and Williams, C. J. A comparison of two doses of for the chick oviduct oestrogen receptor. Biochem. Biophys. Res. Commun., tamoxifen (Nolvadex) in postmenopausal women with advanced breast 91: 812-818, 1979. cancer: 10 mg bd versus 20 mg bd. Br. J. Cancer, 50: 199-205, 1984. 147. Borgna, J. L., and Rochefort, H. High affinity binding to the estrogen 120. Patterson, J. S., Settatree, R. S., Adam, H. K., and Kemp, J. V. Serum receptor of [3H] 4-hydroxytamoxifen, an active antiestrogen metabolite. concentrations of tamoxifen and major metabolites during long-term Nol Mol. Cell. Endocrinol., 20: 71-85, 1980. vadex therapy, correlated with clinical response. In: H. T. Mouridsen and 148. Rochefort, H., and Borgna, J. L. Differences between oestrogen receptor T. Palshoff (eds.). Breast Cancerâ€”Experimental and Clinical Aspects, pp. activated by oestrogen and antioestrogen. Nature (Lond.), 292: 257-259, 89-92. Oxford, United Kingdom: Pergamon Press, 1980. 1981. 121. Furr, B. J. A., and Jordan, V. C. The pharmacology and clinical uses of 149. Jordan, V. C., and Bowser-Finn, R. A. Binding of [3H] monohydroxyta- tamoxifen. Pharmacol. Ther., 25: 127-205, 1984. moxifen by immature rat tissues in vivo. Endocrinology, 110: 1281-1291, 122. Robinson, S. P., and Jordan, V. C. The metabolism of steroid modifying 1982. anticancer agents. Pharmacol. Ther., 36: 41-103, 1988. 150. Tate, A. C., Greene, G. L., DeSombre, E. R., Jensen, E. V., and Jordan, V. 123. Jordan, V. C., Bain, R. R., Brown, R. R., Gosden, B., and Santos, M. A. C. Differences between estrogen and antiestrogen-estrogen receptor com Determination and pharmacology of a new hydroxylated metabolite of plexes from human breast tumors identified with an antibody raised against tamoxifen observed in patient sera during therapy for advanced breast the estrogen receptor. Cancer Res., 44: 1012-1018, 1984. cancer. Cancer Res., 43: 1446-1450, 1983. 151. Lieberman, M. E., Gorski, J., and Jordan, V. C. An estrogen receptor model 124. Kemp, J. V., Adam, H. K., Wakeling, A. E., and Slater, R. Identification to describe the regulation of prolactin synthesis by antiestrogens in vitro. J. and biological activity of tamoxifen metabolites in human serum. Biochem. Biol. Chem., 258: 4741-4745, 1983. Pharmacol., 32: 2045-2052, 1983. 152. Jordan, V. C., Lieberman, M. E., Cormier, E., Bagely, J., and Ruenitz, P. 125. Jordan, V. C., Collins, M. M., Rowsby, L., and Prestwich, G. A monohy- Structural requirements for the pharmacological activity of non-steroidal droxylated metabolite of tamoxifen with potent antioestrogenic activity. J. antiestrogens in vitro. Mol. Pharmacol., 26: 272-278, 1984. Endocrinol., 75: 305-316, 1977. 153. Jordan, V. C., and Lieberman, M. E. Estrogen-stimulated prolactin synthe sis /'/; vitro: classification of agonist, partial agonist and antagonist actions 126. Lein, E. A., Solheim, E., Kvinnsland, S., and Veland, P. M. Identification of 4-hydroxy W-desmethyltamoxifen as a metabolite of tamoxifen in human based on structure. Mol. Pharmacol., 26: 279-285, 1984. bile. Cancer Res., 48: 2304-2308, 1988. 154. Jordan, V. C., Koch, R., Mittal, S., and Schneider, M. R. Oestrogenic and 127. Robinson, S. P., Langan-Fahey, S. M., and Jordan, V. C. Implications of antioestrogenic actions in a series of triphenylbut-1-enes: modulation of tamoxifen metabolism in the athymic mouse for the study of antitumor prolactin synthesis in vitro. Br. J. Pharmacol., 87: 217-220, 1986. effects upon human breast cancer xenografts. Eur. J. Cancer Clin. Oncol., 155. Jordan, V. C., Koch, R., Langan, S., and McCague, R. Ligand interaction 25: 1769-1776, 1989. at the estrogen receptor to program antiestrogen action: a study with 128. Jordan, V. C., Phelps, E., and Lingren, J. U. Effects of antiestrogens on nonsteroidal compounds in vitro. Endocrinology, 122: 1449-1454, 1988. bone in castrated and intact female rats. Breast Cancer Res. Treat., 10: 31- 156. Bignon, E., Pons, M., Crastes de Paulet, A., Dore, J. C., Gilbert, J., 35, 1987. Abecassis, J., Miquel, J. F., Ojasoo, T., and Raynaud, J. P. Effect of 129. Turner, R. T., Wakely, G. K., Hannon, K. S., and Bell, N. H. Tamoxifen triphenylacrylonitrile derivatives on estradiol-receptor binding and on hu prevents the skeletal effects of ovarian deficiency in rats. J. Bone Miner. man breast cancer cell growth. J. Med. Chem., 32: 2092-2103, 1989. Res., 2:449-456, 1987. 157. Murphy, C. S., and Jordan, V. C. Structural components necessary for the 130. Love, R. R., Mazess, R. B., Tormey, D. C., Barden, H. S., Newcomb, P. antiestrogenic activity of tamoxifen. J. Steroid Biochem., 34: 407-411, A., and Jordan, V. C. Bone mineral density in women with breast cancer 1989. treated for at least two years with tamoxifen. Breast Cancer Res. Treat., 12: 158. Jordan, V. C. Biochemical pharmacology of antiestrogen action. Pharmacol. 297-301, 1988. Rev., 36: 245-276, 1984. 4188

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1990 American Association for Cancer Research. ANTIESTROGENS AND BREAST CANCER 159. Wakeling, A. E., and Bowler, J. Steroidal pure antioestrogens. J. Endocri- Cancer Clin. Oncol., 25:493-497, 1989. noi //2-R7-R10 1987 '63. Jordan, V. C. Chemosuppression of breast cancer with tamoxifen: labora- 160. Cormier, E. M., and Jordan, V. C. Contrasting ability of antiestrogens to toI7 evidence and future clinical investigations. Cancer Invest., 6: 5-11, inhibit MCF-7 growth stimulated by estradiol or epidermal growth factor. ÃŒ?â€¢â€¢'i , â€¢ - - â€¢<â€¢, , , , ,-Eur. J. , ^Cancer Clin.?-,â€¢r. Oncol., i 25: ir 57-63,t--, f-, ,oon1989. 164.Â¿.282 Cuzik, J., 1985 and Baum, M. Tamoxifen and contralateral breast cancer. Lancet, 161. Jordan, V. C., and Koch, R. Regulation of prolactin synthesis in vitro by ,65 Powle's T Â¿ Hardy j R Ash,ey s E Farringtoni G. H., Cosgrove, D., estrogenic and antiestrogemc derivatives of estradici and estrone. Endocri- Davey, J. B., Dowsett, M., McKinna, J. A., Wash, A. G., Sinnett, H. p., nology, 124: 1717-1726, 1989. Tillyer, C. R., and Treleaven, J. G. A pilot trial to evaluate the acute toxicity 162. Robinson, S. P., and Jordan, V. C. The paracrine stimulation of MCF-7 and feasibility of tamoxifen for prevention of breast cancer. Br. J. Cancer, cells by MDA-MB-231 cells: possible role in antiestrogen failure. Eur. J. 60: 126-131, 1989.

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Leonard J. Lerner and V. Craig Jordan

Cancer Res 1990;50:4177-4189.

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