Development of Antiestrogens and Their Use in...

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

Special Lecture

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

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

Abstract

This paper describes the laboratory discovery and clinical testing ofthe first nonsteroidal antiestrogen, MER-25 (ethantoxytriphetol). Thecompound blocks estrogen action in all species tested and has only slightbut transient estrogenic effects. No other antisteroidal actions are noted.MER-25 is antiestrogenic in primates and was investigated in the clinicsin a wide range of gynecological conditions, including breast and endo-metrial cancer. Unfortunately toxic side effects (hallucinations, etc.)precluded further investigation. A derivative of triphenylethylene, clom-iphene, has some partial agonist (estrogen-like) actions in laboratoryanimals and following clinical evaluation is now an established agent forthe induction of ovulation in subfertile women. Although clomiphene isactive in advanced breast cancer, it was not developed further. In the late1960s a related compound, tamoxifen, was evaluated to treat a numberof estrogen-responsive disorders but was successfully introduced in the1970s for the treatment of advanced breast cancer. Although there wasonly modest initial interest in the palliative use of tamoxifen, an enormousincrease in basic and applied studies with antiestrogens resulted in adefinition of the target site-specific and tumoristatic actions of tamoxifen.Close cooperation between laboratory and clinical evaluation has guidedthe subsequent development of tamoxifen which is now available to treatall stages of breast cancer. Long-term adjuvant tamoxifen therapy, aconcept developed in the laboratory, is currently the treatment strategyof choice. The considerable success of tamoxifen has focused attentionon new antiestrogens with different pharmacological properties for otherpotential clinical applications.

Introduction

Estrogen is involved in a large number of physiological phenomena throughout the body. The mechanism by which thishormone produces its effects is complex and may not be universal for all of the activities reported to be estrogenic. Estrogenis also involved in some pathologies. Here too, its role has notbeen completely defined. The removal of estrogen or interference with its action is helpful in elucidating the role of estrogenand the mechanism of its activity in both physiological andpathological processes.

Perhaps the earliest association of reproductive tissue andbehavioral responses to a hormonal secreting organ was thecessation of estrus in domesticated animals following ovariec-tomy and the diminution of aggressive behavior in male animalsand humans after castration. Indeed, almost 100 years ago therewas recognition that the ovary was involved in mammary carcinoma and that removal of that endocrine gland could bebeneficial to the patient (1).

Ablation of other endocrine organs including adrenalectomy(2) and hypophysectomy (3) were also found to be beneficial insome breast cancers. Surgical removal of these glands or theovaries, however, did more than decrease or eliminate endogenous estrogen from the body. A number of hormones andnonhormonal substances were removed, some of which were

Received 4/5/90.1Presented at the 80th Annual Meeting of the American Association for

Cancer Research, May 24, 1989, San Francisco, CA.

not involved in the pathology. However, it is reasonable toexpect a complex tissue such as the mammary gland, whichrequires a number of hormones and probably growth factorsfor its normal growth and function, to have the involvement ofmore than one hormone in its pathology.

Certainly there are a number of possible ways of interferingwith estrogen interaction with its target tissue other than byremoving the steroid-secreting glands. Reduction of pituitarygonadotropin secretion by sex steroids or by blocking the bio-conversion of androgen to estrogen are ways of decreasing theendogenous pool of estrogens. Biologically available estrogencan also be reduced by increased catabolism or altering theplasma level of estrogen-binding protein. Another way to interfere with estrogen activity is to block it at the level of itsreceptor in estrogen target tissues.

Development of Nonsteroidal Estrogens

The most common of the wide spectrum of biological activities associated with estrogens, regardless of chemical classification or source, is the uterotrophic response and the cornifi-cation of vaginal epithelium. These activities have been incorporated into the bioassays that have defined every natural andsynthetic compound designated as an estrogen. Developmentof a sensitive bioassay in rodents by Allen and Doisy (4) enabledcharacterization and classification according to potency of thenatural steroidal estrogens and nonsteroidal plant estrogens.This formed the basis to identify the structural requirementsfor activity, as well as potency.

Cook et al. (5) found that the phenanthrene nucleus was notnecessary for estrogenic activity. The laboratory of Dodds andhis collaborators therefore synthesized a large number of di-phenolic and triphenolic compounds resulting in the development of potent, clinically important estrogenic substances suchas diethylstilbestrol (6), hexestrol, and dienestrol (7). At thissame time in the mid-1930s, Robson and Schonberg reported

that triphenylethylene had estrogenic activity (8) and that oralpotency and prolongation of activity could be achieved bybromination (9). This finding led, in the 1940s, to the synthesisof a number of triphenylethylene derivatives including morepotent halogenated compounds by the William S. Merrell Co.in the United States (10, 11) and by Imperial Chemical Industries in the United Kingdom.

While all of the compounds called estrogens increase uterineweight and stratify and cornify the vaginal epithelium, thespectrum of other biological activities associated with estradiolmay or may not be present in all of these estrogens. Moreover,the quantitative aspect of one of the activities may not be relatedto other activities of the same compound.

Knowledge of structure-activity relationships allows for therational design of chemical agents with greater specificity for aparticular pharmacological action.

Some of the physiological interactions of the steroidal hor-

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ANTIESTROGENS AND BREAST CANCER

mones were known in the first half of the 20th century. Theseinteractions could be additive, augmentative, synergistic, orantagonistic depending upon the particular steroids, dosage ofeach, order and time course of administration, as well as theend point and species of animal under study. A small dose ofan estrogen administered to an immature rabbit prior to treatment with progesterone allows for maximal growth and development of the uterus.

It is now known that this effect is a result of increasedestrogen-induced uterine hyperemia and induction of progesterone receptors. Large doses of the estrogen, however, canreduce the subsequent effect of the progestational agent in therabbit.

Estrogen Antagonism

Treatment of an immature or ovariectomized rodent with aprogestogen, androgen or corticoid can antagonize the utero-trophic effect of estrogens (12-16). Antagonism, however, isclosely linked to the relative and absolute dose of the agonistand the antagonist.

The first estrogens that demonstrated antagonism of thestimulation of another estrogen were estriol (17) and 16-epies-triol (18). These weak estrogenic steroids partially inhibited theuterotrophic effect of estradiol in the rat. This antiestrogenicactivity was manifested only over a narrow range of doses (19),and at lower doses the two estrogens induced additive uterinegrowth responses (20). Moreover, for the end point of vaginalcornification in rats and mice, estriol was always additive withestradiol (21).

The nonsteroidal synthetic estrogens and their structurallyrelated weak or inactive relatives were an attractive source inthe search for modulators or inhibitors of estrogenic activity.Several weakly estrogenic stilbene and triphenylethylene compounds demonstrated partial antagonism of estrone- or estra-diol-induced uterotrophic activity in mice in my laboratory (L.J. L.). At a presentation of data (22) on the antiestrogenicactivity of one of the diphenolic compounds Dr. Roy Hertzfrom the NIH remarked that the findings were interesting buta clinically useful antiestrogen would have to be much morepotent.

Discovery and Development of Ethamoxytriphetol (MER-25)

In 1954, a compound related to triphenylethylene and chlo-

rotrianisene was synthesized at Merrell to study its possiblecardiovascular effects, but it was found to be inactive. Thestructural similarities ofthat compound to those of the triphen-ylethylenes that were being studied for antiestrogenic activityin my laboratory (L. J. L.) prompted us to test its endocrineactivity.

That compound, l-/?-2-(diethylamino)ethoxyphenyl-2-(/>-methoxyphenyl)-l-phenylethanol was later given the genericname of ethamoxytriphetol and the reference designation ofMER-25 (Fig. 1). The initial dose of MER-25 was selected onthe basis of experience that showed that even the most activeof the compounds tested up to that time were of low potencyand only partially inhibited the uterotrophic effect of estroneor estradiol.

Weanling mice were given s.c. injections of MER-25 in oliveoil for 3 days at a daily dose of 1.7 mg (170 mg/kg). One-halfof the animals were also given daily injections of estradiolbenzoate (0.1 ^g/animal). The results of this experiment weresurprising since the uterine weights of the mice that had been

ETHAMOXYTRIPHETOL (MER 25)

N1TROMIPHENE (CN-55, 945) NAFOXIDINE(U-11,100A)

TAMOXIFEN (ICI 46, 474)

Fig. 1. Structures of MER-25 and antiestrogens derived from triphenylethylene.

given MER-25 with or without estradiol benzoate were identicalto those of animals given injections of the olive oil alone. Inaddition, uterine vascularity and intraluminal fluid, which alsoincreases with estrogen treatment, were absent in the MER-25-treated groups. Moreover, body weight remained normal. Thestudy was repeated several times and the complete blockade ofthe estrogen-induced uterotrophic response was confirmed.

The inhibition of estradiol action in the mouse uterus byMER-25 was dose related and simultaneous administration ofgraded doses of estradiol was able to prevent the antiestrogeniceffects of MER-25 in a dose-related manner (Fig. 2). This studydemonstrated that MER-25 is a competitive antagonist of es

trogen action.The nature of the competitive mechanism of this and deriv

ative antiestrogens was elucidated when Jensen et al. (23, 24)found that they prevented binding of labeled estradiol to the ratuterus. Numerous studies in vivo and in vitro have confirmedthat the nonsteroidal antiestrogens act at the level of the estrogen receptor. However, the exact nature of the mechanism(s) isstill incompletely understood. Moreover, other activities havebeen documented for this class of pharmacological agents.These include inhibition of estrogen-induced protein synthesis,thymidine incorporation, DNA synthesis, cell cycle blockage atthe GÃ¬phase, inhibition of calmodulin, and non-estrogen-re

lated events.Not all of these activities, however, have been found univer

sally with all antiestrogens and results from studies in vivo donot necessarily support findings in vitro. Some of these effectsmay be a result of the relative estrogenic to antiestrogenicactivities of the compound studied or to the biological half-lifeof each activity. A comparison of an estrogenic antiestrogen

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50

~. 40O)

Fig. 2. Uterotrophic and antiestrogenic activities of MER-25 in immature mice. Groups ofanimals were treated for 3 days with (A) estradicibenzoate (0.3 ng) and MER-25 (O) or MER-25alone (A) or (B) different doses of estradicibenzoate with (d) or without (â€¢)5 mg MER-25.All doses are 3-day total dosages. Data in parentheses, number of animals exhibiting full (/"),

partial (P), or no (AOuterine intraluminal fluid.

30

20

Estradiol0.3 Mg

33 (16F,3P,4N)

l

'Control

n (25N) (1P.33N)

I(38N)

(9N,1P,1F)

rControl

(11F)

(11F)

(1N,3P,7F)

(1N.7P.2F)

^/>4N.4P.3F)

0.2 1.0 5.0 0.03 0.3 30.0 3000

TOTAL DOSE MER-25 (mg) TOTAL DOSE ESTRADIOL <M9>

(partial agonist) with a nonestrogenic antiestrogen (pure antagonist) for each of the activities may help resolve this problem.

The antiuterotrophic effect of MER-25 was also demonstrated in immature, ovariectomized, and adult rats (25-30). Inthe immature rat, a MER-25:estradiol benzoate ratio of 400:1reduced the Uterotrophic effect of the estrogen by approximately45% and complete suppression of the estrogen activity wasobtained with a ratio of 10,000:1 (Fig. 3). Uterine as well asoviductal growth stimulated by estradiol was inhibited in adose-related fashion as were a number of biochemical parameters elevated by estrogen. These included nucleic acids, phos-pholipids, glucose-6-phosphate dehydrogenase and other enzyme activities, nitrogen, and water.

These initial studies prompted my colleagues and I (L. J. L.)to investigate the pharmacological spectrum of activities ofMER-25 in different species in various stages of maturity. Ourstudies evaluated the antihormonal and antifertility propertiesof MER-25 in acute and long-term studies (15, 26). Each studybrought new information but also raised new questions. MER-25 was an antiuterotrophic agent regardless of the route ofadministration or the estrogen used as the agonist, but it hadlittle or no activity as an antagonist of androgen or progesterone-induced Uterotrophic activity.

140

Total Dose of MER-25 - mg

Fig. 3. Uterotrophic and antiestrogenic activities of MER-25 in ovariectomized immature rats. Groups of animals were treated for 4 days with a total doseof 2.0 fig estradiol benzoate ( ), MER-25 <&\,estradiol benzoate plus MER-25 (â€¢)or injection vehicle (sesame oil) alone ( ). Animals were killed on day5 and blotted uteri were weighed wet.

MER-25 was also a complete inhibitor of the effect of endogenous estrogen or administered estrogen at the level of thevagina. Cornification of vaginal epithelium by estrogen wasblocked and MER-25 did not induce any cornification whenadministered to immature or ovariectomized mice or rats, demonstrating a lack of inherent estrogenicity.

Further Studies with MER-25

There was some evidence that this compound did posses veryweak and short-lived estrogen activity at other end points. Atrelatively low doses, it induced a weak Uterotrophic effect which,many times, was only borderline in significance. When a singledose of this agent was administered to immature mice, a slightincrease in uterine weight was noted 48 h after treatment butwas not seen on subsequent days (26). Those doses that wereweakly Uterotrophic also elevated uterine alkaline phosphatase(27).

In immature rats, lower doses induced minimal increases inuterine weight (28). Low doses of MER-25 induced a shortlived increase in glucose-6-phosphate dehydrogenase, glucoseisomerase, and lipids (30). In the immature rabbit pretreatedwith estrogen to allow for progesterone-induced endometrialdevelopment, MER-25 effectively inhibited the estrogen activity(presumed induction of progesterone receptors), thereby blunting the effect of the progesterone (26). MER-25, however, whenadministered instead of estradiol, permitted a weak uterinegrowth response to subsequent progesterone treatment. Otherindications of weak estrogenic activity were the acceleration ofvaginal opening in prepuberal rats and mucification of the adultrat vagina (26, 27). Offspring of rats treated with MER-25during the last third of gestation demonstrated early openingof their vaginas (27).

MER-25 has only weak antigonadotropin activity, but iteffectively blocks estrogen- or castration-induced pituitary hypertrophy and gonadotroph degranulation (26). It was found tobe devoid of any other hormonal activity.

Mouse uteri stimulated by testosterone were unaffected bysimultaneous treatment with this estrogen antagonist (26, 27).High doses of MER-25, however, partially inhibited androgen-stimulated uterine weight increase and biochemical changes inthe immature rat (29). Immature castrated male rats giveninjections of MER-25 and testosterone propionate demonstrated no antagonism of the androgen-induced changes in theseminal vesicles although there was a partial inhibition of the

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augmented weight and nucleic acids of the ventral prostate (32).Antiestrogenic activity was also found in the chick. MER-25

inhibited estrogen-induced oviductal growth and elevatedplasma phospholipids (26). MER-25 did not alter these sameestrogen target parameters at any dose used, indicating a lackof estrogenicity in this species. Tamoxifen and other triphen-ylethylene antiestrogens (33, 34) were later also found to bedevoid of estrogen activity in the chick oviduct. This is confirmatory evidence for a species-related hormonal profile for thischemical class of compounds.

MER-25, if it were to have clinical potential, had to haveantiestrogenic activity in a primate. Ovariectomized Macacamulatta monkeys, maintained on estradici, were periodicallygiven varying p.o. doses of MER-25. The antagonist induced asharp reduction in the cornified vaginal cells of these estrogen-ized animals and at higher doses the decrease in cornified cellswas to the castration level. Accompanying this was a lesseningof the sex skin swelling and reddening and eventually estrogenwithdrawal bleeding (26). Other studies in intact cycling monkeys demonstrated that endogenous estrogen was inhibited byantagonism or via reduced gonadotropin secretion since themenstrual cycles were lengthened and the percentage of cornified cells in the vaginal smear decreased.

These studies paralleled those in intact rats where a daily p.o.dose of 1 mg lengthened the estrous cycle and 5 mg/day stoppedcycling (26). The rats resumed normal cycling of 2-6 daysfollowing the last dose.

This prototype antiestrogen was further investigated in orderto determine its pharmacological properties especially in relation to those biological phenomena where endogenous estrogenor gonadotropins had been shown to be involved.

Effect of Antiestrogens on Reproduction

The reproductive system and all of its accessory tissues require a changing balance of hormones for their developmentand function. Changes in the absolute and relative quantities ofthese hormones may lead to functional disorders and pathology.Treatments that increase or decrease the available pool of theparticular hormone or alter its activity may correct the problem.Similarly a physiological process such as ovulation can beinterrupted by addition of hormones.

It was interesting to explore the actions of MER-25, anantiestrogen having weak gonadotropin-inhibitory and veryweak selective estrogenic activities, on reproduction. The compound was administered to female rats at various times priorto or after mating (26, 27). None of the pretreated animalsmated, since none of the animals exhibited estrus behavior orshowed vaginal cornification. Treatments that were initiatedafter mating on day 1 of pregnancy probably inhibited nidationor altered ova transport since no fetuses or products of conception were found. Administration of the compound on days 4-7postmating prevented blastocyst implantation. When treatmentwas delayed until the 13th day of pregnancy, gestation continued and live pups were delivered, but parturition was delayedand was difficult with several of the young dying probably dueto the effects of estrogen deprivation on uterine contraction.

The postcoital antifertility effect of MER-25 was confirmedby other investigators (35, 36) and similar results were obtainedfor other triphenylethylene antiestrogens including clomipheneand MRL-37 in my laboratories (L. J. L.) and by other investigators (37, 38). None of the antiestrogenic agents were sufficiently efficacious in primates, although ovum implantation inthe monkey uterus was inhibited by several (39) and at least

one such agent was developed for postcoital antifertility utility(40), but it was never marketed.

MER-25 was also shown to augment pregnancy maintenanceby progesterone in Ovariectomized rats similar to that obtainedwith estrone (27). However, in rats fed a protein-free diet whereestrone helps maintain pregnancy, the administration of MER-25 negated the supportive effect of the estrogen (27).

The activities of MER-25 were expressed relative to thehormonal and developmental state of the animal. In adultcycling rats, this compound decreased the weights and functionof the ovaries, pituitary, and uterus. It also antagonized theeffect of estrogen on the latter two organs (26). Administrationof MER-25 to pubertal rats, however, produced different results. Ovarian weights, nucleic acids, and enzyme activities wereelevated after 4 days of treatment (41). The increases in theovaries were similar to that obtained by treatment with humanchorionic gonadotropin (41). Estradici treatment at the sametime prevented the MER-25-induced augmentation of ovarianweight and biochemical activities.

The increase in ovarian activity following MER-25 treatmentof the pubertal rat may parallel the ovarian stimulation inwomen after administration of antiestrogens including MER-25 and clomiphene citrate.

Utility of Antiestrogens for Ovulation Induction

In the 1950s Dr. James H. Leathern used the human chorionic gonadotropin-treated hypothyroid rat as a model forpolycystic ovaries. His work stimulated us to study the effectof MER-25 in this model. The antiestrogen blocked the uterinehypertrophy induced by the hyperactive ovary and reducedovarian weight; however, cystic follicles were still evident. Thesestudies were repeated and extended by Leathern and his graduate students. MER-25 prevented the induction of cystic ovariesand alteration in ovarian ascorbic acid concentration (42). Furthermore, pretreatment with MER-25 prior to induction of thehypothyroid state followed by gonadotropin administration wasalso effective in blocking cystic development (43).

A preliminary study in nymphomaniac cows bearing cysticovaries was performed by W. Hansel and R. M. Melampy usinga low use of MER-25 that I (L. J. L.) had suggested. Thesecows promptly resumed normal behavior; however, no measurement of ovarian follicular size or ovulation was recorded.

These findings in animals encouraged us to initiate exploratory studies on selected patients to determine the effects ofMER-25 on menstrual or ovarian disorders. Kistner and Smith(44) reported that patients treated for endometrial hyperplasiaand carcinoma with MER-25 demonstrated higher urinary excretion of estrogen and follicle-stimulating hormone and hadtemperature changes indicative of ovulation. These investigators also induced ovulation with this compound in patients withStein-Leventhal syndrome (45). At this same time Tyler et al.(46) reported that 6 of 18 patients with severe anovulatoryproblems ovulated within 7 days of starting MER-25 therapy.

This antiestrogen, however, induced undesirable and unexpected central nervous system symptoms leading to cessationof its use. Fortunately my laboratory (L. J. L.) had been developing, at the same time, another related but more potentantiestrogen that had some qualitative differences from MER-25. That compound, MRL-41 or clomiphene citrate (47) (originally called chloramiphene) (Fig. 1), was, in comparison toMER-25, weakly estrogenic (25). Clomiphene was provided toR. W. Kistner, E. T. Tyler, and R. B. Greenblatt to study itsability to induce ovulation. Shortly thereafter Greenblatt et al.

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(48) reported that this compound successfully induced ovulationin women and similar successes were reported by the otherinvestigators. Ovulation was induced with doses that weresmaller than those used for MER-25 and no central nervoussystem disturbances were found.

These studies ushered in a new mode for treating anovulatoryand infertile patients having functional ovaries which has hadwidespread use in in vitro fertilization technology.

Effect of Antiestrogens on Lipids

Estrogens are known to regulate the synthesis and metabolism of lipids. This produces a change in the relative concentration of various lipid fractions in the plasma and disposition intissues. Indeed endogenous estrogen is a normal protectiveregulator for the cardiovascular system and the decreased synthesis of these steroids at menopause is associated with anincrease in atherosclerosis and coronary disease. Men are moreprone to these disorders than are premenopausal women. Achemical compound that demonstrates an estrogen-like activityon lipids without affecting the reproductive system would therefore be desirable. Another possible partitioning of the biologicalspectrum of activities of estrogen might result from the administration of an estrogen such as estradiol and of a "nonestro-genic" antiestrogen.

An experiment in 1-week-old chicks showed that estradiol

benzoate could increase oviductal weight by 1500% while doubling the plasma phospholipid concentration. MER-25 administration did not alter either end point even at doses 300 timeslarger than that of the estrogen. When both substances wereadministered to these birds the estrogenic effect on the plasmaphospholipids as well as on the oviduct were blocked (26).

Even though MER-25 failed to show an estrogen-like effecton the chick plasma phospholipids, this antiestrogen and anumber of other compounds having different spectra of estrogenic and/or antiestrogenic activities were investigated in therat. MER-25, clomiphene citrate, and a related compound,triparanol, were hypocholesterolemic (41) with the latter compound being 5 times more potent than the other two. Studiesin vitro with rat liver homogenates demonstrated that MER-25and clomiphene inhibited the conversion of mevalonate tocholesterol whereas triparanol produced its major blockingeffect after mevalonic acid (41, 49).

The nonestrogenic weak antiestrogen 2,2'"-(l-methyl-4,4-diphenylbutylidene)bis(/>-phenyleneoxy)bistriethylamine oxa-late lowered plasma cholesterol in rats fed normal and hyper-lipemic diets by blocking cholesterol biosynthesis between mevalonate and lanosterol (49). It also reduced serum free fattyacids and triglycÃ©rides.

It may be interesting to investigate the relationship of cholesterol biosynthesis inhibition and anticancer potential of theantiestrogens in light of present knowledge concerning thecatalogue of factors required for cellular growth, development,regulation, and multiplication.

Demonstration of the Efficacy of an Antiestrogen in BreastCancer

Laboratory studies with MER-25, clomiphene, and manyother related compounds suggested a variety of potential clinicalutilities based on the particular pharmacological spectrum ofeach compound. In mid-1956 a list of some of these utilitieswas suggested for evaluation with MER-25. These included,"1) precocious development, 2) anti-fertility, 3) habitual abor

tion, 4) fertility in the female, 5) dysmenorrhea, 6) all kinds ofmenstrual disturbances including menorrhagia or prolongedmenstrual periods, 7) menopause, 8) endometriosis, 9) acne,10) cancerâ€”a) to test for or substitute for adrenalectomy incancer, b) as a substitute for ovariectomy, c) mammary carcinoma, d) prostatic cancerâ€”alone or in combination with anestrogen, in an attempt to maintain the effects of estrogen onthe prostate and avoid side reactions of estrogens, 11) benignprostatic hypertrophy, 12) postpartum breast engorgement, and13) precipitate the menopause when desirable."

While that list was compiled largely on the basis of laboratorydata on MER-25 presented by L. J. L., it is interesting that theanticancer indications were not derived from animal tumormodels since such models were not available. The stimulationfor such evaluation in human cancers came largely from theavailability of a unique compound that could block the effect ofestrogen completely and also have weak gonadotropin inhibition activity.

Several well known competent clinical investigators weresuggested for each of the areas. The two who were asked toinvestigate MER-25 in breast disorders including cancers wereDr. Roy Hertz at the NIH and Dr. Robert Kistner at HarvardUniversity. The dosages suggested were derived from the laboratory studies in animals including those performed in themonkey. Dr. Kistner also investigated other diseases of thebreast, uterus, and ovaries.

Both investigators confirmed antiestrogenic activity in theirpatients. Kistner and Smith (44) reported that patients withchronic cystic mastitis not only had pain relief but also hadreduced glandular proliferation and breast size. He also foundthat two of four patients with breast carcinomas experiencedpain relief and a reduction of calcium excretion while on MER-25 therapy. Hertz also treated several patients with metastaticbreast cancer and obtained similar favorable results. Thesestudies demonstrated the utility of an antiestrogen in breastcancer.

Unfortunately the investigation of MER-25 as a therapeuticagent had to be discontinued because Drs. Hertz, Kistner, andTyler reported that their patients were experiencing hallucinations, psychotic episodes, nightmares, disturbed sleep, or otherindications of undesirable central nervous system activity.

Clomiphene which was the next most advanced in preclinicalinvestigation or another of the structurally related antiestrogenscould have been evaluated for anticancer activity in the early1950s. However, the clinical evaluation of these compounds forthose indications was never undertaken due to the concentrationof effort on the use of clomiphene in induction of ovulation inanovulatory infertile women. In fact clomiphene citrate waslater shown to have efficacy in breast cancer in a few patientsin 1964(50).

Resumption of investigation of similar antiestrogens forbreast cancer had to wait for more than a decade. Chemists andbiologists in the United Kingdom reexplored the triphenylethy-lenes for antiestrogenic and antifertility activity and eventuallyfor antitumor activity. The clinical efficacy of one of thesecompounds, tamoxifen, established antiestrogens as the standard endocrine treatment in breast cancer.

Development of Tamoxifen

Tamoxifen (ICI 46,474; Nolvadex) (Fig. 1) is a nonsteroidalantiestrogen with antifertility properties in laboratory animals(51-54). The compound exhibits an interesting species-specificpharmacology (55); it is a pure antiestrogen in chicks (33) and

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an antiestrogen with partial estrogenic activity in rats (52) but,in short-term tests, it is classified as an estrogen in mice (51,52, 56). However, it appears that the prolonged administrationof tamoxifen to ovariectomized mice causes the uterus andvagina to become refractory to the stimulatory effects of estradici (37, 57, 58).

Tamoxifen is a competitive inhibitor of estradici binding toestrogen receptors (59, 60) and the compound inhibits thebinding of estrogen to estrogen target tissues in vivo (57, 61-64).

Antiestrogens can inhibit estrogen-regulated events in cellsin culture; e.g., tamoxifen slows the growth of MCF-7 breastcancer cells [in phenol red containing media (65, 66)]; however,the addition of increasing concentrations of estradiol will reverse the effects of tamoxifen and the cells will continue togrow ("estrogen rescue") (67). Nevertheless, high concentra

tions of antiestrogens will cause estrogen irreversible actions(68, 69). An "antiestrogen binding site" has been described (70)

but its relevance to the cytocidal actions of tamoxifen is notclear. It is clear, however, that the high affinity of tamoxifen toa variety of proteins causes ubiquitous binding of the drug invivo and probably accounts for its long biological half-life

Current research is focused on the action of growth factorsto regulate the breast cancer cell cycle. Estrogens decrease cellcycle time and tamoxifen causes a reversible blockade at the G,phase (71, 72). Antiestrogens inhibit estrogen-stimulated increases in transforming growth factor a, a stimulatory factor(73), and there is a complementary rise in transforming growthfactor ÃŸ,an inhibitory growth factor (74). Although the actualregulatory mechanisms that orchestrate the inhibitory and stimulatory actions of growth factors are unclear, this is an area ofintense investigation.

The knowledge that tamoxifen is an inhibitor of estrogenaction focused attempts to develop an appropriate clinical application. Tamoxifen was initially evaluated in a number ofendocrine-responsive conditions (75-80) and the drug subse

quently became available in some countries for the induction ofovulation in subfertile women. However, the discovery that thepresence of the estrogen receptor may predict the hormoneresponsivness of advanced breast cancer (81) naturally led tothe evaluation of tamoxifen to control tumor growth [tamoxifeninhibits the binding of estrogen to estrogen receptors derivedfrom breast tumors (82)]. Much of the credit for steering theuse of tamoxifen toward the treatment of advanced breastcancer must go to the late Dr. Arthur L. Walpole of ICI,Pharmaceuticals Division, Macclesfield, United Kingdom. Earlier in his career Dr. Walpole had been particularly interestedin carcinogenesis and cancer therapy but in the 1960s hisattention was directed, as head of the fertility control program,to the development of antifertility agents (83). Much of theearly preclinical antitumor work was subsequently conductedin my (V. C. J.) laboratory funded through the good offices ofArthur Walpole at ICI.

Antitumor Actions of Tamoxifen in Laboratory Models

The first systematic laboratory study of the antitumor actionof tamoxifen was conducted at the Worcester Foundation forExperimental Biology in Shrewsbury, MA (82, 84-88). One ofthe results of this research (89, 90) was to encourage the clinicalevaluation of tamoxifen in the United States by the EasternCooperative Oncology Group.

Most of the early research on tamoxifen used carcinogen-induced rat mammary tumor models (85-90) but during the

past half-decade interest has focused on an evaluation of theantitumor action of tamoxifen in athymic mice, hetero-transplanted with breast cancer cell lines (91-95).

Carcinogen-induced Models. Mammary tumors can be induced in 50-day-old Sprague-Dawley rats by a single feeding(20 mg in 2 ml peanut oil) of DMBA. Tamoxifen will inhibitthe initiation (85, 87) and growth (61, 88, 96-98) of DMBA2-

induced tumors. The model system could be likened to thetreatment of advanced breast cancer but the tumors do notmetastasize and therefore a direct analogy with breast cancer isimpossible. This fact is important inasmuch as the fashion forthe treatment of breast cancer was changing in the mid-1970s.A strategy was devised to use adjuvant hormonochemotherapyto destroy micrometastases following mastectomy, a treatmentaimed at curing a majority of patients. Unfortunately no animalmodel system was, or is, available to duplicate the clinicalsituation to test the value of this treatment strategy in thelaboratory. Nevertheless the DMBA- and yV-methylnitrosourea-induced rat mammary carcinoma models were adapted to evaluate whether antiestrogens could "cure" animals with a micro

scope tumor burden.When DMBA is administered to Sprague-Dawley rats, the

initiated mammary cells are promoted by circulating hormonesto effect transformation. Palpable tumors appear 100-150 daysafter DMBA. There is therefore a period when microscopicdisease can be treated to establish whether it is possible to curethe animals with antiestrogens. If different doses are administered for 4 weeks starting at different times after DMBA (99,100), a delay occurs in the appearance of tumors. In contrastcontinuous therapy causes the majority of animals to remaintumor free (101-103). Nevertheless of tamoxifen therapy isstopped, tumor growth recurs (104).

A similar situation occurs with the carcinogen jV-methylni-trosourea; a short course of tamoxifen delays the appearanceof tumors but the animals remain tumor free during a continuous treatment regimen (105, 106).

Athymic Mouse Models. Breast cancer cell lines can be het-erotransplanted into athymic immunodeficient mice. Estradiolencourages the growth of hormone-responsive lines (e.g., MCF-7) but hormone nonresponsive lines (e.g., MDA-MB-231) willgrow with or without estrogen treatment. Tamoxifen and itsmetabolites inhibit estrogen-stimulated growth of estrogen receptor-positive tumors but in general do not inhibit the growthof receptor-negative tumors (91, 92). However, this model hasbeen used successfully to determine whether tamoxifen produces a tumoristatic or tumoricidal effect on implanted hormone-responsive breast cancer cells. If animals are treated for1, 2, or 6 months with sustained release preparations of tamoxifen the tumor cells are not specifically destroyed. When theanimals are treated with estradiol to identify surviving tumorcells, tumors regrow in every case (92, 93).

Overall the experimental evidence points to the fact thattamoxifen is a tumoristatic rather than a tumoricidal agent.Long-term adjuvant tamoxifen treatment, or treatment untilrelapse, is predicted to be the best therapeutic strategy to controlthe recurrence of breast cancer following mastectomy. Thisapplication of tamoxifen can be called chemosuppression.

Long-Term Adjuvant Tamoxifen Therapy

Despite the developing laboratory data that demonstratedthat tamoxifen is a tumoristatic agent (99-102), the vast ma-

2The abbreviation used is: DMBA, dimethylbenzanthracene.

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jority of clinical trials initially evaluated 1 or 2 years of adjuvanttamoxifen therapy. There were several sound reasons for this:(a) a short course of tamoxifen might produce a tumoricidaleffect before the onset of resistance since it was known thatpatients with advanced disease respond to tamoxifen for only1-2 years; (b) the adjuvant use of tamoxifen might encouragethe early outgrowth of receptor-negative disease (the rationalewas that it would be wiser to save the drug until recurrence sothat physicians would have something available to treat thepatient); and (c) the long-term side effects of tamoxifen wereunknown (thousands of women with advanced disease had beentreated, but only until relapse, 1-2 years later).

Although tamoxifen was initially used conservatively therecent overview of all randomized clinical trials (107) hasdemonstrated the survival advantage of postmenopausal, node-positive women who received 1 or 2 years of adjuvant tamoxifentherapy. Swayed by the encouraging clinical trial results andthe laboratory findings (101, 107) clinical trials are currentlyfocused on an evaluation of 5, 10, or in some cases indefiniteadjuvant tamoxifen therapy in pre- and postmenopausal patients with node-positive and node-negative disease.

In 1977, on the basis of encouraging laboratory data (101),Dr. Douglass C. Tormey organized a pilot nonrandomizedadjuvant clinical study to identify any unusual side effectsproduced by long-term adjuvant tamoxifen therapy (5 years)and to evaluate the potential efficacy of the treatment strategy(108). The results demonstrated the safety of tamoxifen andprovided promising preliminary data to establish the EasternCooperative Oncology Group protocols EST 4181 and EST5181 to evaluate chemotherapy and different lengths of tamoxifen therapy in randomized clinical trials (109).

The National Surgical Adjuvant Breast and Bowel Projecthas chosen a similar course of action. Based upon their successful evaluation of 2 years of adjuvant tamoxifen and chemotherapy (110) they conducted a registration study of chemotherapy and tamoxifen for 2 years followed by an additionalyear of tamoxifen for those patients who did not have recurrence. The results are encouraging and support the view thatlong-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 notreatment. Tamoxifen produced a survival advantage especiallyin receptor-positive patients. Similarly a large clinical trial inScotland (113) has evaluated whether it is an advantage to treatpatients for 5 years with adjuvant tamoxifen. This clinical studyis particularly interesting because it addresses the question ofwhether to use tamoxifen as an adjuvant therapy or to use thedrug only when the patient develops advanced disease. In theScottish study, patients in the control arm received tamoxifenat first recurrence. The results demonstrate that long-termadjuvant therapy with tamoxifen provides an effective controlof disease recurrence and a survival advantage for the patient.There appears to be no need to delay the drug until recurrencebecause early drug resistance does not develop.

The National Surgical Adjuvant Breast and Bowel Project iscurrently conducting a trial of long-term adjuvant tamoxifentherapy (10 years) in pre- and postmenopausal patients withnode-negative estrogen receptor-positive disease. The preliminary results are again encouraging (114) inasmuch as tamoxi-fen-treated women have an extended disease-free survival.

Overall the clinical trials community has demonstrated someadvantages for long-term adjuvant tamoxifen therapy. To datethe trial population has been diverse with both pre- and post

menopausal women receiving tamoxifen alone and women withnode-negative disease who might be treated for decades. Thisexpanded and extended use of tamoxifen requires an ongoingevaluation of the pharmacology and potential toxicologicalconsequences of indefinite therapy. Obviously it is importantto establish the safety of tamoxifen to reassure both physiciansand patients and to avoid unwarranted restrictions on thisvaluable chemosuppressive therapy.

Clinical Pharmacology

The clinical pharmacology of tamoxifen was initially investigated in patients with advanced breast cancer (115-118). Thedrug is readily absorbed following p.o. administration andaccumulates to steady-state levels by the end of the first monthof treatment (116). The serum levels of tamoxifen at steadystate are extremely variable (50-300 ng/ml) but blood levelsare not related to response (119). The principal metabolite oftamoxifen is ,/V-desmethyltamoxifen which produces blood levels approximately twice as high as those of tamoxifen by theend of the first month of treatment. The serum half-lives oftamoxifen and W-desmethyltamoxifen are extremely long (7and 14 days, respectively) (120), probably because the compounds are highly protein bound.

Tamoxifen is extensively metabolized in patients (121, 122)(Fig. 4) but to date no significant quantities of estrogenicmetabolites have been noted that would cause concern duringlong-term adjuvant tamoxifen therapy. ,/V-Desmethyltamoxifenis further metabolized to metabolite Y which has a glycol sidechain (123, 124). Tamoxifen is also converted to 4-hydroxyta-moxifen but this is observed only at low concentrations inserum (118). However, this metabolite has an extremely highbinding affinity for the estrogen receptor (the same as that ofestradici) (125); therefore it probably plays a significant role insupporting the antitumor action of tamoxifen. Recently 4-hydroxy-yV-desmethyltamoxifen has been identified in patients(126). This metabolite has been shown in the laboratory to be

N-DESMETHYLTAMOXIFEN 4-HYDROXY N-DESMETHYLTAMOXIFEN

N.N. DIDESMETHYLTAMOXIFEN METABOLITE Y

Fig. 4. Metabolites of tamoxifen identified in patients.

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formed from either 4-hydroxytamoxifen or jV-desmethyltarnox-ifen(127).

The blood levels of tamoxifen and its metabolites are stablefor up to 10 years of adjuvant therapy and no metabolic tolerance seems to occur. This fact and the low incidence of reportedside effects (121) makes tamoxifen an ideal long-term chemo-suppressive agent for the adjuvant treatment of breast cancer.

Potential Side Effects of Long-Term Adjuvant Tamoxifen Therapy

Tamoxifen therapy is being extended beyond a decade. Indeednode-negative patients with only a moderate risk of recurrencemay in fact receive the drug indefinitely. This raises importantquestions about the potential long-term side effects of nonste-roidal antiestrogens (Table 1).

Estrogen is important in prevention of the development ofosteoporosis in women; therefore the long-term administrationof an "antiestrogen" would seem to imply that women may

become predisposed to osteoporosis. Similarly estrogen protects women from coronary heart disease by altering the circulating levels of cholesterol. Again it would seem that the long-term administration of an "antiestrogen" might not be beneficial. However, tamoxifen exhibits some estrogen-like effectsthat appear to have target site specificity (55). Tamoxifen is anantiestrogen in the rat uterus (52) but displays estrogen-likeeffects in bone (128, 129). There are only few clinical data (130,131) to support these beneficial laboratory findings but tamoxifen is clearly not detrimental to bone in patients. The beneficialeffects of nonsteroidal antiestrogens on serum cholesterol hasbeen noted previously in animals (41) and tamoxifen has beenshown to lower serum cholesterol (mainly, low density lipopro-tein) in patients (132, 133).

Obviously the beneficial estrogen-like effects of tamoxifen insome target sites may prove to be a disadvantage in others.Estrogens can cause an increase in thromboembolic disordersand estrogen replacement therapy has been associated with anincrease in endometrial carcinoma. Tamoxifen does not, however, cause a significant increase in thromboembolic disorderswhen administered alone but there is a small decrease in anti-thrombin III during long-term adjuvant tamoxifen therapy(132, 134). The decrease (generally not more than 30%) is,however, not great enough to cause clinical concern. Nevertheless patients with a history of thromboembolic disease probablyshould not be treated with tamoxifen.

There is very little information about the effects of tamoxifenon the human uterus although tamoxifen does have someefficacy for treating endometrial carcinoma (135). Neverthelesstamoxifen has been implicated (anecdotally) in the developmentof endometrial carcinoma (136). Realistically, however, thereis little possibility that tamoxifen could control the growth ofall endometrial tumors as only about 1 of 3 are hormoneresponsive. Interestingly enough there is one provocative laboratory observation (137) that tamoxifen can encourage thegrowth of a human endometrial carcinoma in athymic mice.Indeed this effect appears to be tumor specific (138). If athymicmice are bitransplanted with the endometrial tumor EnCalOland the breast tumor MCF-7 and treated with estrogen andtamoxifen, the endometrial tumor is stimulated to grow whereas

Table 1 Potential side effects of long-term adjuvant tamoxifen therapy

Antiestrogenic actionsEstrogenic actionsReproductive

Osteoporosis, coronary heart diseaseClotting, uterine stimulationOvarian, teratogenesis

the growth of the breast tumor is controlled by tamoxifen. Onelarge clinical trial (139) has shown that a similar phenomenoncan occur during long-term adjuvant tamoxifen therapy; Le,second breast tumors are reduced but there is an increase inendometrial carcinoma. To date these data have not been confirmed by any other major clinical trials organization and it isimportant to state that adjuvant tamoxifen therapy should notbe denied to a patient on the basis of this potential side effect.It should be obvious that tamoxifen is of proven benefit (107)for the treatment of breast cancer, a disease that is invariablyfatal, and endometrial carcinoma, should it occur, has a goodprognosis. Routine gynecological examination must be performed to monitor the health and well-being of the patientsduring adjuvant tamoxifen therapy.

Finally, since more and more premenopausal patients arereceiving adjuvant tamoxifen therapy, it should be stressed thatthey are at risk for pregnancy and require counseling aboutbarrier contraceptives. Short-term tamoxifen treatment causesan increase in ovarian estrogen synthesis (140) and premenopausal patients on long-term adjuvant tamoxifen regimens (withor without chemotherapy) continue to menstruate and haveelevated circulating ovarian steroid hormone levels (141, 142).It is currently not known what the long-term effects of tamoxifen on the ovary will be; this is a topic for future clinicalinvestigation.

Clearly the elevated levels of estrogen, a known tumor mito-gen, might be able to impair the antitumor action of theantiestrogen. Tamoxifen is, however, an effective antitumoragent in premenopausal women with advanced disease (121).This clinical situation has been investigated in the athymicanimal model, and similar high circulating levels of estradici(500-800 pg/ml) do not reverse the antitumor action of tamoxifen (50 ng/ml).3 However, compliance issues with tamoxifenwill be very important in premenopausal patients during long-term adjuvant tamoxifen. Strategies to lower circulating estrogen like oophorectomy or the administration of luteinizinghormone-releasing hormone agonists during adjuvant tamoxifen therapy should be evaluated in future clinical trials.

New Antiestrogens

A study of the structure-activity relationships of tamoxifenhas provided an important insight into the mechanism of actionof both estrogens and antiestrogens. Early work demonstratedthat antiestrogens had a weak interaction with the estrogenreceptor (59, 143) and it was widely believed that this propertywas essential to inhibit hormone action (144); i.e., an estrogenwould bind tightly to the receptor and the resulting complexcould initiate estrogen-dependent events whereas an antiestrogen would bind to the receptor to block the access of estrogenbut the complex would "fall apart" before it could itself initiate

estrogen action. The discovery of the pharmacological properties of monohydroxytamoxifen (4-hydroxytamoxifen) (125,145) changed this view of antiestrogen action. 4-Hydroxyta-moxifen has an affinity for the estrogen receptor similar to thatof estradici but it is a potent inhibitor of estrogen action.Subsequent confirmation of this work (146, 147) and the availability of radiolabeled material for studies in vitro and in vivo(147-150) has established the drug as a standard laboratorybiochemical.

During the 1980s a number of assay systems in vitro wereused to describe the structure-function relationships of anties-

3Y. lino and V. C. Jordan, unpublished observation.

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trogens (151-157). These model systems are important as theyprovide information about the direct effects of ligands on thecell without metabolic transformation. In the future this knowledge will undoubtedly dovetail with current research on themolecular biology of the estrogen receptor to understand thecomplex conformation changes that occur in the receptor protein to program cell replication.

As a result of the successful use of tamoxifen to treat breastcancer, several new antiestrogens are now being evaluated inthe laboratory and the clinic. The structures of several of thenew compounds are shown in Fig. 5. The literature on newantiestrogens is increasing dramatically and interested readersshould consult recent reviews (122, 158).

One notable advance is the investigation of nonestrogenicantiestrogens for potential clinical applications. The discoveryprocess appears to have gone full circle (MER-25 has fewestrogenic properties) to find compounds without the estrogen-like properties of the triphenylethylenes. At present only onecompound (ICI 164,384) is available for evaluation and it hasdemonstrated activity as an inhibitor of estradiol action both invivo and in vitro (159-162). Interestingly the steroid also hasthe ability to inhibit tamoxifen-stimulated tumor growth in thelaboratory (Ref. 94; Fig. 6). Various pure antiestrogens arecurrently being evaluated in the laboratory and some areundergoing toxicology testing. When (or if) any of these newcompounds become available for clinical application, they mayprovide some additional clues about estrogen-regulated breastcancer growth. One question has been whether the antitumoractivity of tamoxifen is related to its estrogenic or antiestrogenicproperties (or neither?) This could soon be resolved. It ispossible that pure antiestrogens could produce longer responsesin advanced disease or be useful as a second line therapy afterlong-term adjuvant tamoxifen treatment. This presupposes thata significant proportion of patients who eventually fail tamoxifen therapy do so because of the estrogen-like properties oftamoxifen. Unfortunately the potential of pure antiestrogens toproduce osteoporosis or early atherosclerosis may precludetheir use as a front line long-term adjuvant therapy in node-

negative disease.

TRIOXIFENE

TOREMIFENE DROLOXIFENE

'"ICH j),0C N(CMjljCH,

ICI 164, 384 CH,

O

111K

DCO

D

Fig. 5. Structures of new antiestrogens that are being evaluated in the laboratory and the clinics.

WEEKSFig. 6. Effect of the nonestrogenic antiestrogen ICI 164,384 on tamoxifen-

stimulated growth of endometrial tumor EnCa 101 in the athymic mouse. Animalswere treated with a sustained release preparation of tamoxifen (2-cm Silasticcapsule) (D), a control capsule (O), or ICI 164,384, 1 mg/0.1 ml peanut oil s.c. 3times weekly (â€¢)or tamoxifen plus ICI 164,384 (â€¢).

Current and Future Uses of Antiestrogens

In the 30 years since the first publication of the pharmacological properties of MER-25, the clinical application of anti-estrogens has proved to be a remarkable success. Tamoxifen isnow the antihormonal treatment of choice for advanced breastcancer (pre- and postmenopausal). As valuable as this application is as a palliative treatment, the remarkable advances haveundoubtedly been made in the application of antiestrogens totreat all stages of breast cancer. Tamoxifen is the antihormonaltreatment of choice for the adjuvant therapy of postmenopausalnode-positive women. Two years of treatment is known toproduce a survival advantage, and longer treatment schedulesare being evaluated to exploit the known tumoristatic qualitiesof tamoxifen. We already know that long-term adjuvant tamoxifen therapy (5 years) is beneficial compared to saving tamoxifenuntil first recurrence, so 10-year and indefinite schedules arebeing evaluated.

The proven efficacy and low incidence of side effects haveencouraged the evaluation of adjuvant tamoxifen therapy inpre- and postmenopausal women with node-negative disease.At present the long-term toxicological concerns appear to benegligible compared to the known course of breast cancerwithout treatment. Hundreds of thousands of women could bebeing treated indefinitely with tamoxifen by the turn of thecentury. Indeed it has even been suggested that antiestrogenscould be used to prevent breast cancer. However, the timingand cause of the carcinogenic insult are unknown; therefore theuse of antiestrogens as true preventives seems impractical. Abetter term, also applicable to adjuvant therapy, is chemosup-pressive; i.e., the drug can be used to suppress subclinical diseaseand prevent the appearance of a primary tumor (163). In someadjuvant trials tamoxifen inhibits the development of secondprimary breast tumors (114, 139, 164) and an early clinicalevaluation of the toxicology of tamoxifen in normal women hasbeen reported (165) as a first step in a prevention study.Nevertheless there is a real concern about being able to targetthe right population. We cannot predict who will develop breastcancer; we can only guess at the probability. Furthermore "highrisk" women are, in fact, only a minority of those who will

develop breast cancer so any success must be balanced againstas yet unknown accumulative toxicities.

Is this the end of the possible applications for antiestrogens?Certainly not. We have obtained valuable clinical information

4185




about this group of drugs that can be applied in other diseasestates. Research does not travel in straight lines and observations in one field of science often become major discoveries inanother. Important clues have been garnered about the effectsof tamoxifen on bone and lipids so it is possible that derivativescould find targeted applications to retard osteoporosis or atherosclerosis. The ubiquitous application of novel compounds toprevent diseases associated with the progressive changes aftermenopause may, as a side effect, significantly retard the development of breast cancer. The target population would be post-menopausal women in general, thereby avoiding the requirement to select a high risk group to prevent breast cancer.

Acknowledgments

This paper is not intended to be an encyclopedic review article but isoffered only as a personal recounting of research conducted in ourrespective laboratories during a period that has spanned almost 40years. We sincerely acknowledge the support of colleagues, students,and assistants in our respective laboratories. We also acknowledge theimportant additional contributions made to the understanding of antiestrogen action by James H. Clark, Kathryn B. Horwitz, Benita andJohn Katzenellenbogen, Marc E. Lippman, C. Kent Osborne, RobertI. Nicholson, Henri Rochefort, Robert L. Sutherland, and Alan E.Wakeling. The influence of our teachers the late James H. Leathamand the late James B. Allison (L. J. L.) and Edward R. Clark (V. C. J.)was important for the direction and development of our investigativeapproach. Dr. Lerner is ever grateful for the encouragement he receivedas a developing scientist from Roy O. Creep, Roy Hertz, the late AlbertSegaloff, and the late Ralph Dorfman. Dr. Jordan wishes to acknowledge William L. McGuire, Jack Gorski, Elwood V. Jensen, the lateArthur L. Walpole, and colleagues at ICI for their invaluable help andsupport and Paul P. Carbone, Richard R. Love, Bernard Fisher, SydneyE. Salmon, and Douglass C. Tormey for the clinical application ofmany of the concepts and results described here. The EnCalOl tumorwas kindly provided by Dr. P. G. Satyaswaroop, Department of Obstetrics and Gynecology, Hershey School of Medicine, Hershey, PA,for the experiment in Fig. 6 performed by Dr. Marco M. Gottardis andMichelle Ricchio.

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53. Harper, M. J. K., and Walpole, A. L. Mode of action of ICI 46,474 inpreventing implantation in rats. J. Endocrinol., 37: 83-92, 1967. 84.

54. Labhsetwar, A. P. Role of estrogens in ovulation. A study using the estrogenantagonist ICI 46,474. Endocrinology, 87: 542-551, 1970. 85.

55. Jordan, V. C., and Robinson, S. P. Species-specific pharmacology of anti-estrogens: role of metabolism. Fed. Proc., 46: 1870-1874, 1987.

56. Terenius, L. Structure-activity relationships of anti-oestrogens with regard 86.to interaction with 17ÃŸ-oestradiolin the mouse uterus and vagina. ActaEndocrinol. Copenh., 66: 431-447, 1971. 87.

57. Jordan, V. C. Prolonged antioestrogenic activity of ICI 46,474 in theovariectomized mouse. J. Reprod. FÃ©rtil.,52: 251-258, 1975.

58. Jordan, V. C., Lababidi, M. K., and Mirecki, D. M. Inhibition of mouse 88.mammary tumorigenesis by the antiestrogen tamoxifen. In: Proceedings ofthe British Pharmacological Society, King College, London, Abstract 22, 89.December 1988.

59. Skidmore, J. R., Walpole, A. L., and Woodburn, J. Effect of some triphenylethylenes on oestradiol binding in vitro to macromolecules from uterus 90.and anterior pituitary. J. Endocrinol., 52: 289-298, 1972.

60. Jordan, V. C., and Prestwich, G. Binding of [3H] tamoxifen in rat uterine

cytosols. A comparison of swinging bucket and vertical tube rotor sucrose 91.density gradient analysis. Mol. Cell. Endocrinol., 8: 179-188, 1977.

61. Jordan, V. C., and Dowse, L. J. Tamoxifen as an antitumour agent: effecton oestrogen binding. J. Endocrinol., 68: 297-303, 1976. 92.

62. Jordan, V. C., Dix, C. J., Rowsby, L., and Prestwich, G. Studies on themechanisms of action of the non-steroidal antioestrogen (ICI 46,474) in therat. Mol. Cell. Endocrinol., 7: 177-192, 1977. 93.

63. Jordan, V. C., Rowsby, L., Dix, C. J., and Prestwich, G. Dose-related effectof non-steroidal antioestrogens and non-steroidal oestrogens on the measurement of cytoplasmic oestrogen receptors in the rat and mouse uterus. J. 94.Endocrinol., 78: 71-81, 1978.

64. Jordan, V. C., and Naylor, K. E. Binding of [3H]oestradiol in the immature

rat uterus during the sequential administration of antioestrogens. Br. J. 95.Pharmacol., 65: 167-173, 1979.

65. Berthois, Y., Katzenellenbogen, J. A., and Katzenellenbogen, B. S. Phenolred in tissue culture media is a weak estrogen: implications concerning the 96.study of estrogen-responsive cells in culture. Proc. Nati. Acad. Sci, USA,Â«5:2496-2500, 1986.

66. Bindal, R. D., Carlson, K. E., Katzenellenbogen, B. S., and Katzenellenbogen, J. A. Lipophilic impurities, not phenosulfophthalein, account for the 97.estrogenic activity in commercial preparations of phenol red. J. SteroidBiochem., 31: 287-293, 1988.

67. Lippman, M. E., and BolÃ¡n,G. Oestrogen-responsive human breast cancer 98.in long-term tissue culture. Nature (Lond.), 256: 592-595, 1975.

68. Reddel, R. R., Murphy, L. C., and Sutherland, R. L. Effects of biologicallyactive metabolites of tamoxifen on the proliferation kinetics of MCF-7 99.human breast cancer cells in vitro. Cancer Res., 43: 4618-4614, 1983.

69. Taylor, C. M., Blanchard, B., and Zava, D. T. Estrogen receptor mediatedand cytotoxic effects of the antiestrogens tamoxifen and 4-hydroxytamoxi-fen. Cancer Res., 44: 1409-1414,1984. 100.

70. Sutherland, R. L., Murphy, L. C., Foo, M. S., Green, M. D., Wybourne, A.M., and Krozowski, Z. S. High affinity antioestrogen binding site distinctfrom the oestrogen receptor. Nature (Lond.), 288: 273-275, 1980. 101.

71. Sutherland, R. L., Green, M. D., Hall, R. E., Reddel, R. R., and Taylor, I.W. Tamoxifen induces accumulation of MCF-7 human mammary carcinoma cells in the Go/G] phase of the cell cycle. Eur. J. Cancer Clin. OncoL, 102.19: 615-621, 1983.

72. Osborne, C. K., Boldt, D. H., Clark, G. M., and Trent, J. M. Effect of 103.tamoxifen on human breast cancer cell cycle kinetics: accumulation of cellsin early G, phase. Cancer Res., 43: 3583-3586, 1983.

73. Bates, S. E., Davidson, N. E., Vilverius, E. M., FrÃ©ter,C. E., Dickson, R. 104.B., Tarn, J. P., Kudlow, J. E., Lippman, M. E., and Salomon, D. S.

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Expression of transforming growth factor a and its messenger ribonucleicacid in human breast cancer: its regulation by estrogen and its possiblefunctional significance. Mol. Endocrino!., 2: 543-555, 1988.Knabbe, C., Lippman, M. E., Wakefield, L. M., Flanders, K. C., Kasid, A.,Derynck, R., and Dickson, R. B. Evidence that transforming growth factorÃŸis a hormonally regulated negative growth factor in human breast cancercells. Cell, 48: 417-428, 1987.Cole, M. P., Jones, C. T. A., and Todd, I. D. H. A new antioestrogenicagent in late breast cancer: an early clinical appraisal of ICI 46,474, Br. J.Cancer, 25: 270-275, 1971.Ward, H. W. C. Antioestrogen therapy for breast cancer: a trial of tamoxifenat two dose levels. Br. Med. J., /: 13-14, 1973.O'Halloran, M. J., and Maddock, P. G. ICI 46,474 in breast cancer. J. Ir.Med. Assoc., 67: 38-39, 1974.Klopper, A., and Hall, M. New synthetic agent for the induction of ovulation. Preliminary trial in women. Br. Med. J., /: 152-154, 1971.El-Sheikha, Z., Klopper, A., and Beck, J. S. Treatment of menometrorrhagiawith antioestrogen. Clin. Endocrinol., /: 275-282, 1972.Williamson, J. G., and Ellis, J. D. The induction of ovulation by tamoxifen.J. Obstet. Gynaecol. Br. Commonw., 80: 844-847, 1973.Jensen, E. V., Block, G. E., Smith, S., Kyser, K., and DeSombre, E. R.Estrogen receptors and breast cancer response to adrenalectomy. Nati.Cancer Inst. Monogr., 34: 55-70, 1971.Jordan, V. C., and Koerner, S. Tamoxifen (ICI 46,474) and the humancarcinoma 8S estrogen receptor. Eur. J. Cancer, 11: 205-206, 1975.Jordan, V. C. The development of tamoxifen for breast cancer therapy: atribute to the late Arthur L. Walpole. Breast Cancer Res. Treat., 11: 197-209, 1988.Investigational Drug Brochure for ICI 46,474. Clinical Research Department, ICI Americas Inc., Wilmington, DE, March 1974.Jordan, V. C. Antitumour activity of the antioestrogen ICI 46,474 (tamoxifen) in the dimethylbenzanthracene (DMBA)-induced rat mammary carcinoma model. J. Steroid. Biochem., 5: 354, 1974.Jordan, V. C., Koerner, S., and Robison, C. Inhibition of oestrogen-stimulated prolactin release by antioestrogens. J. Endocrinol., 65:151-152,1975.Jordan, V. C. Effect of tamoxifen (ICI 46,474) on initiation and growth ofDMBA-induced rat mammary carcinomata. Eur. J. Cancer, 12: 419-424,1976.Jordan, V. C., and Koerner, S. Tamoxifen as an antitumour agent: role ofoestradiol and prolactin. J. Endocrinol., 68: 305-310, 1976.Jordan, V. C. The antiestrogen tamoxifen (ICI 46,474) as an antitumoragent. Proceedings of the Eastern Cooperative Oncology Group, Miami,FL, February 11-12, 1974.Jordan, V. C. Tamoxifen: mechanism of antitumor activity in animals andman. Proceedings of the Eastern Cooperative Oncology Group, Jaspar,Alberta, Canada, June 22-25, 1974.Osborne, C. K., Hobbs, K., and Clark, G. M. Effect of estrogens andantiestrogens on growth of human breast cancer cells in athymic mice.Cancer Res., 45: 584-590, 1985.Gottardis, M. M., Robinson, S. P., and Jordan, V. C. Estradiol-stimulatedgrowth of MCF-7 tumors implanted in athymic mice: a model to study thetumoristatic action of tamoxifen. J. Steroid. Biochem., 30: 311-314, 1988.Gottardis, M. M., and Jordan, V. C. Development of tamoxifen-stimulatedgrowth of MCF-7 tumors in athymic mice after long-term antiestrogenadministration. Cancer Res., 48: 5183-5187, 1988.Gottardis, M. M., Jiang, S. Y., Jeng, M. H., and Jordan, V. C. Inhibitionof tamoxifen-stimulated growth of an MCF-7 tumor variant in athymicmice by novel steroidal antiestrogens. Cancer Res., 49:4090-4093, 1989.Jordan, V. C., Gottardis, M. M., Robinson, S. P., and Friedl, A. Immune-deficient animals to study "hormone-dependent" breast and endometrialcancer. J. Steroid. Biochem., 34: 169-176, 1989.Jordan, V. C. The antitumour affect of tamoxifen in the dimethylbenzan-thracene-induced mammary carcinoma model. Proceedings of a Symposiumon the Hormonal Control of Breast Cancer, Alderley Park, pp. 11-17.Macclesfield, United Kingdom: ICI Pharmaceuticals Division PLC, 1975.Nicholson, R. I., and Golder, M. P. Effect of synthetic antioestrogens onthe growth and biochemistry of rat mammary tumours. Eur. J. Cancer, //:571-579, 1975.Jordan, V. C., and Jaspan, T. Tamoxifen as an antitumour agent: oestrogenbinding as a predictive test for tumour response. J. Endocrinol., 68: 453-460, 1976.Jordan, V. C., Dix, C. J., and Allen, K. E. The effectiveness of long-termtreatment in a laboratory model for adjuvant hormone therapy of breastcancer. In: S. E. Salmon and S. E. Jones (eds.), Adjuvant Therapy for CancerII, pp. 19-26. New York: GruÃ±eand Stratton, 1979.Jordan, V. C., and Allen, K. E. Evaluation of the antitumor activity of thenonsteroidal antioestrogen monohydroxytamoxifen in the DMBA-inducedrat mammary carcinoma model. Eur. J. Cancer, 16: 239-251, 1980.Jordan, V. C. Use of the DMBA-induced rat mammary carcinoma systemfor the evaluation of tamoxifen treatment as a potential adjuvant therapy.Rev. Endocrinol. Rei. Cancer, October Suppl., 49-55, 1978.Jordan, V. C., Allen, K. E., and Dix, C. J. The pharmacology of tamoxifenin laboratory animals. Cancer Treat. Rep., 64: 745-759, 1980.Robinson, S. P., and Jordan, V. C. Reversal of the antitumor effect oftamoxifen by progesterone in the 7,12-dimethylbenzanthracene-induced ratmammary carcinoma model. Cancer Res., 47: 5386-5390, 1987.Robinson, S. P., Mauel, D. A., and Jordan, V. C. Antitumor actions oftoremifene in the 7,12-dimethylbenzanthracene (DMBA)-induced rat mam-




mary tumor model. Eur. J. Cancer Clin. Oncol., 24: 1817-1821, 1988. 131.105. Wilson, A. J., Tehrani, F., and Baum, M. Adjuvant tamoxifen therapy for

early breast cancer: an experimental study with reference to oestrogen andprogesterone receptors. Br. J. Surg., 69: 121-125, 1982. 132.

106. Gottardis, M. M, and Jordan, V. C. The antitumor actions of keoxifeneand tamoxifen in the A'-nitrosomethylurea-induced rat mammary carcinomamodel. Cancer Res., 47: 4020-4024, 1987. 133.

107. Early Breast Cancer Trialists' Collaborative Group. Effect of adjuvant

tamoxifen and of cytotoxic therapy on mortality in early breast cancer. N.Engl. J. Med., 319: 1681-1692, 1988.

108. Tormey, D. C., and Jordan, V. C. Long-term tamoxifen adjuvant therapy 134.in node positive breast cancer: a metabolic and pilot clinical study. BreastCancer Res. Treat., 4: 297-302, 1984.

109. Falkson, H. C., Gray, R., and Wolberg, W. M. Adjuvant therapy of post- 135.menopausa! women with breast cancerâ€”an ECOG phase III study. Abstract67. Proc. ASCO San Francisco May, 1989. 136.

110. Fisher, B., Redmond, C., Brown, A., Fisher, E. R., Wolmark, N., Bowman,D., Plotkin, D., Wolter, J., Bornstein, R., Legault-Poisson, S., Saffer, E.A., and Other NSABP Investigators. Adjuvant chemotherapy with and 137.without tamoxifen in the treatment of primary breast cancer: 5 year resultsfrom the National Surgical Adjuvant Breast and Bowel Project Trial. J.Clin. Oncol., 4: 459-471, 1986. 138.

111. Fisher, B., Brown, A., Wolmark, N., Redmond, C, Wickerham, L., Wittliff,J., Dimitrov, N., Legault-Poisson, S., Schipper, H., Prager, D., and OtherNSABP Investigators. Prolonging tamoxifen therapy for primary breast 139.cancer. Ann. Intern. Med., Â¡06:649-654, 1987.

112. Delozier, T., Julien, J-P., Juret, P., Veyret, C., Couette, J-E., Grai, Y.,Olliver, J-.M.. and de Ranier, E. Adjuvant tamoxifen in postmenopausalbreast cancer: preliminary results of a randomized trial. Breast Cancer Res. 140.Treat., 7: 105-110, 1986.

113. Breast Cancer Trials Committee, Scottish Cancer Trials Office (MRC).Adjuvant tamoxifen in the management of operable breast cancer: theScottish Trial. Lancet, 2: 171-175, 1987. 141.

114. Fisher, B., Constantino, J., Redmond, C., and Other Members of theNSABP. A randomized clinical trial evaluating tamoxifen in the treatmentof patients with node-negative breast cancer who have estrogen receptor- 142.positive tumors. N. Engl. J. Med., 320: 479-484, 1989.

115. Fromson, J. M., Pearson, S., and Bramali. S. The metabolism of tamoxifen(ICI 46,474) Part II in female patients. Xenobiotica, 3: 711-713, 1973.

116. Fabian, C., Sternson, L., and Barrett, M. Clinical pharmacology of tamox- 143.ifen in patients with breast cancer: comparison of traditional and loadingdose schedules. Cancer Treat. Rep., 64: 765-773, 1980. 144.

117. Adam, H. K., Gay, M. A., and Moore, R. H. Measurement of tamoxifen inserum by thin layer densitometry. J. Endocrinol., 84: 35-42, 1982.

118. Daniel, C. P., Gaskell, S. J., Bishop, H., and Nicholson, R. I. Determination 145.of tamoxifen and a hydroxylated metabolite in plasma from patients withadvanced breast cancer using gas chromatography-mass spectrometry. J.Endocrinol., 83: 401 -408, 1979. 146.

119. Bratherton, D. G., Brown, C. H., Buchanan, R., Hall, V., Kinglsey Pillers,E. M., Wheeler, T. K., and Williams, C. J. A comparison of two doses oftamoxifen (Nolvadex) in postmenopausal women with advanced breastcancer: 10 mg bd versus 20 mg bd. Br. J. Cancer, 50: 199-205, 1984. 147.

120. Patterson, J. S., Settatree, R. S., Adam, H. K., and Kemp, J. V. Serumconcentrations of tamoxifen and major metabolites during long-term Nolvadex therapy, correlated with clinical response. In: H. T. Mouridsen and 148.T. Palshoff (eds.). Breast Cancerâ€”Experimental and Clinical Aspects, pp.89-92. Oxford, United Kingdom: Pergamon Press, 1980.

121. Furr, B. J. A., and Jordan, V. C. The pharmacology and clinical uses of 149.tamoxifen. Pharmacol. Ther., 25: 127-205, 1984.

122. Robinson, S. P., and Jordan, V. C. The metabolism of steroid modifyinganticancer agents. Pharmacol. Ther., 36: 41-103, 1988. 150.

123. Jordan, V. C., Bain, R. R., Brown, R. R., Gosden, B., and Santos, M. A.Determination and pharmacology of a new hydroxylated metabolite oftamoxifen observed in patient sera during therapy for advanced breastcancer. Cancer Res., 43: 1446-1450, 1983. 151.

124. Kemp, J. V., Adam, H. K., Wakeling, A. E., and Slater, R. Identificationand biological activity of tamoxifen metabolites in human serum. Biochem.Pharmacol., 32: 2045-2052, 1983. 152.

125. Jordan, V. C., Collins, M. M., Rowsby, L., and Prestwich, G. A monohy-droxylated metabolite of tamoxifen with potent antioestrogenic activity. J.Endocrinol., 75: 305-316, 1977. 153.

126. Lein, E. A., Solheim, E., Kvinnsland, S., and Veland, P. M. Identificationof 4-hydroxy W-desmethyltamoxifen as a metabolite of tamoxifen in humanbile. Cancer Res., 48: 2304-2308, 1988. 154.

127. Robinson, S. P., Langan-Fahey, S. M., and Jordan, V. C. Implications oftamoxifen metabolism in the athymic mouse for the study of antitumoreffects upon human breast cancer xenografts. Eur. J. Cancer Clin. Oncol., 155.25: 1769-1776, 1989.

128. Jordan, V. C., Phelps, E., and Lingren, J. U. Effects of antiestrogens onbone in castrated and intact female rats. Breast Cancer Res. Treat., 10: 31- 156.35, 1987.

129. Turner, R. T., Wakely, G. K., Hannon, K. S., and Bell, N. H. Tamoxifenprevents the skeletal effects of ovarian deficiency in rats. J. Bone Miner.Res., 2:449-456, 1987. 157.

130. Love, R. R., Mazess, R. B., Tormey, D. C., Barden, H. S., Newcomb, P.A., and Jordan, V. C. Bone mineral density in women with breast cancertreated for at least two years with tamoxifen. Breast Cancer Res. Treat., 12: 158.297-301, 1988.

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TÃ¼rken,S., Siris, E., Seidin, E., Seidin, D., Plaster, E., Hyman, G., andLindsay, R. Effects of tamoxifen on spinal bone density in women withbreast cancer. J. Nati. Cancer Inst., */: 1086-1088, 1989.Bertelli, G., Pronzoto, P., and Amoroso, D. Adjuvant tamoxifen in primarybreast cancer: influence on plasma lipids and antithrombin III. Breast CancerRes. Treat., 12: 307-310, 1988.Love, R. R., Newcomb, P. A., Wiebe, D. A., Surawicz, T. S., Jordan, V. C.,Carbone, P. P., and DeMets, D. C. Lipids and lipoprotein effects oftamoxifen therapy in postmenopausal women with node negative breastcancer. Breast Cancer Res. Treat., 13: Abstract 204, 1989.Jordan, V. C., Fritz, N. F., and Tormey, D. C. Long-term adjuvant therapywith tamoxifen effects on sex hormone binding globulin and antithrombinIII. Cancer Res., 47: 4517-4519, 1987.Swenerton, K. D. Treatment of advanced endometrial adenocarcinoma withtamoxifen. Cancer Treat. Rep., 64: 805-811, 1980.Killackey, M. A., Hakes, T. B., and Pierce, V. K. Endometrial adenocarcinoma in breast cancer patients receiving tamoxifen. Cancer Treat. Rep., 69:237-238, 1985.Satyaswaroop, P. G., Zaino, R. J., and Mortel, R. Estrogen-like effects oftamoxifen on human endometrial carcinoma transplanted into nude mice.Cancer Res., 44: 4006-4010, 1984.Gottardis, M. M., Robinson, S. P., Satyaswaroop, P. G., and Jordan, V. C.Contrasting actions of tamoxifen on endometrial and breast tumor growthin the athymic mouse. Cancer Res., 48: 812-815, 1988.Fornander, T., Rutquist, L. E., Cedermark, B., Glas, U., Mattson, A.,Siljversward, J. D., Skoog, L., Somell, A., Theve, T., Wilking, N., Asker-gren, J., and Hjolmar, M. L. Adjuvant tamoxifen in early breast cancer:occurrence of new primary cancers. Lancet, 1: 117-120, 1989.Groom, G. V., and Griffiths, K. Effect of the antioestrogen tamoxifen onplasma levels of luteinizing hormone, follicle-stimulating hormone, prolac-tin, oestradiol and progesterone in normal premenopausal women. J. Endocrinol., 70:421-428, 1976.Jordan, V. C., Fritz, N. F., and Tormey, D. C. Endocrine effects of adjuvantchemotherapy and long-term tamoxifen administration on node positivepatients with breast cancer. Cancer Res., 47: 624-630, 1987.Ravdin, P. M., Fritz, N. F., Tormey, D. C., and Jordan, V. C. Endocrinestatus of premenopausal node positive breast cancer patients followingadjuvant chemotherapy and long-term tamoxifen. Cancer Res., 48: 1026-1029, 1988.Korenman, S. G. Relation between estrogen inhibiting activity and bindingto cytosol of rabbit and human uterus. Endocrinology, 87:1119-1123,1970.Bouton, M. M., and Raynaud, J. P. The relevance of interaction kinetics indetermining biological response to estrogens. Endocrinology, 705:509-575,1979.Jordan, V. C., Dix, C. J., Naylor, K. E., Prestwich, G., and Rowsby, L.Nonsteroidal antiestrogens: their biological effects and potential mechanisms of action. J. Toxicol. Environ. Health, 4: 364-390, 1978.Binan. N., Catelli, M. H., Geynet, Puri, V., Hahnel, R., Mester, J., andBaulieu, E. E. Monohydroxytamoxifen: an antiestrogen with high affinityfor the chick oviduct oestrogen receptor. Biochem. Biophys. Res. Commun.,91: 812-818, 1979.Borgna, J. L., and Rochefort, H. High affinity binding to the estrogenreceptor of [3H] 4-hydroxytamoxifen, an active antiestrogen metabolite.Mol. Cell. Endocrinol., 20: 71-85, 1980.Rochefort, H., and Borgna, J. L. Differences between oestrogen receptoractivated by oestrogen and antioestrogen. Nature (Lond.), 292: 257-259,1981.Jordan, V. C., and Bowser-Finn, R. A. Binding of [3H] monohydroxyta-moxifen by immature rat tissues in vivo. Endocrinology, 110: 1281-1291,1982.Tate, A. C., Greene, G. L., DeSombre, E. R., Jensen, E. V., and Jordan, V.C. Differences between estrogen and antiestrogen-estrogen receptor complexes from human breast tumors identified with an antibody raised againstthe estrogen receptor. Cancer Res., 44: 1012-1018, 1984.Lieberman, M. E., Gorski, J., and Jordan, V. C. An estrogen receptor modelto describe the regulation of prolactin synthesis by antiestrogens in vitro. J.Biol. Chem., 258: 4741-4745, 1983.Jordan, V. C., Lieberman, M. E., Cormier, E., Bagely, J., and Ruenitz, P.Structural requirements for the pharmacological activity of non-steroidalantiestrogens in vitro. Mol. Pharmacol., 26: 272-278, 1984.Jordan, V. C., and Lieberman, M. E. Estrogen-stimulated prolactin synthesis /'/; vitro: classification of agonist, partial agonist and antagonist actions

based on structure. Mol. Pharmacol., 26: 279-285, 1984.Jordan, V. C., Koch, R., Mittal, S., and Schneider, M. R. Oestrogenic andantioestrogenic actions in a series of triphenylbut-1-enes: modulation ofprolactin synthesis in vitro. Br. J. Pharmacol., 87: 217-220, 1986.Jordan, V. C., Koch, R., Langan, S., and McCague, R. Ligand interactionat the estrogen receptor to program antiestrogen action: a study withnonsteroidal compounds in vitro. Endocrinology, 122: 1449-1454, 1988.Bignon, E., Pons, M., Crastes de Paulet, A., Dore, J. C., Gilbert, J.,Abecassis, J., Miquel, J. F., Ojasoo, T., and Raynaud, J. P. Effect oftriphenylacrylonitrile derivatives on estradiol-receptor binding and on human breast cancer cell growth. J. Med. Chem., 32: 2092-2103, 1989.Murphy, C. S., and Jordan, V. C. Structural components necessary for theantiestrogenic activity of tamoxifen. J. Steroid Biochem., 34: 407-411,1989.Jordan, V. C. Biochemical pharmacology of antiestrogen action. Pharmacol.Rev., 36: 245-276, 1984.




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€¢, , , ,,- , ^ ?-,â€¢r. i ir t--, f-, ,oon 164. Cuzik, J., and Baum, M. Tamoxifen and contralateral breast cancer. Lancet,Eur. J. Cancer Clin. Oncol., 25: 57-63, 1989. Â¿.282 1985

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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1990;50:4177-4189. Cancer Res Leonard J. Lerner and V. Craig Jordan Eighth Cain Memorial Award LectureDevelopment of Antiestrogens and Their Use in Breast Cancer:

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