Molecular mechanisms and future uses of antiestrogens

Molec. Aspects Med. Vol. 18, pp. 187-247, 1997 0 1997 Elsevier Science Ltd. All rights resetved Printed in Great Britain 0098-2997197 $32.00+0.00

PII: SOO98-2997(96)00015-5

MOLECULAR

MECHANISMS AND FUTURE ANTIESTROGENS

USES

OF

V. Craig Jordan and William J. Gradishar The Robert H. Lurie Cancer Center and Medical Oncology, Northwestern Medical School, Chicago, IL 60611, USA

University

Contents

PREFACE

168

DEDICATION

169

CHAPTER 1

The Development of Tamoxifen

171

CHAPTER 2

Current Clinical Use of Tamoxifen

177

CHAPTER 3

Concerns About Tamoxifen

181

CHAPTER 4

New Directions in Research

187

CHAPTER 5

Molecular Mechanisms of Antiestrogen Actions

195

CHAPTER 6

Triphenylethylenes

201

CHAPTER 7

Pure Antiestrogens

217

CHAPTER 8

Targeted Antiestrogens

221

CHAPTER 9

Comparison of New Antiestrogen

REFEREiNCES

(Tamoxifen Analogs)

and Conclusions

223 225

Preface Twenty five years ago there was no tamoxifen for the treatment of breast cancer, only ICI 46,747 an antiestrogen with potent antifertility properties in laboratory rats. Today tamoxifen is the most prescribed cancer medicine and the endocrine treatment of choice for all stages of breast cancer. The low incidence of side-effects and the fact that tamoxifen increases the survival of breast cancer patients has revolutionized the approach to the treatment of breast cancer in general practice. However, the success of tamoxifen as a cancer therapy and the finding that tamoxifen can maintain bone density and lower circulating cholesterol has provided the incentive to develop new drugs for the treatment of osteoporosis and coronary heart disease. These drugs may have the added advantage of preventing breast cancer as a beneficial side-effect. This issue describes the development of antiestrogens as clinically useful agents from the time when Dr Leonard Lerner published his pioneering paper on the first non-steroidal antiestrogen MER-25 (Lerner et al., 1958) to the present. Lerner’s discovery was greeted with intense interest by the pharmaceutical industry because one of the fascinating effects of the drug was its action as a postcoital contraceptive. Regrettably MER-25 did not achieve its promise as a clinically useful antiestrogen but a successor compound MRL-41, also described by Dr Lerner (Holtkamp et ul., 1960), was successfully developed, not as a contraceptive but as an inducer of ovulation in subfertile women. MRL-41 or clomiphene became established as the therapeutic cornerstone in reproductive endocrinology, for the induction of ovulation. The reason for the intense interest in the value of antiestrogens as regulators of reproduction was because the oral contraceptive, conceived by Pincus and Chang at the Worcester Foundation for Experimental Biology (now the Worcester Foundation for Biomedical Research) in Shrewsbury, Massachusetts, had been so successful clinically. A ‘morning after pill’ would have been a valuable innovation but this was not to be. Nevertheless, the new antiestrogens, nafoxidine (Upjohn), CI 628 (Park-Davis), clomiphene (Merrel), and ICI 46,474 (ICI now Zeneca) were all investigated extensively in the laboratory throughout the 1960s and became standard laboratory reagents to investigate estrogen action. During the 196Os, there was only modest interest in cancer research in general and almost no interest in breast cancer therapeutics in particular. This was all to change in the 1970s with a ‘declaration of war on cancer’. However, several clinical trials with antiestrogens were completed in the 1960s but toxicity and an overall lack of interest by the pharmaceutical industry retarded progress. Only by a series of fortunate circumstances did ICI 46,474 become tamoxifen and this issue is dedicated to those who helped to play the key roles in this success story. 0 1997 Elsevier Science Ltd

168

Dedication Dr Leonard Lerner provided the scientific breakthrough that proved it was possible to design a compound to block estrogen action. Dr Elwood Jensen described the target site specific effects of estrogen in a laboratory model and developed the ‘estrogen receptor assay’ to predict which breast cancer patients would respond to antiestrogen therapy. Dr Jack Gorski first reported the isolation of the estrogen receptor as a soluble protein and in parallel with Jensen’s group developed intracellular models of estrogen action. Dr Arthur Walpole, head of the Reproduction Research program at Zeneca (then ICI), was the driving force behind the testing of ICI 46,474 (tamoxifen) as a therapy for advanced breast cancer. Finally Dr Bill McGui:re, whose clinical application of the steroid receptor concept, provided the rationale and target for therapeutic intervention at all stages of breast cancer. On a personal note, Dr Edward Clark, the former acting head of the Department of Pharmacology at the University of Leeds, UK deserves credit for preparing one of us (VCJ) for his subsequent career. Dr Clark was convinced that Jensen and Gorski’s reports of the isolation of a soluble receptor protein for estrogen was going to be pivotal for the future development of antiestrogens. Dr Clark is a synthetic bioorganic chemist who had a particular interest in Lerner’s work and convinced me in 1969 to read for a PhD degree in his laboratory in Leeds. My topic was to be simple. All I had to do was isolate the estrogen receptor and crystallize it with an estrogen and an antiestrogen. I was then to discover the change in shape of the complex by Xray diffraction in the Astbury Department of Biophysics. I encountered some difficulties and changed my project to the ‘structure-activity relationships of some triphenylethylenes and triphenylethanes.’ As it turned out this was a good strategic decision as no-one has yet succeeded in doing my first project! I am personally grateful to Dr Clark, my PhD supervisor, for the rigorous training and education I received. The late Dr Arthur Walpole was the examiner for my PhD in 1972 and provided me with all the opportunities to turn ICI 46,474 into tamoxifen. I met my friends and mentors Elwood Jensen (Chicago) and the late Bill McGuire (San Antonio) whilst I was a visiting scientist at the Worcester Foundation, conducting the first systematic laboratory study of tamoxifen as a antitumor agent. Their assistance and council over the past 20 years is truly appreciated. When I was recruited to develop a breast cancer research program at the University of Wisconsin in 1980: one of the reasons I accepted the challenge was the opportunity to collaborate with Jack Gorski, who is a Professor in the Biochemistry Department. Over the 1.5 years of my association with the University of Wisconsin I enjoyed tremendous help from Jack and his staff. Jack willingly served on the committees for all eleven of my PhD students at Wisconsin from Anna Riegel (n&e Tate) in 1983, who came to Madison as a Fulbright/Hays Scholar from Leeds University, to John Pink in 1995. I 169

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V. C. Jordan and W. J. Gradishar

am extremely grateful for all the help Jack has given so much of his intellect.

to my students

and for sharing

Finally I want to thank my friend Len Lerner who sees me as a ‘younger brother.’ I am grateful for his inspiration and his desire to ‘pass the baton’ to me to realize a dream of developing antiestrogens as therapeutic agents with applications throughout medicine. Although circumstances have never permitted us to work together we were both delighted to be linked by the award of the 8th Bruce F. Cain Memorial Award by the American Association for Cancer Research in 1989. The award recognized our preclinical work that spanned 40 years, and lead to the application of antiestrogens as an effective cancer therapy.

Northwestern

Robert University

V. Craig Jordan H. Lurie Cancer Center Medical School Chicago

Top left to right: Drs Len J. Lerner, Elwood V. Jensen, Jack Gorski Bottom left to right: Drs Arthur L. Walpole, William L. McGuire, Edward R. Clark

Chapter 7

The Development of Tamoxifen

Historical Perspective In 1896 George Beatson (Beatson, 1896) demonstrated that removal of the ovaries from premenopausal women could cause the regression of breast cancer. By the turn of the century it was established (Boyd, 1900) that about l/3 of all premenopausal women with advanced breast cancer could benefit from oophorectomy and from that time, a principal strategy for the treatment and prevention of breast cancer has been either to block or to restrict the action of estradiol in its target tissue, the breast. However, the successful clinical development of antiestrogenic drugs did not initially focus on the therapy for breast cancer but drew upon expertise in several unrelated disciplines. Most of the early interest in antiestrogens was focused on reproductive endocrmology but it was clear from the beginning of clinical studies that the effects of the (drugs on cholesterol biosynthesis would play a pivotal role in assessing safety considerations for long-term therapy. Ultimately the discovery of the estrogen receptor (ER) (J ensen and Jacobson, 1962; Gorski et al., 1968) in the 1960s and the application of this basic knowledge to understand hormone-dependent breast cancer growth (McGuire et al., 1975) focused interest on the development of antiestrogens as targeted agents to block estrogen action in the tumor directly. In 1958, Lerner and coworkers reported the biological properties of the first nonsteroidal antiestrogen MER-25 (Fig. 1). The compound was found to be an antiestrogen in all species tested and was found to have no other hormonal or antihormonal properties. However, the discovery was of importance because MER-25 was found to be a post coital contraceptive in laboratory animals. Obviously one application could have been as a ‘morning after pill’ but after clinical evaluation in numerous situations the results were disappointing. MER-25 underwent initial evaluation for the induction of ovulation (Tyler et al., 1960) and the treatment of chronic cystic mastitis, breast and endometrial carcinoma (Kistner and Smith, 1960; Smith et al., 1963), but the low potency and severe CNS side-effects prohibited further clinical development (Lerner, 1981). It is relevant to point out that the antiestrogen MER-25 is a structural derivative of the cholesterol lowering drug triparanol (MER-29). In the late 1950s there was initial enthusiasm about the potential benefits of triparanol as a hypocholestremic drug (Hollander et al., 1960). However, the finding that triparanol caused an accumulation of desmosterol (Avigan et al., 1960; Gaylor, 1963; Frantz et al., 1966; Steinberg et al., 171

V. C. Jordan and W. J. Gradishar

172

1961); (an intermediate in cholesterol biosynthesis) and the linking of this biochemical effect to cataract formation (Laughlin and Carey, 1962; Kirby et al., 1962; von Sallman et al., 1963), caused withdrawal of the drug in 1962. Nevertheless triparanol was first evaluated as a potential therapy for breast cancer (Kraft, 1962) but again the results were disappointing. A successor compound to MER-25, MRL-41 or clomiphene, was a more potent antiestrogen but drug development for long-term use was to be retarded because of toxicological concerns. Clomiphene is an effective antifertility agent in laboratory animals (Holtkamp et al., 1960) but paradoxically induces ovulation in subfertile women (Greenblatt et al., 1961, 1962; Kistner, 1965). Again the prospect of developing a ‘morning after pill’ for women was not realised. Although clomiphene showed some activity in the treatment of advanced breast cancer (Herbst et al., 1964; Hecker et al., 1974), the drug was only developed for short-term use for the induction of ovulation (Huppert, 1979) because desmosterol was noted in patient sera during prolonged treatment (W. S. Merrill Company, 1967).

MER 25

r /\/“\_ P

MER 29 (TRIPARANOL)

Fig. 1.

A comparison

of the structures of the first non-steroidal antiestrogen the hypocholestremic drug triparanol (MER-29).

MER-25

and

Molecular Mechanisms and Future Uses of Antiestrogens

173

Clomiphene is marketed as an impure mixture of geometric isomers (Fig. 2) that have opposing biological activities: one isomer is an estrogen and one isomer is an antiestrogen (Palopoli et aE., 1967). Unfortunately the isomers were initially (1967) given the incorrect designation but this was corrected by 1976 (Ernst et al., 1976). Although breast cancer clinical trials were still being reported with the impure mixture of isomers in 1974 (Hecker et al., 1974), the antiestrogenic isomer eventually entered into clinical trial for breast cancer treatment but the studies were dropped by the NIH due to the interest in tamoxifen (Holtkamp, 1987). derivative In contrast, the compound nafoxidine (U- 11, lOOA) is a dihydronaphthalene that cannot isomerize (Fig. 2). The drug is a potent antiestrogen with antifertility 1965). Nafoxidine properties in laboratory animals (Duncan et al., 1963; Greenwald, exhibits antitumor properties in laboratory models (Bloom et al., 1967) but following extensive testing as a treatment for breast and renal cancer, the drug was not developed further because of unacceptable side-effects experienced by all patients (Legha et al., 1976). Although the discovery of ICI 46,474 (tamoxifen), the antiestrogenic, pure trans isomer of a substituted triphenylethylene, was made by Drs Harper, Richardson and Walpole (Harper and Walpole, 1966; Bedford and Richardson, 1966) as part of the Fertility Control Program at ICI Pharmaceuticals (now Zeneca), Cheshire, UK, the

I-

(CLOMIPHENE (mbdure of cl8 and trans isomers)

TAMOXIFEN (trans isomer only)

Fig. 2. The triphenylethylene derivatives, clomiphene and tamoxifen, that were developed into clinically useful agents in the 1960s and 1970s. The clinically unsuccessful compound nafoxidine is shown for comparison.

174

V. C. Jordan and W. J. Gradishar

study of cancer therapies was Dr Walpole’s long-term interest (Jordan, 1988). The story of ICI’s involvement with the hormonal treatment of breast cancer goes back to the early 1940s following the laboratory discovery of chlorotriphenylethylene as an orally active estrogen (Robson et al., 1938). ICI supplied the triphenylethylenes initially used by Haddow, Watkinson and Paterson in their landmark study of the antitumor effects of synthetic estrogens in advanced breast and prostate cancer (Haddow et al., 1944). These studies paved the way for the standard use of high dose estrogen therapy to treat both breast and prostate cancer for the next two decades. In 1949, Walpole and Paterson (Walpole and Paterson, 1949) studied the antitumor actions of non-steroidal estrogen therapy on breast cancer in the hope of finding the reason why some patients responded and others did not. These early studies by Walpole were unsuccessful but the subcellular mechanism of hormonal-dependent growth was eventually discovered by Dr Elwood Jensen with his pioneering work on the ER (Jensen and Jacobson, 1962). Although Harper and Walpole (1967) s h owed that tamoxifen was an antifertility agent and an antiestrogen in the laboratory rat, like clomiphene it induced ovulation in subfertile women (Williamson and Ellis, 1973). Fortunately preliminary clinical studies demonstrated efficacy for tamoxifen in the treatment of advanced breast cancer (Cole et al., 1971, 1972; Ward, 1973) and the stage was now set for the successful development of tamoxifen as the first clinically acceptable antiestrogen for the treatment of breast cancer.

Clinical Development Tamoxifen (Nolvadexm) had several advantages at the outset: (1) higher antitumor potency compared with other compounds being evaluated at the time (Table 1); (2) a low reported incidence of side-effects for the drug itself (Ward, 1973); (3) a lower incidence of side-effects compared with other endocrine therapies available to the clinician (i.e. high-dose estrogens or androgens (Cole et al., 1971)); and (4) the laboratory finding (Harper and Walpole, 1967), confirmed in the clinic (Cole et al., 1972) that desmosterol levels were not affected by tamoxifen. Long-term therapy (i.e. years) could be considered without the fear of cataract formation. This has proved to be true after 20 years of usage. The evolution of strategies to determine the optimal application of tamoxifen for the treatment of breast cancer has taken more than 20 years but the process has been facilitated by a dialogue between the laboratory and the clinical trials community. The principal areas of contact are illustrated in Fig. 3. The clinical development of tamoxifen from the first preliminary clinical reports, suggesting its efficacy as an agent for the palliation of advanced breast cancer in postmenopausal women, to the present has been extensively reviewed (Furr and Jordan, 1984; Lerner and Jordan, 1990; Jordan, 1993; Jaiyesimi et al., 1995; Osborne, in press). However, the laboratory studies investigating the biology of tamoxifen have played, and continue to play, a pivotal role in the clinical investigation of the treatment strategies, concepts and concerns with antiestrogens. Long-term adjuvant therapy (i.e. greater than 1 year) (Jordan, 1978, 1983, 1994; Jordan et al., 1979), prevention (Jordan, 1974, 1976; Jordan et al., 1991), the association of tamoxifen with endometrial cancer (Satyaswaroop et

Molecular Mechanisms Table 1. Preliminary

and Future Uses of Antiestrogens

clinical trials of antiestrogens

for the treatment

of metastatic

Total patients (% response)

Toxicities

Compound Reference

Daily dose

Triparanol, Kraft (1962) MER-2.5, Kistner and Smith (1960) Clomiphene, Herbst et al. (1964) Hecker et al. (1974) Nafoxidine, Legha et al. (1976)

250-1000 500-4500 100-300

8 (11) 4 (25) 56 (34)

180-240

198 (31)

20-40

114 (31)

(mg)

TamoxiFen, Cole et al. (1971) Ward (1973)

175 breast cancer

None” Acute psychotic episodeb None reported or mild’

Bilateral cataracts Ichthyosis Cutaneous photophobiad Transient thrombocytopenia’

“Withdrawn from the market by the William S. Merrel Co. in cooperation with the FDA in April 1962. hDoes not include patients treated by Dr Roy Hertz when therapy was stopped due to hallucinations (Lerner, 1981). ‘Visual rsymptoms (W. S. Merrill Company, 1967). ‘Eighty to one hundred percent of patients affected (Legha et al., 1976). “The particular advantage of this drug is the low incidence of side effects’ (Cole et al., 1971) ‘The side effects were usually trivial’ (Ward, 1973).

al., 1984; Gottardis et al., 1988), carcinogenesis and tamoxifen (Han and Liehr, 1992; Williams et al., 1993; Greaves et al., 1993) and the targeting of novel antiestrogens to prevent osteoporosis (Jordan et al., 1987; Lerner and Jordan, 1990) are all laboratory concepts that have had a profound impact on the clinical applications of

CLINICAL TESTS TAMOXIFEN Advanced Disease Adjuvant Single Agent

Tamoxifen

Pure Antiestrogens PREVENTION Raloxifene for Osteoporosis

High Affinity Preserve Bone Long Term Tamoxlfen

Pure Antiestrogens

LABORATORY TESTS Fig. 3. The development of tamoxifen during the 1970s with laboratory results being translated into clinically useful treatment strategies culminating in the current evaluation of tamoxifen as a preventive. An investigation of tamoxifen’s pharmacology in the laboratory establislhed the concept of antiestrogens with high affinity for the estrogen receptor, pure antiestrogens and the target site specific actions on bones that has culminated in the testing of raloxifene to treat and prevent osteoporosis.

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V. C. Jordan and W. J. Gradishar

The enormous clinical success of tamoxifen and the discovery of its target site-specific actions has provided an enormous incentive to develop new and improved compounds with applications to treat breast cancer and diseases associated with the menopause (Tonetti and Jordan, 1996). antiestrogens.

We will first describe the effectiveness of tamoxifen in the treatment of breast cancer and focus on the potential concerns that have been raised about the use of extended durations of adjuvant tamoxifen therapy and the testing of tamoxifen as a breast cancer preventive in high risk women. The broad application of tamoxifen has encouraged far ranging studies of its toxicology and side-effects. At present there are several concerns regarding tamoxifen, but there is little clear cut clinical evidence or description of serious unpredicted side-effects, to support the hypothetical toxicities. Nevertheless with the reported concerns in mind we will describe the properties of an ideal antiestrogen to be used as a preventive agent, a long-term adjuvant therapy or as a treatment for osteoporosis. We will then evaluate each available new compound, based upon the criteria required for the ideal therapeutic agent, so that clinicians can make an objective assessment of the information that is used to support the claims for new antiestrogens currently under investigation.

Chapter2

Current Clinical Use of Tamoxifen

Tamoxifen is the endocrine treatment of choice for all stages of breast cancer. There are currently 8 million women years of clinical experience with the drug and it is the world’s most prescribed cancer medicine. In 1973 Nolvadex@, the ICI brand of tamoxifen was approved for the treatment of breast cancer by the Committee on the Safety of Medicines in the United Kingdom. Similar approval was given in the United States for the treatment of advanced disease in postmenopausal women by the Food and Drug Administration (FDA) on 30 December 1977. Tamoxifen was approved for the treatment of advanced ER-positive breast cancer in premenopausal women in 1989. With more than 20 years of clinical experience world-wide, tamoxifen is established as the endocrine therapy of choice for metastatic breast cancer in pre- and postmenopausal patients. Extensive testing as an adjuvant therapy and proven efficacy to enhance survival (NATO, 1983, 1988; Fisher et al., 1983, 1986, 1989; Breast Cancer Trials Committee, Scottish Trials Office, 1987) lead to FDA approval for the use of tamoxifen as an adjuvant therapy with chemotherapy (1985), as an adjuvant therapy alone (1986) in node-positive, postmenopausal patients and pre- and postmenopausal patients with ER-pomsitive, node-negative disease (1990). Tamoxifen was approved for the treatment of male breast cancer in 1993. The currently accepted strategy for the adjuvant treatment of breast cancer is to employ at least 5 years of therapy. This duration is the standard used in clinical trials in the United States. However, based upon theoretical considerations and its safety profile., tamoxifen treatment has been used until relapse in both a clinical trials setting (Falkson et al., 1990; Tormey et al., 1992) and in clinical practice on a case by case basis. If all the published clinical trials data kept are considered it is not possible to define the optimal duration of tamoxifen treatment (Bilimoria et al., 1996). Nevertheless a recent clinical alert from the National Cancer Institute has recommended stopping tamoxifen at 5 years in ER-positive, node-negative women because no improvement can be detected in an National Surgical Adjuvant Breast and Bowel Program (NSABP) study by continuing tamoxifen to 10 years. However, we believe that additional published data are required before rigid guidelines can be establi’shed. Furthermore, the small Scottish trial of 5 years versus indefinite 177

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V. C. Jordan and W. J. Gradishar

tamoxifen shows no benefit for indefinite therapy but strongly encourages further evaluation in clinical trial (Stewart et al., 1996). The Cancer Research Campaign in the United Kingdom has opened a registration trial (ATTOM) of stopping tamoxifen trials at 2 years or continuing for at least 5 additional years. Similarly the clinical trials center in Oxford is preparing a study, ATLAS, with sites around the world, to define the optimal duration of adjuvant tamoxifen treatment. Despite our inability to define the optimal duration for tamoxifen treatment under all circumstances it is reassuring that the reporting of major toxicities has remained so low during a period when the duration of tamoxifen treatment has not been restricted (Jordan, 1994).

The success of tamoxifen as a therapeutic agent has been demonstrated convincingly through the Oxford overview analysis of randomized clinical trials (Early Breast Cancer Trialists’ Collaborative Group, 1988, 1992). However, the direct investigation of potential side-effects has demonstrated additional benefits for patients treated with tamoxifen that have ultimately resulted in the widespread testing of new antiestrogens as potential preventives for osteoporosis, coronary heart disease, breast and endometrial cancer (Lerner and Jordan, 1990; Jordan, 1995a; Tonetti and Jordan, 1996).

Additional Benefits of Tamoxifen A decade ago when the strategy of long-term tamoxifen therapy was being incorporated into treatment arms of all clinical trials using tamoxifen, preliminary studies were undertaken to evaluate the possibility that an ‘antiestrogen’ might have a detrimental effect on bone density and also place patients at risk for coronary heart disease. However, it was known that tamoxifen is not a pure antiestrogen but has estrogen-like effects in animals and postmenopausal women (Furr and Jordan, 1984; Lerner and Jordan, 1990; Jordan, 1993). Tamoxifen maintains bone density in et al., 1987, 1988) and has been ovariectomized rats (Jordan et al., 1987; Turner shown to have either a beneficial effect in maintaining bone density in the lumbar spine and neck of the femur in postmenopausal women (Turken et al., 1989; Love et al., 1992, 1994; Ward et al., 1993; Kristensen et al., 1994) or have no detrimental effects (Fornander et al., 1990). These data have been summarized recently (Bilimoria et al., 1996) and suggest that tamoxifen has an estrogen-like effect on maintaining bone density in postmenopausal women. Similarly, tamoxifen lowers circulating cholesterol. However, high density lipoprotein (HDL) cholesterol levels are unaffected (Bertelli et al., 1988; Love et al., 1991, 1995) and only low density lipoprotein (LDL) cholesterol is reduced. Overall these data suggest that tamoxifen could lower the incidence of coronary heart disease if long-term treatment schedules are used. Preliminary studies from the Scottish trial demonstrate that 5 years of tamoxifen will reduce the risk of fatal myocardial infarction (McDonald and Stewart, 1991) and there is a reduced incidence of hospitalizations from any cardiac condition in the Stockholm trial (Rutqvist and Matteson, 1993). A recent update of the Scottish Trial (McDonald ef al., 1995) has shown a clear decrease in coronary heart disease if women have ever taken tamoxifen. However, most protection is seen for current users.

Molecular

Mechanisms

and Future Uses of Antiestrogens

179

Antiestrogens and Lipids: Molecular Mechanisms There is intense interest in the ability of antiestrogens to decrease coronary heart disease and two lines of investigation have demonstrated possible mechanisms. First, the decrease in circulating cholesterol has recently been shown to be caused by a block in the conversion of delta 8 cholestenol to lathosterol (Gylling et al., 1995). (Fig. 4). Both tamoxifen and toremifene cause an increase in circulating delta 8 cholestenol but there is very little effect on the conversion of desmosterol to cholesterol. This is the primary mechanism for triparanol blocking cholesterol biosynthesis that was associated with cataract formation in treated patients (Avigan et al., 1960). Some of the beneficial effects of tamoxifen may be related to its antioxidant ability. Tamoxifen protects rat cardiac membranes from damage caused by lipid peroxidation (Wiseman et al., 1993a). Oxidative damage to LDL is also thought to play a role in the development of atherosclerosis and Wiseman et al. (1993b) have shown that tamoxifen and its metabolites can protect human LDL against copper-ion dependent lipid peroxidation.

Contralateral Breast Cancer If the beneficial effects of tamoxifen on bones and cholesterol are expressions of the estrogen-like effects of tamoxifen, the effects of tamoxifen on reducing contralateral breast cancer must be considered to be an expression of the antiestrogenic/antitumor properties of the drug. The incidence of second primary breast cancer in patients treated with tamoxifen is reduced by approximately 40% compared to

Lanosterol

Dihydrolanosterol + AS-dimethylsterol 1 A*-monomethylsterol + A*-cholestenol X+Lathosterol

TAMOXIFEN & TOREMIFENE

4

Desmosterol

Cholesterol Triparanol

Fig. 4.

The inhibition

of cholesterol biosynthesis by tamoxifen, Adapted from Gylling et al. (1995).

toremifene

and triparanol.

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V. C. Jordan and W. J. Gradishar

not receiving tamoxifen (Fisher et al., 1989; Early Breast Cancer Trialists’ Collaborative Group, 1992; Fornander et al., 1989). A significant reduction in contralateral breast cancer (0.5 matched odds ratio) for patients receiving 2 years of adjuvant tamoxifen has recently been reported by Cook et al. (1995) based on an epidemiology study. patients

In summary tamoxifen has been shown to produce significant benefits to patients as an anticancer agent by increasing patient survival and reducing the risk of developing contralateral breast cancer (Early Breast Cancer Trialists’ Collaborative Group, 1992). Furthermore, the estrogen-like effects of tamoxifen appear to provide benefits to postmenopausal patients who would normally be denied a hormone replacement therapy to prevent osteoporosis or coronary heart disease (Bilimoria et al., 1996). Nevertheless there are legitimate concerns about the potential toxicities associated with long-term tamoxifen treatment that have required consideration by the clinical community.

Chapter 3

Concerns About Tamoxifen

During the past half decade several important aspects of the pharmacology of tamoxifen have emerged that have had an impact on the clinical use of the drug. There are three principal areas of concern. First, an increased incidence in endometrial cancer has been reported (Fornander et al., 1989; Fisher et al., 1994) in tamoxifsen treated patients and one study found higher grade disease and patients with a poorer prognosis associated with tamoxifen treatment compared to patients with de nova disease (Magriples et al., 1993). Based on laboratory evidence (Satyaswaroop et al., 1984; Gottardis et al., 1988), an increased detection of endometrial cancer could be ascribed to the estrogen-like effects of tamoxifen. Second, laboratory studies have shown that tamoxifen produces liver tumors in rats (Williams et al., 1993; Greaves et al., 1993) and concerns have been raised that tamoxifen could produce second primary tumors in the liver, stomach, colon and rectum of women (Rutqvist et al., 1995). Finally, laboratory evidence (Osborne et al., 1987; Gottardis and Jordan, 1988; Gottardis et al., 1989a; Wolf et al., 1993) shows that tamoxifen-stimulated breast tumors can develop so the antitumor action of long-term adjuvant tamoxifen therapy could eventually fail. Tamoxifen might ultimately encourage tumor growth in patients. However, each of these concerns must be placed in perspective based on the current clinical data base of 8,000,OOO woman-years of experience accumulated from the worldwide use of tamoxifen in patients for nearly a quarter of a century.

Endomietrial Cancer Much controversy has surrounded the associations between tamoxifen use and the detection of endometrial cancer. However, it is now possible to provide a reasonable picture of the actual incidence of endometrial cancer and provide a balanced view of the concerns. Recent reviews (Assikis and Jordan, 1995; Assikis et al., 1996; Jordan and Assikis, 1995) of the literature have only identified about 400 cases of endometrial cancer associated with tamoxifen use world-wide and our original reviews (Assikis and Jordan, 1995; Jordan and Assikis, 1995) has now been updated to the end of 1995 in Table 2. The disease is found predominantly in postmenopausal women and there is not a strong association between the duration of tamoxifen use and the risks of developing endometrial carcinoma. Indeed it is interesting to re-evaluate earlier studies that claim an association between long-term tamoxifen and 181

182

V. C. Jordan and W. J. Gradishar Table 2. The incidence and characteristics of endometrial tumors associated with the use of tamoxifen. The cases accumulated are those reported up to the end of 1995

Endometrial carcinomas Premenopausal patients Postmenopausal patients Stage 1 disease Good Grade (1+2) More than 2 years tamoxifen Less than 2 years tamoxifen

349 2 200 184/234 (79%) 185/225 (82%) 108 91

The information is reported in full elsewhere (Assikis et al., 1996). It should be noted that disease and patient characteristics are not provided by the reporting authors uniformly.

endometrial cancer. Re-analysis of the Stockholm study (Fornander et al., 1989) that originally concluded that randomization to 2 years of adjuvant tamoxifen did not cause an increase in endometrial cancer, but randomization to 5 years of adjuvant tamoxifen caused a six-fold increase in the risk of endometrial cancer actually demonstrates that 12 of 16 received only 2 years of drug (Jordan and Morrow, 1994). Clearly pre-existing disease was being detected. Based on the known long genesis of cancer in humans it would be inappropriate to suggest that early detection of endometrial cancer was caused by short courses of tamoxifen. It is also important to point out that DNA adducts are absent from uterine samples of patients taking tamoxifen (Carmichael et al., 1996). Detection bias, through the investigation of symptoms, is almost certainly responsible for the disease found in many patients receiving tamoxifen. It is important to appreciate that epidemiology studies do not show a statistically relevant increase in the incidence of endometrial cancer after a short (2 year) course of tamoxifen (Cook et al., 1995; van Leeuwen et al., 1994). The finding, by Magriples et al. (1993), that tamoxifen use is associated with poor prognosis disease has not been confirmed by any other study (Fisher et al., 1994; Rutqvist et al., 1995; Barakat et al., 1994). Overall, the stage and grade of endometrial cancer associated with tamoxifen use is proportionally the same as Surveillance, Epidemiology, and End Results (SEER) data (Assikis and Jordan, 1995). Therefore, it is fair to say that the overall consensus is that the benefits of tamoxifen in the treatment of breast cancer far outweigh the risks associated with a two-fold elevation in early stage low grade endometrial carcinoma (Jaiyesimi et al., 1995; Osborne, in press; Bilimoria et al., 1996; Early Breast Cancer Trialists’ Collaborative Group, 1992; Jordan, 1995b). However, as a precaution, patients should be screened to determine whether they have pre-existing endometrial carcinoma before starting a course of adjuvant tamoxifen therapy. Additionally patients who present with spotting and bleeding during treatment must be investigated with a thorough gynecological examination. There is, however, no justification for an extensive screening program to detect endometrial cancer in asymptomatic women taking tamoxifen (Barakat, 1997).

Molecular

Mechanisms

and Future Uses of Antiestrogens

183

Hepatocellular Carcinoma and Other Malignancies Several investigators report that tamoxifen is both an initiator and a promoter of rat liver carcinogenesis (Williams et al., 1993; Greaves et al., 1993; Hard et al., 1993; Dragan et al., 1994, 1995, 1996). Tamoxifen, at high doses, causes DNA adducts in rat liver (Han and Liehr, 1992; Hard et al., 1993; White et al., 1992). However, only low adduct formation is noted in mouse liver DNA, (White et al., 1992) a species that does not produce tumors in response to high daily doses of tamoxifen (Furr and Jordan, 1984). It is also reassuring to note that there is no increase in DNA adduct formation in the livers of patients receiving tamoxifen (Martin et al., 1995). As a result, it has been argued that the rat studies are not relevant to human usage (Jordan and Morrow, 1994; Jordan, 1995b, 199%). An exarnination of the data from the rat carcinogenesis studies demonstrate that the animals receive tamoxifen (S-SO mg/kg daily) from puberty for more than 50% of their life (Jordan and Morrow, 1994). In contrast, the therapeutic dose of tamoxifen, as an anticancer agent in rats, is 250 pg/kg (Jordan, 1983) which is comparable to the therapeutic dose in a 70 kg patient of 285 ,ug/kg or 20 mg of tamoxifen administered twice daily. The duration of adjuvant therapy for postmenopausal patients is usually 5 years. This would be equivalent to 8% of a woman’s life. Thus, the animal experiment at the lowest dose to produce tumors, 5 mg/kg, is equivalent to a teenage girl (i.e. 14 years of age) receiving 20 times the daily dose of tamoxifen until she is 40 years old. This is 40 tablets a day. The reason that such large doses have to be administered to the rat to produce drug levels comparable to the human is that the drug is cleared from the rat ten times faster than in humans (Jordan and Morrow, 1994). Thus, artificially high levels of drug must be given, far outside the therapeutic range, that ultimately cause damage in the rat liver. In recent years, concerns about carcinogenesis with tamoxifen have lead to a report of increases in co10 rectal cancer and stomach cancer (Rutqvist et al., 1995). These results have not been supported by either individual reports from clinical trials (Fisher et al., 1994) or from the current (1996) Oxford overview analysis. The finding of liver carcinogenesis in the rat would be cause for concern with any new drug that is about to go into clinical trial. However, tamoxifen had been used extensively for 20 years before the investigation of rat liver carcinogenesis. There has not been a significant increase in hepatocellular carcinoma since the two initial cases reported. in 1989 (Fornander et al., 1989). Similarly epidemiology studies (Muhleman et al., 1994) have not shown a rise in hepatocellular carcinoma in breast cancer patients since tamoxifen was approved for use in the United States in 1978. In contrast., oral contraceptives cause a ten-fold increase in the risk for the development of hepatocellular carcinoma (Prentice, 1991), but this risk is considered to be acceptable to regulatory authorities because of the rarity of the disease.

Molecular Mechanism of Carcinogenesis During the past 5 years there has been intense interest in discovering event for tamoxifen-induced rat liver carcinogenesis and determining

the initiating the relevance

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for humans. Han and Liehr (1992) first noted an accumulation of DNA adducts in the liver of Sprague-Dawley rats on repeated injections of 20 mg/kg (cf. human dosage of 0.3 mg/kg). This has been adequately confirmed by numerous investigators and the focus of investigation has been the identification of the actual DNA adduct. Several candidates have been proposed: an epoxide (Styles et al., 1994; Lim et al., 1994; Phillips et al., 1994a), 4-hydroxytamoxifen (Randerath et al., 1994; Moorthy et al., 1996), Metabolite E (Pongracz et al., 1995) or cr-hydroqamoxifen (Potter et al., 1994; Phillips et al., 1994b, 1994~). Recently, Osborne et al. (1996) prepared a-acetoxytamoxifen which is able to react with DNA to a greater extent (1 in 50 bases) than a-hydroxytamoxifen (1 in lo5 DNA bases). The products of the reaction were identical to those isolated from DNA of rat hepatocytes or the livers of rats treated with tamoxifen. The adduct of tamoxifen and DNA has been identified at the nucleoside deoxyguanosine in which the a position of tamoxifen is linked covalently to the exocyclic amino of deoxyguanosine (Fig. 5). These important observations have provided a framework to study the metabolic activation of tamoxifen in human systems and to identify any DNA adducts in human tissues. The metabolic activation of tamoxifen and its metabolite cc-hydroxytamoxifen has been compared using primary cultures of rat, mouse and human hepatocytes (Phillips et al., 1996a). Although DNA adducts are readily identified in rat and mouse

Fig. 5. The formula of cc-hydroxytamoxifen (I), a-acetoxytamoxifen (II) and the adduct formed between a-hydroxytamoxifen and deoxyguanosine (III) Adapted from Osborne er al. (1996).

Molecular Mechanisms and Future Uses of Antiestrogens

185

hepatocytes (90 and 15 adducts/lOa nucleotides, respectively), DNA adducts were not detected in tamoxifen-treated human hepatocytes. Additionally human hepatocytes also appeared to produce SO-fold lower levels of a-hydroxytamoxifen from tamoxifen compared to rat hepatocytes. Further studies showed that if cells were treated with had 300-fold lower levels of adducts a-hydroxytamoxifen human hepatocytes compared to rat hepatocytes. Studies in humans have confirmed that the human is not as susceptible as the rat to DNA adduct formation with tamoxifen. The pattern of DNA adducts found in the rat liver is not found in humans treated with tamoxifen (Martin et al., 1995), DNA adducts are not found in lymphocytes (Phillips et al., 1996b) and there is a lack of genotoxicity of tamoxifen in human endometrium (Carmichael et al., 1996). In the latter studies, DNA adducts could be produced in endometrial samples with a-hydroxytamoxifen but not with tamoxifen. The authors proved that tissue was capable of metabolizing tamoxifen to cr-hydroxytamoxifen but apparently it is incapable of producing adducts. Endometria from patients taking tamoxifen for up to 9 years were analyzed for DNA adducts. No evidence for any DNA adducts induced by tamoxifen was found in any of the patients examined. The authors concluded that the genotoxic events observed with tamoxifen in the rat may not apply to the human endometrium (Carmichael et al., 1996). This conclusion supports the previous suggestl.on that tamoxifen, or indeed any new antiestrogen which has partial agonist actions, will cause the activation and detection of pre-existing disease (Jordan and Morrow, 1994).

Drug Resistance Drug resistance to tamoxifen therapy can take many forms (Morrow and Jordan, 1993; Tonetti and Jordan, 1995). Obviously if tumors are ER-negative there is only a small probability of a response to antiestrogen therapy. In the case of metastatic breast cancer about 10% of ER- and progesterone receptor (PR)-negative patients respond to any form of endocrine modulation (Jordan et al., 1988). Similarly the overview analysis (Early Breast Cancer Trialists’ Collaborative Group, 1992) of clinical trials suggest that postmenopausal, node-positive patients with receptor poor disease will only benefit from adjuvant tamoxifen with a small survival advantage compared with highly receptor-positive disease. Adjuvant tamoxifen produces the best survival benefits in patients with receptorpositive disease, however, the duration of therapy may have a profound effect on the overall effectiveness of treatment. One goal of current clinical studies is to determine the optimal duration of tamoxifen. Two (NATO, 1983) and five (Breast Cancer Trials Committee, Scottish Trials Office, 1987) years of tamoxifen treatment will produce a survival advantage in unselected postmenopausal patients with predominately nodepositive disease. However, the emergence of drug resistance to tamoxifen will limit the effectiveness of indefinite tamoxifen treatment (Bilimoria et al., 1996). It is interesting to note that the NSABP B-14 trial evaluating 5 versus 10 years of tamoxifen in node-negative patients, was recently stopped, not because significant drug resistance was observed with 10 years of therapy, but because 10 years of therapy

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no significant benefit over 5 years of therapy. The issue, therefore, and a consideration of a possible increase in iatrogenic diseases.

becomes

Nonetheless, there are ongoing clinical trials in place in the United Kingdom (ATTOM and ATLAS) that are addressing the duration of tamoxifen in large patient populations. Each study is recruiting 20,000 patients to establish guidelines for the duration of tamoxifen therapy in node-positive and node-negative populations. In the laboratory, tamoxifen will inhibit estrogen-stimulated growth of MCF-7 breast tumors implanted into athymic mice (Osborne et al., 1985). Nevertheless, continuous therapy with tamoxifen results in the emergence of tamoxifen-stimulated breast tumors that will grow in response to either estrogen or tamoxifen (Osborne et al., 1987; Gottardis and Jordan, 1988; Gottardis et al., 1989a; Wolf et al., 1993). Since there are clinical reports of tamoxifen-stimulated tumors that have a withdrawal response to tamoxifen (Canney et al., 1987; Howell et al., 1992), new second-line agents (or first-line agents) are necessary to control tumors that grow after extended tamoxifen treatment.

Chapter 4

New Directions in Research

The Ideal Agents for Therapeutic Evaluation There are three distinct goals for drug discovery to exploit the current therapeutic applications of antiestrogens. The opportunities are illustrated in Fig. 6. Tamoxifen is being used experimentally as a preventive for breast cancer in high risk women and

BIOLOGY INITIATION

ANTIESTROGEN TREATMENT STRATEGY

1

-

? PRGMOTlON WllH ESTROGEN

I I I I I I

+

@ DETECTION

--)

@O@

O@O O@

-

SURGERY

@O

PURE ANllESTRGGENS

w Fig. 6. Treatment opportunities with antiestrogens. New antiestrogens could be discovered that do not develop antiestrogen-stimulated tumor growth and are not cross-resistant with tamoxifen. Pure antiestrogens could be used for advanced disease but targeted antiestrogens could be used as preventives for breast cancer and as specific hormone replacement therapies for post-menopausal women. 187

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V. C. Jordan and W. J. Gradishar

also as a treatment for all stages of breast cancer. One strategy is to develop agents that are not cross-resistant with tamoxifen and have a safer toxicity profile. Another strategy is to develop a pure (i.e. non-estrogenic) antiestrogen that does not have stimulatory effects in the uterus and does not cause premature drug resistance. These agents could find use as second-line therapies after the failure of tamoxifen or as optimal estrogen antagonists for the first-line treatment of metastatic breast cancer. Finally, an ideal agent for adjuvant therapy, and as a preventive, could be designed to exploit the beneficial effects of tamoxifen on bones and lipids but solve the problems suggested by an increased detection of endometrial cancer and the theoretical concerns about DNA adduct formation in rat liver. However, quality of life issues will become of primary importance for the successful application of antiestrogens in women without a diagnosis of breast cancer. Clearly, if a new agent is unpleasant to take, compliance will be a problem. As a result a critical area for research consideration must be the control of hot flashes and menopausal symptoms; a feeling of well-being is essential in any new agent. A compound that mimics estrogen in the brain would be a major advantage for patient compliance and in the long run, an estrogen-like effect may also prevent Alzheimers syndrome. (Tang et al., 1996). The properties of an ideal agent that could be employed more widely, for example as a hormone replacement therapy, or a prevention maintenance therapy in postmenopausal women, are illustrated in Fig. 7. If these properties can be achieved,

Pfopertiesofldeal TargetedAntiestrogens - rnk*

Value of ideal Targeted Andestogens Eliminate6 hd Hashea

Fig. 7. Properties of an ideal targeted antiestrogen to be developed as a new hormone replacement therapy, or prevention maintenance therapy, for postmenopausal women.

Molecular

Mechanisms

and Future Uses of Antiestrogens

189

the agents could be applied to a host of novel situations throughout medicine as treatments and preventatives for osteoporosis and coronary heart disease. To this end we will describe the progress that has been made during the past decade to achieve the three separate goals: the development of a safer antiestrogen, a pure antiestrogen and a targeted antiestrogen.

Discovery of New Antiestrogens The intense investigation of the pharmacology of tamoxifen during the 1970s and 1980s established a data base for the development of new compounds to treat breast cancer and for broader therapeutic applications. During the 1960s and 1970s all the availablle antiestrogens were noted to have a low affinity for the ER (Jordan, 1984). This observation lead to the widely held belief that antiestrogens had to be compounds with low affinity for the ER so that the receptor complex would break up before the functions required for estrogen action could be completed (Jordan, 1984). The compounds were thought of as weak estrogens that inhibited full estrogen action by saturating all available ER. The discovery that the metabolites of tamoxifen, 4-hydroxytamoxifen and 3,4_dihydroxytamoxifen, have a high affinity for the ER and inhibit full estrogen action, changed the understanding of antiestrogen action (Jordan

METABOLITE

MIMICRY N’

P-’

4-HYDROXYTAMOXIFEN

DROLOXIFENE

Fig. 8. The mono- and dihydrovlated been copied to develop the antiestrogen

antiestrogenic metabolites of tamoxifen that have TAT-59 (a pro drug) and droloxifene (3-hydroxytamoxifen).

190 et al.,

V. C. Jordan and W. J. Gradishar

Receptor binding and biological in the same molecule.

1977).

functions

activity

are now viewed

as two separate

The principle of an antiestrogen with high affinity for the ER has been exploited with the antiestrogens, TAT-59 and 3-hydroxytamoxifen (droloxifene), that mimic the metabolites of tamoxifen (Fig. 8). TAT-59 is a phosphorylated derivative of 4-hydroxytamoxifen that requires dephosphorylation to the active antiestrogen (Toko et al., 1992). In contrast, droloxifene is derived from the tamoxifen metabolite 3,4_dihydroxytamoxifen. This metabolite of tamoxifen is unusual because it has antiestrogenic activity in both rat and the mouse uterine weight tests (Jordan et al., 1977, 1978), whereas tamoxifen and 4-hydroxytamoxifen are both classified as estrogen in short-term mouse assays (Jordan et al., 1977). 3,4_Dihydroxytamoxifen is very unstable and is readily deactivated by the enzyme catechol ortho methyl transferase so this tamoxifen metabolite would be unpromising as a therapeutic agent (Jordan et al., 1984). However, removal of the 4-hydroxy to produce droloxifene retains antiestrogenic properties (Roos et al., 1983) and, most importantly, antitumor properties. The Eli Lilly Company has made triphenylethylene structure but

a systematic study of compounds that avoid the retain potent antiestrogenic properties. The

NAFOXIDINE

TRIOXIFENE Fig. 9.

A comparison

of the structures

of nafoxidine

and trioxifene.

Molecular Mechanisms and Future Uses of Antiestrogens

191

antiestrogen trioxifene was discovered (Jones et al., 1979) during the late 1970s and a comparison of the structure of trioxifene with nafoxidine illustrates that the main differe:nce is a methanone bridge that alters the triphenylethylene-like structure (Fig. 9). Trioxifene is active as an antitumor agent in the laboratory (Rose et al., 1981) and in the .treatment of advanced breast cancer (Manni et al., 1981; Witte et al., 1986; Lee et al., ‘1986), but it does not have the severe side-effects noted earlier with nafoxidine (Legha et al., 1976). Although the drug showed some promise it has not been developed as a clinically useful agent because side-effects were believed to be a potential problem when compared to tamoxifen.

In contrast to tamoxifen the novel antiestrogens, LY117018 and LY156758 (Fig. lo), have a high affinity for the ER and are less estrogenic in the rat and mouse uterus than tamoxifen (Black et al., 1983; Jones et al., 1984; Jordan and Gosden, 1983a, 1983b) as well as potent inhibitors of estrogen-stimulated effects in vitro (Lieberman et al., 1.983; Scholl et al., 1983; Poulin et al., 1989). The compounds exhibit antitumor properties in animal models but are not superior to tamoxifen (Clemens et al., 1983; Wakeli,ng and Valcaccia, 1983; Gottardis and Jordan, 1987). However, and perhaps most importantly, the weakly estrogenic, antiestrogen keoxifene (LY1.56758) was found to maintain bone density in the ovariectomized rat (Jordan et al., 1987). The

4-HYDROXYTAMOXIFEN (1977)

2) /

0

LY117018 (1980)

r/0

0

LY156,758

LY 139,481

KEOXIFENE (1982)

RALOXIFENE (1994)

Fig. 10. The development of the novel, weakly estrogenic, antiestrogen LY 117018 from the tamoxiffzn metabolite 4-hydroxytamoxifen Subsequent structure-activity relationships improved the estrogenic/antiestrogenic ratio while retaining high affinity for the estrogen receptor.

V. C. Jordan and W. J. Gradishar

192

drug has subsequently been renamed treatment of osteoporosis following 1994; Sato et al., 1995) of the original

raloxifene and has entered clinical trials for the confirmation (Evans et al., 1994; Black et al., animal studies (Jordan et al., 1987).

The possibility that a pure antiestrogen could be developed with high binding affinity for the ER combines the observation that MER-25, the first antiestrogen, has virtually no estrogenic properties in any animal species (Lerner et al., 19.58), with the knowledge that binding affinity and biological activity are separate functions of the same molecule (Jordan et al., 1977). The antiestrogens ICI 164,384 and ICI 182,780 are derivatives of estradiol with an optimal binding affinity for the ER, but these structural analogues are unique because they do not have any estrogenic properties

Alkylating Antiestrogens

Affinity Chromatography

Long Alkylating Side Chains

Pure Antiestrogens

Laboratory Studies

HO

I

I

OH

ICI164,364

0 OH

Clinical Studies

HO

ICI 182,780 Fig. 11. The discovery of pure antiestrogens occurred via an investigation of 6,7 substituted alkylating derivatives of estradiol and attempts to purify the estrogen receptor using affinity chromatography linked to estradiol through the 7-a position. ICI 164,384 has been used extensively in laboratory studies to describe the unique pharmacology of this class of drugs. ICI 182,780 is being evaluated clinically to treat advanced breast cancer.

Molecular

Mechanisms

and Future Uses of Antiestrogens

193

and they have a novel subcellular mechanism of action (Wakeling, 1994) (see below). The serendipitous discovery of pure antiestrogens occurred through two essentially unsuccessful research endeavors that converged thus providing the optimal intellectual environment for new drug discovery (Fig. 11). Derivatives of estradiol or estrone substituted in the 6 and 7 position were being evaluated as potential alkylating antiestrogens in the late 1970s through an ICI-Leeds University joint research scheme (Jordan et al., 1981). Independently, scientists in France were attempting to purify the ER using estradiol linked at the 7 position through a tenmembered carbon side chain to Sephadex columns (Bucourt et al., 1978). Dr Alan Wakeling brought both of these independent ideas together to discover the structurefunction relationships of a new class of compounds that have no estrogenic properties in any test system (Wakeling and Bowler, 1987; Bowler et al., 1989; Wakeling et al., 1991). The pure antiestrogen ICI 164,384 has been used extensively in laboratory studies (Wakeling, 1994), but the more potent ICI 182,780 (Wakeling et al., 1991) is

TAMOXIFEN o-N< I

TOREkFENE

IDOXIFENE Fig. 12.

Substituted

tamoxifen

analogs.

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V. C. Jordan and W. J. Gradishar

being evaluated as a second-line therapy for the treatment of breast failure of long-term adjuvant tamoxifen (Howell et al., 1995).

cancer

after the

Finally, there has been a search for the past 1.5 years for tamoxifen derivatives that might have less clinical toxicity (Fig. 12). T oremifene, or chlorotamoxifen (Kangas et al., 1986; Kangas, 1990a), is less potent than tamoxifen as an antiestrogen (DiSalle et al., 1990) and an antitumor agent (Robinson et al., 1988) and this has translated into higher daily doses of toremifene being used in clinical trials to treat advanced breast cancer (Hayes et al., 199.5). The most interesting feature of the toxicology is that the compound does not produce liver tumors in rats (Hard et al., 1993; Hirsimaki et al., 1993). Clearly this property of toremifene could become important if tamoxifen is proven to be carcinogenic in the human liver. In contrast to toremifene, idoxifene (Fig. 12) was designed to be metabolically resistant so that there would be less likelihood of carcinogenic potential. Substitution of a halogen atom at the 4 position of tamoxifen to prevent metabolic activation to 4-hydroxytamoxifen is known to reduce antiestrogenic potency (Allen et al., 1980). It is therefore an advantage, but not a requirement, for the antiestrogen tamoxifen to be metabolically activated (Allen et al,, 1980; Lieberman et al., 1983). To exploit this concept, idoxifene was designed with an iodine atom at the 4 position of tamoxifen to prevent toxicity through 4-hydroxylation and a pyrrolidino side chain to avoid theoretical toxicities associated with demethylation (McCague et al., 1989, 1990). The compound is entering clinical trial but it is unclear whether idoxifene will be protected from carcinogenic potential. In summary numerous new compounds have been discovered that are now actively being evaluated in clinical trial and should be approved for different treatment applications within the next 5 years. However, before we consider the current status of the evaluations of each new agent we will briefly describe the subcellular mechanism of actions of the two principal classes of agents: compounds based on the tamoxifen molecule and the pure antiestrogens. This is important because it provides insight into the current problems of understanding target tissue responses and the complex issue of drug resistance to antihormones.

Chapter

5

Molecular Mechanisms of Antiestrogen Actions

Subcellular Mechanism of Action of Antiestrogens in Breast Cancer The recognition that the ER is a nuclear transcription 1991) which bind estrogens or antiestrogens and can enormous interest replicate or not, has focused antiestrogen action. A generally accepted model for Fig. 12;. Estradiol (or indeed any estrogen) binds produces a change in shape to expose fully the DNA

factor program on the estrogen to the binding

(Green and Chambon, breast cancer cells to basic mechanism of action is illustrated in nuclear ER that then domain on the protein

STOP GROWTH lTamoxifen Analogues

Fig. 13. Molecular model of estrogen actions in the breast cancer cell. Estradiol (E2) binds to the nuclear estrogen receptor (ER) which changes shape to bind to estrogen response elements (EREs) that control the transcription of estrogen responsive genes. All antiestrogens can bind to the ER but cause an inappropriate change in shape so that binding to the EREs is imperfect. The pure antiestrogens have an additional mechanism of action by promoting the cytoplasmic destruction of the newly synthesized ER.

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complex. The activated receptor complex then dimerizes and binds to an estrogen response element (ERE) located in the promoter region of estrogen responsive genes. Once bound to the ERE, the ER acts as an anchor for other transcription factors that when assembled and associated with RNA polymerase, produces a transcription complex. The estrogen responsive gene is then transcribed and subsequently translated to proteins that are either involved in growth responses or differentiation responses, for example progesterone receptor synthesis. The regulation of the ER is complex and not clearly understood, although some general principles can be stated that appear to be important for the development of hormone independent growth. Only estrogens will cause synthesis of the ER while other steroid hormones (e.g. progesterone) are inactive. Nevertheless, there appear to be two models of receptor regulation (Pink and Jordan, 1996) (Fig. 14). In Model 1, MCF-7 breast cancer cells have their ER downregulated by estradiol. Conversely, in times of estrogen deprivation ER levels increase (Welshons and Jordan, 1987; Katzenellenbogen et al., 1987) and estrogen independent clones emerge (Jiang et al., 1992; Pink et al., 1995, 1996a, in press). However, antiestrogens are still effective at regulating growth in short-term laboratory experiments. Ultimately the drug resistance and tumor growth that is observed during indefinite tamoxifen therapy occurs despite the fact the tumor remains ER-positive. It is clear that the drugreceptor complex has discovered new ways of activating growth through the ER. In Model II, exemplified by the T47D cell line, estrogen is necessary to induce the synthesis of the ER and maintain the sensitivity of the cell to the influences of estrogen and antiestrogens. During prolonged periods of estrogen deprivation, the ER

Model I (Inhibitory) E2

,_,---mRNA +--___.\ /’ 1’

IiR

-

Ek

*

\

\

REGULATORY MECHANISM

ER ER

Model II (Stimulatory) E2

-

REGULATORY * MECHANISM

ER ER ERER

ER

Fig. 14. The model systems for the regulation of estrogen receptor function in target tissues. Either the estrogen receptor is down regulated by estrogen (Model I) or upregulated by estrogen (Model II). There would clearly be a physiological need, in rapidly responding tissues, to sequester estrogen rapidly. High concentration of receptor would then be down regulated once estrogen appears. In contrast, sustained, protein synthetic responses of a target tissue, that occur periodically, would require a receptor mechanism that is switched off in the absence of estrogen (Model II) (Pink and Jordan, 1996).

Molecular Mechanisms and Future Uses of Antiestrogens

197

can be lost (Murphy et al., 1992; Pink et al., 1996b) with an associated refractoriness to antiestrogens. This method of drug resistance may be important during long-term total estrogen withdrawal in patients (i.e. sequential combinations of aromatase inhibitors and pure antiestrogens). It is possible that the two mechanisms for regulating the ER may be different stages in the evolution of complete hormone independent growth. Unfortunately we currently have little information about the true state of affairs in breast tumors, but it would be reasonable to assume that because breast tumors are heterogeneous they will contain equilibrium mixtures of cells each seeking to survive through selection in an antiestrogenic environment. The current molecular model of estrogen action provides several potential points of weakness that can be exploited by antiestrogens. It is now apparent that antiestrogens can be divided into two major categories based on their mechanism of action. Type I antiestrogens are the analogs of tamoxifen or structural derivatives of the triphenylethylene type of drug. Type II are the pure antiestrogens. All compounds are competitive inhibitors of the binding of estradiol to the ER but there the similarity ends. Type I antiestrogens appear to form a receptor complex that is incompletely converted to the fully activated form (Tate et al., 1984; Martin et al., 1988; Pham et al., 199 1; Tzukerman et al., 1994; McDonnell et al., 1995; Allan et al., 1992). As a result of the imperfect changes in the tertiary structure of the protein, the complex is only partially active in initiating the programmed series of events necessary to orchestrate gene activation (Metzger et al., 1988; Jordan, 1984). Studies in vitro demonstrate that very low concentration of triphenylethylene-type antiestrogens can cause a single round of replication in breast cancer cells but high concentration of these antiestrogens are completely inhibitory (Berthois et al., 1986). It is possible that the modest partial estrogen-like action at low concentrations causes the tamoxifen flare that is sometimes observed when therapy is started in patients with boney metastases (Reddel and Sutherland, 1984). Once steady state levels of the drug have been achieved (approximated 4-8 weeks with 20 mg/day), symptoms will have dis’appeared and the patient will experience a response to therapy (Furr and Jordan, 1984). It is important therefore, to be able to identify tumor flare and not prematurely terminate a beneficial therapy. Nevertheless, a recent report (Vogel et al., 1995) has demonstrated that clinicians often prematurely terminate antiestrogen treatment based on changes in bone scintigraphy misinterpreted as progressive disease. Since there are clear toxicological advantages in disease control with antiestrogens, a premature change to chemotherapy may be inappropriate. Several type II antiestrogens are available for study in the laboratory (Wakeling, 1994; Van de Velde et al., 1994; Dukes et al., 1994; von Angerer et al., 1990) but only ICI 182,780 is being developed clinically (Wakeling et al., 1991). Initially it was believed that pure antiestrogens prevent the dimerization of receptor complexes thereby preventing binding to EREs (Fawell et al., 1990). Clearly if receptor complexes do not bind to any EREs then no genes can be activated and the compound would be a ‘pure’ antiestrogen. However, numerous reports (Pink and Jordan, 1996; Pham et al., 1991; Salbbah et al., 1991) now demonstrate that pure antiestrogen-ER complexes can

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bind to EREs but the transcriptional unit is inactive. What is unique about the type II antiestrogens is the observation that they provoke the destruction of the ER in breast cancer cells in culture (Dauvois et al., 1992), mouse uterus (Gibson et al., 1991) and in breast tumors in situ (DeFriend et al., 1994). The ER is synthesized in the cytoplasm and transported to the nucleus where it functions as a transcription factor. A pure antiestrogen binds to the newly synthesized receptor in the cytoplasm and prevents transport to the nucleus (Davois et al., 1993). The paralyzed receptor complex is then rapidly destroyed (Davois et al., 1993). The complete destruction of available ER will prevent any estrogen regulated events from occurring. Normal cells will become quiescent whereas hormone dependent tumors will rapidly regress because senescent tumor cells cannot be replaced by replication. The elucidation of the subcellular model for the molecular mechanism of action of estrogen has provided an insight into the inhibitory actions of antiestrogens in breast cancer. Most of the work has been completed on tumor cells in culture and extrapolated to the clinic. However, the pharmacology of antiestrogens is complex and the target site specific effects observed in viva on bones and lipids are not adequately explained by studies in breast cancer cells. Alternate explanations for the estrogenic effects of tamoxifen are currently being sought at the molecular level.

Target Site Specific Effects of Antiestrogens Although studies of the molecular biology of the ER have gone some way in explaining the inhibitory effects of antiestrogens there is currently no universal explanation for the diverse pharmacological effects observed with tamoxifen in women. Tamoxifen appears to exhibit inhibitory effects on breast cancer growth but produce estrogen-like effects in postmenopausal women that maintains bone density (Turken et al., 1989; Love et al., 1992, 1994; Ward et al., 1993; Kristensen et al., 1994)) reduces circulating gonadotropins, luteinizing hormone (LH) and folliclestimulating hormone (FSH) (J or d an et al., 1987) and produces estrogen-like effects on vaginal cytology (Boccardo et al., 1981). The ER has been cloned and sequenced (Green et al., 1986; Greene et al., 1986), and, until the discovery of ER,8, there was no evidence that there were different ER receptors located at different sites around a woman’s body. However, at least two alternative mechanisms, illustrated in Fig. 15, could be used by the antiestrogen-ER complex to stimulate the selective activation of estrogen responsive genes. We will consider each hypothesis in turn: the role of associated proteins in the cell nucleus and the concept of an ‘antiestrogen response element’ (AERE). ER-associated proteins (Halachmi et al., 1993) may be differentially located at target sites throughout a woman’s body and these could be selectively used by the cells to govern estrogen regulated growth or differentiation. The molecular events needed to provoke cells to pass through the cell cycle have to be quantitatively and qualitatively precise. Any disruption of the correct sequence or defect in the amounts of required catalysts would lead to inappropriate scheduling of replication which clearly would be catastrophic for a growing tumor that depends for its survival on the equilibrium between replicating and dying cells. It is possible that the changes in the tertiary

Molecular

Mechanisms

199

and Future Uses of Antiestrogens

No replication

)’

*Differentiation, prolonged response

_)

\;-_

7

differentiation only

replication and differentiation

Fig. 15. A model of estradiol (E,) action at physiological sites around a woman’s body. (a) The nuclear estrogen receptor (ER) may bind to associated proteins (ASP) to modulate the rapid response of replication or the prolonged effects of differentiation. There are currently two potential models to describe the estrogen-like effects of tamoxifen at physiological sites. (b) The antiestrogen (AE) binds to the ER but the ASP have only a weak affinity for the receptor complex and cannot support transcription of genes necessary to support replication. types of receptor The relentless signaling may, however, support other differentation responses because the EREs are less specific. (c) The AE binds to the ER, but the AER complex interacts with an antiestrogen response element (AERE) that triggers differentiation only. The AER blocks EREs but the AERE plays a dominant role.

200

V. C. Jordan and W. J. Gradishar

shape of the antiestrogen-ER complex may be inappropriate to bind to associated proteins to accomplish the rapid responses needed for replication. The low affinity, and perhaps the inability of the cells to create higher concentrations of associated proteins, might cause a paralysis in the ability of the breast cancer cell to survive. In contrast, the physiological effects of tamoxifen on bones, cholesterol and gonadotropins depend on long-term signaling that lasts days, months or even years for completion. Normal cells may be able to respond to the signal from the altered ER complex, by either synthesizing more associated proteins over days and months to establish the appropriate signal transduction pathway, or by producing the correct expansion of estrogen responsive pathways. Overall the partial estrogen-like response would build over time in selected normal tissues to produce an estrogenic effect. In contrast, cell replication in a tumor could only succeed through selection of resistant clones that can exploit the weak estrogen-like effects of antiestrogens by expansion of the pool of associated proteins necessary for replication. The second potential mechanism to explain the estrogen like effects of antiestrogens depends on the antiestrogen-ER complex using an alternate response element from the ERE in the promoter region of estrogen responsive genes. The concept depends upon the ability of the antiestrogen-ER complex to alter the shape of the protein so that binding to the ERE is inappropriate for gene activation but the complex can seek out an alternate site in the promoter region to initiate gene transcription (Yang et al., 1996). This could be an AERE. It is possible that the explanation for the target site specific action of type I antiestrogens could be any one or a combination of the hypotheses illustrated in Fig. 15. However, it is important to elucidate the precise mechanism because the idea of being able to target antiestrogens to switch on or switch off gene function in specific sites has fundamental importance for the prevention of osteoporosis, coronary heart disease, breast and endometrial cancer. With this background of tamoxifen’s toxicology, novel drug discovery mechanisms of actions of antiestrogens we will now consider the progress been made in the development of new clinically useful antiestrogens.

and that

the has

Numerous compounds are being evaluated in clinical trial but for convenience have divided them into three major categories based on their proposed applications.

we

Triphenylethylenes

(tamoxifen

analogs)

Pure antiestrogens Targeted

antiestrogens

We will consider each new compound by discussing support the clinical testing currently in progress.

the

laboratory

evidence

to

Chapter

6

Triphenylethylenes (Tamoxifen Analogs)

Toremifene Toremifene (Fig. 12) or chlorotamoxifen began development in Finland by Farmos Pharmalceuticals (Orion) in 1978. The drug is FDA approved for the treatment of metastatic breast cancer and is marketed in the US under the trade name of Farnesdonm by Scheering Plough, New Jersey. The recommended treatment regimen for metastatic breast cancer in postmenopausal women is at least 60 mg daily.

Laboratory Studies Toremifene has a biphasic effect on the growth of ER-positive, MCF-7 breast tumor cells in vitro (Kangas et al., 1986; Kangas, 1990a). At low concentrations (10h7 to 10W6 M) toremifene is an estrogen antagonist but at high concentration ( > lop6 M) toremifene is oncolytic and the effects cannot be reversed by estrogen (Warri et al., 1993; Grenman et al., 1991; Robinson et al., 1990). Nevertheless, toremifene can be washed out of the cells and estradiol can re-stimulate growth. Toremifene and other antiestrogens increase the production of TGF-/? (Colletta et al., 1990; Knabbe et al., 1991) and toremifene-induced cell death has been documented as apoptotic (Warri et al., 1993). Toremifene is an antiestrogen in the immature rat uterine weight test and exhibits the properties of a partial estrogen antagonist (Kangas, 1990b; DiSalle et al., 1990) (i.e. toremifene causes modest increases in uterine weight when administered alone). Toremifene is effective in controlling the growth of DMBA-induced rat mammary carcinomas (DiSalle et al., 1990; Huovinen et al., 1993, 1994, 1995), but appears to be approxirnately l/3 less potent than tamoxifen (Robinson et al., 1988). This is consistent with the larger dose of toremifene (60 mg daily) that is used clinically compared with tamoxifen (20 mg daily). The MCF-7 (ER-positive) and MDA-MB-231 (ER-negative) breast cancer cell lines will grow in athymic mice. However, toremifene will only block the estrogenstimulatmed growth of MCF-7 cells but does not inhibit the growth of MDA-MB-231 cells (Robinson and Jordan, 1989). The drug does not control the growth of mixed tumors containing both MDA-MB-231 and MCF-7 cells (Robinson and Jordan, 1989). Nevertheless, toremifene has been reported to have a cytolytic effect on controlling the growth, in vivo, of an ER-negative, glucocorticoid sensitive mouse uterine

V. C. Jordan and W. J. Gradishar

202

can produce acquired resistance sarcoma (Kangas et al., 1986). Toremifene long-term therapy. Athymic animals implanted with MCF-7 cells will eventually tumors in response to toremifene treatment (Osborne et al., 1994).

after grow

Toxicology Toremifene does not produce a mutagenic effect in either the Ames test or the sister chromatid exchange assay (Kangas et al., 1986; Styles et al., 1994). Large daily doses (5-20 mg) o f t oremifene do not produce DNA adducts in the rat liver and long-term therapy does not result in hepatocarcinogenesis (Hard et al., 1993; Hirsimaki et al., 1993). However, recent studies demonstrate that large doses of toremifene (750 mg toremifene/kg food) can promote rat liver and kidney carcinogenesis (Dragan et al., 1995).

Clinical Pharmacology and Endocrinology Toremifene is extensively metabolized in animals and patients(Antilla et al., 1990; Kangas, 1990b). The principal metabolites are shown in Fig. 16. Toremifene can be

CI

Toremifene

N-desmethyltoremifene

-a HO

&

4 hydroxytoremifene

c1

T6k IV

TOR VI

Fig. 16. The metabolites of toremifene described in animals and man. The routes appear to be the same as those previously described for tamoxifen (Jordan, 1984), demethylation and deamination of the dimethylaminoethoxy side-chain and 4-hydrovlation to a metabolite with high binding affinity for the ER.

Molecular

Mechanisms

and Future Uses of Antiestrogens

203

measured using high performance liquid chromatography (HPLC) (Wiebe et al., 1990; Webster et al., 1991; Berthou and Dreano, 1993; Bishop et al., 1992) and the time to steady state (Wiebe et al., 1990; Bishop et al., 1992; Kohler et al., 1991) and terminal elimination half-life (Antilla et al., 1990) have been determined as 2 weeks and 5 days, respectively. Toremifene exhibits weak estrogen-like properties in the postmenopausal patient. LH and FSH are slightly depressed during therapy and sex hormone binding globulin (SHBG) is increased (Hamm et al., 1991; Kivinen and Maenpaa, 1990; Szamel et al., 1994). Although toremifene exhibits antiestrogenic effects on the vaginal mucosa of estrogen-primed women (Homesley et al., 1993; Maenpara et al., 1990), there is no effect in blocking short-term estrogen action in the uterus ((Maenpaa et al., 1990). Toremifene and tamoxifen produce the same estrogenlike effects on the histology of the postmenopausal endometrium (Tomas et al., 1995).

Clinical Evaluation The initial phase I studies, started in the early 198Os, demonstrated that toremifene was well1 tolerated with minimal toxicity, and activity in breast cancer was observed (Wiebe et al., 1990; Hamm et al., 1991; Tominga et al., 1990). Several phase II clinical trials of toremifene have been reported (Table 3) in postmenopausal patients with advanced disease, who did not receive prior hormonal or cytotoxic chemotherapy (Valavaa.ra et al., 1988; Gunderson, 1990; Modig et al., 1990; Valavaara and Pyrhonen, 1989; Hietanen et al., 1990; Valavaara, 1990). In 46 previously untreated patients with ER-positive, metastatic breast cancer, Valavaara et al. (1988) reported a 63% objective response rate (CR-37%; PR-26%) following treatment with toremifene (60 mg/day, PO). Responses were observed in soft tissue and visceral sites of disease. No significant differences in response rates could be detected when related to different ER concentrations. Toxicity was mild with hot flashes occurring in 22% of patients. Gunderson (1990) reported a 48% response rate, including six complete responders, using a:n identical treatment schedule of toremifene in a group of 23 patients with advanced disease in whom 20 had received no prior therapy. The median duration of complete and partial responders was 14 and 1.5 months, respectively. Hot flashes were reported in approximately half of the patients. Similar findings have been reported by Modig et al. (1990). In an effort to determine if lower daily doses of toremifene were equally effective as higher doses, toremifene treatment was evaluated by initially administering an oral loading dose: 120 mg on day 1, 60 mg on days 2 and 3, and thereafter 20 mg daily (Valavaara and Pyrhonen, 1989). Of the 14 patients treated according to this schema, three achieved a partial response (21%) and seven achieved stabilization of disease. No complete responses were observed. In a multicenter study involving 38 patients with advanced disease, oral toremifene was administered at a dose of 240 mg/day (Hietanen et al., 1990). An objective response rate of 68% was observed (CR-26%; PR-42%). This study suggested that higher doses of toremifene are more effective in inducing objective responses compared to low-dose toremifene (20 mg/day). Toremifene in patients

has been directly compared with advanced disease (Table

to tamoxifen as first-line hormonal therapy 4) (Hayes et al., 1995; Nomura et al., 1993;

204

V. C. Jordan and W. J. Gradishar

Konstantinova and Gershanovich, 1990; Stenbygaard et al., 1993). Three small randomized trials, lacking the statistical power to make a valid comparison between tamoxifen and toremifene, have been published. In a randomized trial comparing toremifene (40 mg/day, p.o.) to tamoxifen (20 mg/day, p.o.), Nomura et al. (1993) observed similar response rates and median time to disease progression in both treatment arms. Konstantinova and Gershanovich (1990) reported on 47 patients randomized to one of two doses to toremifene (60 mglday, p.o. or 240 mglday, p.o.) or tamoxifen (40 mglday, p.0.). Patients treated with 60 mglday of toremifene had a response rate of 50% compared to 35% in the higher dose toremifene arm and 36% in the tamoxifen arm. In contrast, Stenbygaard et al. (1993) reported inferior response rates for patients treated with toremifene (240 mg/day, p.o.) compared to tamoxifen (40 mg/day, p.0.). In a recently reported international trial (Hayes et al., 1995), 648 previously untreated, hormone receptor-positive or -unknown, metastatic breast cancer patients were randomized between tamoxifen (20 mglday, p.o.) or two different doses of toremifene (60 or 200 mg/day, p.0.). Tamoxifen produced a response rate of 19% and a median survival of 32 months. Toremifene produced a response rate of 21% with the 60 mg dose and 23% with the 200 mg dose. Median survival of toremifene-treated

Table 3. Phase II trials of toremifene in postmenopausal women with advanced breast cancer with no prior endocrine therapy of chemotherapy Study

Patient population

Dose/schedule

Response

Toxicity

Valavaara et al. (1988)

46 postmenopausal ER+, metastatic breast cancer

60mgpoqd

Mild, hot flashes-22%

Valavaara and Pyrhonen (1989) Gunderson (1990) Hietanen et al. (1990) Modig et al. (1990)

14 postmenopausal, ER+ or unknown

20mgpoqd

RR CR SD 54% 17% 26% Soft tissue, visceral sites most likely to respond RR CR SD 21% 0 58%

23 postmenopausal, ER+ or unknown 38 postmenopausal, ER+ or unknown 12 postmenopausal, hormone receptor + or unknown, inoperable or metastatic breast cancer 113, ER+, postmenopausal, metastatic breast cancer. First-line rx for advanced disease.

60mgpoqd

Valavaara (1990)

240 mgpoqd 60mgpoqd

60mgpoqd

RR 48% RR 68% RR 50%

CR 26% CR 26% CR 25%

SD 26% SD SD 42%

RR CR SD 45% 15% 35% Duration of response correlated with ER concentration

Mild, hot flashes-28% Mild Mild None

NA

Abbreviations: rx, treatment; RR, response rate; po, oral; CR, complete response; qd, daily; SD, stable disease; NA, not available.

Molecular

Mechanisms

and Future Uses of Antiestrogens

205

patients was 38 months (60 mg/day) and 30 months (ZOOmg/day). The median time to disease progression was not statistically different between treatment arms (Hayes et al., 1995). Response rates, times to disease progression and overall survival for patients on each arm were superior for ER-positive patients compared to ER-negative patients. However, no statistical difference in any of these endpoints was detected between treatment arms. Furthermore, quality-of-life assessments were not different between treatment arms. Toxicity was mild in all patients, but toremifene-treated patients experienced less nausea (26% versus 37%). The data from this large trial (Hayes et al., 1995) support the use of toremifene as an alternative first-line therapy to tamoxifen in hormone receptor-positive, postmenopausal patients with advanced disease. Toremifene appears to have cross-resistance to tamoxifen, since overall response rates to toremifene following tamoxifen therapy in several phase II studies are low

Table

4. Clinical

trials

Study

comparing

toremifene to tamoxifen advanced breast cancer

Patient population

Dose/ schedule

Konstantinova and Gershanovich (1990)

Nomura

Toxicity

RR

CR

NA

TOR 60 mg

50%

0

17

PO qd TOR240mg

35%

6%

14

PO qd TAM 40 mg

36%

1%

PO qd 57

TOR 40 mg

RR 26%

CR 14%

57

PO qd TAM 20mg

28%

5%

PO qd

et al. (1993)

patients

Response

16

et al. (1993)

Stenbygaard

in postmenopausal

with

NA

31

TOR240mg

RR 29%

CR 3%

31

PO qd TAM 40 mg

42%

16%

RR 19%

CR 5.8

SD 31.7

Mild; nausea 37%

TOR 60 mg

21%

5.6

38

26%

PO qd TOR 200 mg

23%

5.6

30.1

26%

PO qd

Hayes et al. (1995) 648, peri- or post-menopausal hormone receptor+ or unknown metastatic breast cancer

TAM 20 mg

NA

PO qd

PO qd Abbreviations: TAM, tamoxifen; RR, response rate; TOR, oral; SD, stable disease; qd, daily; NA, not available.

toremifene;

CR, complete

response;

po,

V. C. Jordan and W. J. Gradishar

206

(Table 5) (Ebbs et al., 1990; Hindy et al., 1990; Jiinsson et al., 1991; Asaishi et al., et al., 1994). The largest experience was a 1993; Vogel et al., 1993; Pyrhonen multicenter trial reported by Vogel et al. (1993) in which 102 perimenopausal or postmenopausal women with metastatic breast cancer, refractory to tamoxifen, received 200 mg/day of toremifene. Patients in this trial were heavily pretreated with 65% having failed chemotherapy and 72% having failed two or more hormonal therapies. Forty-nine percent of patients had visceral-dominant disease. The objective

Table 5. Clinical trials of toremifene

in patients

with advanced breast cancer refractory

to tamoxifen

Study

Patient population

Dose/schedule

Response

Toxicity

Ebbs et al. (1990)

9 TAM-refractory metastatic breast cancer 17 postmenopausal metastatic breast cancer; prior hormonal rx or RT if d/c 2 months prior 35 TAM-refractory, ER+ or unknown, post-menopausal inoperable or metastatic breast cancer 5 1 TAM-refractory, ER+ or unknown metastatic breast cancer 50 TAM-refractory, ER+ metastatic breast cancer. 48% patients with disease progression during adjuvant TAM 52% patients with advanced disease 102 TAM-refractory, metastatic breast cancer. I: 28-1” refractory II: 43-relapse after TAM response III: 31-relapse while receiving TAM 831102 assessable 81% ER+ and/or PRt 50% visceral disease

200 mg po qd

RR 33%

CR 0%

Mild

60, 120, or 300 mg po

RR 25% 33%

CR 0% 0%

Hindy et al. (1990)

Jonsson et al. (1991)

Asaishi et al. (1993)

Pyrhonen et al. (1994)

Vogel et al. (1993)

qd

240 mg po qd

120 mg po qd

120 mg po bid

(60 mg) (300 mg)

Mild

RR SD 0% 26% TTP in SD 8 months

RR CR 14% 0 TTP in SD 6 months RR CR 4% 2%

Minimal; nausea, hot flashes

SD 19%

NA

SD 22%

Minimal: sweating 12%: nausea 4%

SD 23%

Minimal: nausea, hot flashes, dry eyes

TTP in SD 2 months 200 mg po qd

RR 5%

CR 2%

TTP in SD 8 months

Abbreviations: TAM, tamoxifen; po, oral; CR, complete response; NA, not available; qd, daily; SD, stable disease; rx, treatment; RR, response rate; TTP, time to progression.

Molecular

Mechanisms

and Future Uses of Antiestrogens

response rate was 5% with only two patients achieving a CR. An additional patients maintained stable disease status for a median of 8 months.

207

23% of

Toremifene was observed to have an antitumor effect in mice with ER-negative uterine sarcomas and, as a result, a cytolytic effect independent of the estrogen receptor was postulated (Kangas et al., 1986). In a small trial of nine patients with ER-unknown breast cancer progressing following tamoxifen therapy, 33% of patients (3/9) responded to toremifene (200 mg/day, p.o.) (Ebbs et al., 1990). In an effort to confirm a mechanism of action independent of the ER, the Cancer and Leukemia Group B (CALGB) conducted a phase II trial to test the efficacy of high-dose toremifene (400 mgiday) in a population of patients with hormone receptor-negative, metastatic disease with limited prior chemotherapy exposure (Perry et al., 1995). Twenty patients were enrolled, but no objective responses were observed. These findings reaffirm that toremifene is primarily active in patients with hormone receptor-positive, metastatic breast cancer.

Droloxifene Droloxifene or 3-hydroxytamoxifen (Fig. 8) began development in Germany Pharmaceuticals in the late 1970s and subsequently by Fujisawa in Japan. has been tested for the treatment of metastatic breast cancer but is being by Pfizer for the treatment of osteoporosis in postmenopausal women.

by Klinge The drug developed

Laboratory Studies N-desmethyldroloxifene Droloxifene (Roos et al., 1983) and its major metabolite (Loser et al., 1985a) have a ten-fold higher binding affinity for MCF-7 (ER-positive) breast cancer cells than tamoxifen. Droloxifene inhibits estrogen-stimulated cell et al., 1991; Kawamura et al., 1993) by replication (Loser et al., 1985b; Eppenberger appears to be arresting cells in GdG, (Hasman et al., 1994). In addition, droloxifene a more potent inducer of TGF-/I than either tamoxifen or toremifene (Knabbe et al., 1991). Similarly droloxifene inhibits IGF-l-stimulated growth of MCF-7 cells (Hasman et al., 1994). In contrast, droloxifene does not inhibit the growth of the ERthat negative cell line, MDA-MB-231 (Kawamura et al., 1993). This demonstrates droloxifene is active through the ER. Droloxifene exhibits antiestrogenic activity in the immature rat uterine weight test but also causes a partial increase in uterine wet weight when administered alone action of (Wosikiowski et al., 1993; Loser et al., 1985b). The partial agonist droloxifene is only slightly less potent than tamoxifen (Eppenberger et al., 1991; Wosikowski et al., 1993). However, droloxifene does maintain bone density in the ovariectomized rat (Ke et al., 1995). Droloxifene exhibits antitumor activity in several rat and mouse models. Droxloxifene inhibits the growth of the transplantable rat mammary tumor R3230AC, and both DMBA-(Loser et al., 1985b; Kawamura et al., 1991) and NMU-(Winterfeld et al., 1992) induced rat mammary tumors, but only ERpositive breast tumors transplanted into athymic mice (Kawamura et al., 1993; Wosikowski et al., 1993).

208

V. C. Jordan and W. J. Gradishar

Toxicology Droloxifene does not produce DNA adducts or hepatocellular carcinomas in male or female rats fed daily doses of 36 mg/kg (Hasman et al., 1994). One male and one female rat (2% total) developed a hepatocellular carcinoma following treatment for 24 months with 90 mglkglday. By comparison, tamoxifen produced hepatocellular carcinomas in 100% animals following 36 mg/kg/day for 24 months (Hasman et al., 1994). Droloxifene is inactive in the ability to transform Syrian hamster embryo cells in vitro whereas tamoxifen and 4-hydroxytamoxifen produce a significant level of transformation (Metzler and Schiffmann, 1991).

Clinical Pharmacology and Endocrinology Droloxifene is rapidly absorbed and excreted and does not appear to accumulate like tamoxifen and toremifene. Droloxifene can be monitored in serum using HPLC (Grill and Pollow, 1991; Lien et al., 1995). Under chronic dosing conditions steady state levels of parent drug were 83.5 _+32.5 ng/ml (40 mg daily) and 146 + 115.4 ng/ml (100 mg daily) with a half-life of 28 and 27 hours, respectively. Steady state levels were achieved rapidly within 5 hours (Grill and Pollow, 1991). There are several metabolites of droloxifene (Fig. 17) and all are present in serum as both free and glucuronide conjugates. This profile contrasts with tamoxifen that does not have serum glucuronide metabolites (Grill and Pollow, 1991). Droloxifene causes a dose-related decrease in LH and FSH in postmenopausal patients (Lien et al., 1995). There is only a modest decrease at the 20 and 40 mg dose daily, whereas there is a definite decrease at the 100 mg daily dose. Similarly there is only a marginal rise in SHBG at the 20 and 40 mg daily dose but a moderate increase occurs with 100 mg droloxifene daily. These conclusions have recently been confirmed in patients treated with 40 mg droloxifene daily (Geisler et at., 1995).

Clinical Evaluation Table 6 summarizes the results of clinical trials evaluating droloxifene in patients with metastatic breast cancer (Ahlemann et al., 1988; Abe et al., 1990, 1991; Bellmunt and Sole, 1991; Haarstad et al., 1992; Raushning and Pritchard, 1994; Buzdar et al., 1994). The majority of patients on these trials had been previously treated with chemotherapy and/or hormone therapy (Ahlemann et al., 1988; Abe et al., 1990, 1991; Bellmunt and Sole, 1991; Haarstad et al., 1992; Raushning and Pritchard, 1994; Buzdar et al., 1994). The daily dose of droloxifene ranged from 20 to 300 mg. Response rates ranged from 0 to 70% with most responses occurring in peri- or postmenopausal patients. Trials evaluating different doses of droloxifene have not convincingly demonstrated a dose-response effect (Bellmunt and Sole, 1991; Raushning and Pritchard, 1994; Buzdar et al., 1994). The largest clinical trial involving patients with metastatic breast cancer treated with droloxifene was recently updated by Raushning and Pritchard (1994). This phase II study compared droloxifene in doses of 20, 40 and 100 mg daily in postmenopausal women with metastatic, inoperable recurrent, or primary locoregional breast cancer

Molecular

Mechanisms

and Future Uses of Antiestrogens

who hatd not received prior hormonal eligible: and 268 evaluable for response. 47% in the 40 mg group and 44% occurred within 2 months of starting been extremely well tolerated with flashes, fatigue and nausea.

209

therapy. Of 369 patients randomized, 292 were Response rates were 30% in the 20 mg group, in the 100 mg daily group. Most responses therapy. In all trials reported, droloxifene has the most common toxicities cited being hot

ldoxifene The drug was originally designed Sutton (Surrey, UK) and offered

at the Cancer Research to the pharmaceutical

-“<

Campaign Laboratory in industry through British

-E-

0

0 minor metabolite

HO

HO

DROLOXIFENE

N-DESMETHYLDROLOXIFENE

HO

4-METHOXYDROLOXIEENE Fig. 17 The metabolites of droloxifene described in patient serum. The drug and metabolites are rapidly conjugated to glucuronides that are the major component detected in the blood. ![nterestingly the 4-methoxy metabolite of droloxifene has been identified despite the earlier observation that 3,4_dihydroxytamoxifen is metabolically stable in vitro in the presence of an inhibitor of catechol-o-methyl transferase (Jordan et al., 1984). The enzyme would prefer to direct a methyl to the 3-hydroxyl not the 4-hydroxyl.

10 peri or postmenopausal, ER+ or unknown, metastatic breast cancer. Four received prior hormone or chemotherapy.

Ahlemann

16 pre- or postmenopausal, hormone receptor +, -, or unknown. Metastatic breast cancer. All pretreated.

94 pre- or post-menopausal, hormone receptor +, -, or unknown. Primary or recurrent advanced breast cancer. Majority pretreated.

Abe et al. (1991)

Abe et al. (1990)

et al. (1988)

Patient population

Study

qd

PO qd

20, 40, or 80 mg

120 mgpo

100 mg po q 2 or 3 days

treated patients

CR 0% 4% 0%

RR 20 mg: 14% 40 mg: 5% 100 mg: 17% Most responses in peri- or post-menopausal patients.

SD 32% 46% 48%

CR 0%

RR 29%

Side effects mild in all groups, but fewest occurred in 20 mg group.

Mild in two patients

Mild Hot Flashes

CR 30% 40% 20%

RR 70% 60% 80%

30% RR in perior post-menopausal patients. 40% RR in ER+ or unknown disease.

Toxicity

SD 43%

with advanced breast cancer Response

Overall q 2 days: q 3 days:

in previously

Dose/schedule

Table 6. Clinical trials of droloxifene

Haarstad et al. (1992)

26 postmenopausal women; 1 male. Metastatic disease. All pretreated with endocrine rx and 35% with chemotherapy

124 postmenopausal, hormone receptor + or unknown (65%); metastatic disease; 68% pretreated

Bellmunt (1991)

and Sole

Patient population _ -

Study

PO qd

100 mg

20, 40, or 100 mg PO od

Dose/schedule

20 mg: 40 mg: 100 mg:

Table 6. contd.

CR SD 0% 19% SD duration 22 weeks

RR 15%

SD 36-40%

CR 0% 3% 10%

RR 17% 30% 31%

Response

Nausea 22% Hot flashes 15% Vomiting 15% GI discomfort 18% Anorexia 15% Two patients with severe fatigue.

Toxicity

$-.

5 b 2 E 5 0

response;

20, 40 or 100, 200 or 300 mg PO qd.

20, 40 or 100 mg PO qd.

Dose/schedule

*Updated results from Bellmunt and Sole (1991). Abbreviations: po, oral; qd, daily; RR, response rate; CR, complete

Buzdar et al. (1994)

23 patients with metastatic, refractory breast cancer. ER+ or previously responsive to endocrine therapy; median number prior hormone rx-3; chemotherapy rx-2.

369 postmenopausal, hormone receptor + or unknown, metastatic or inoperable recurrent or primary locoregional breast cancer

Raushning Pritchard

and (1994)*

Patient population

Study

Mild. Hot flashes, headache, fatigue, nausea and leg cramps most common.

Adverse effects mild and equal between groups.

SD 42% 35% 27%

RR 30% 47% 44% CR 6% 8% 12%

Toxicity

Response

PR, partial; SD, stable disease.

No CR or PR 13% minor response

20 mg: 40 mg: 100 mg:

Table 6. contd.

$ ZJ

&

5 0

Molecular Mechanisms and Future Uses of Antiestrogens Biotechnology. Idoxifene development by SmithKline

(Fig. 12) is currently Beecham in the UK

undergoing

213 evaluation

and

Laboratory Studies Idoxifene has a binding affinity for the ER that is about twice that of tamoxifen and this translates to a modest increase, compared to tamoxifen, in the ability to inhibit the growth of ER-positive, MCF-7 breast cancer cells in culture (Chander et al., 1991). As would be expected with a compound that cannot be metabolically activated to the 4-hydroxy derivative (McCague et al., 1990), idoxifene is a less potent (approximately ten-fold) antiestrogen in immature rat uterine weight tests. However, idoxifene has less uterotropic activity when administered alone (Chander et al., 1991). Idoxifene demonstrates antitumor properties in the NMU-induced rat mammary carcinoma model in the dose range l-2 mg/kg (Chander et al., 1991). Although this range is somewhat higher than reported for tamoxifen, this would be expected from a drug that cannot be metabolically activated. The authors did show superior activity to tamoxifen at the 1 mg/kg dose (Chander et al., 1991).

Toxicology The drug was designed to avoid the toxicology problem of liver carcinogenic@ produced by tamoxifen (McCague et al., 1989, 1990). A recent report demonstrates that tamoxifen, toremifene and idoxifene, given as acute doses, will cause aneuploidy in rat liver (Sargent et al., 1996). However, idoxifene has not been evaluated as a rat liver carcinogen. It is known that N-demethylation is the major metabolic route for tamoxifen so a pyrrolidino group was used to resolve liver toxicity through a reduction in formaldehyde production. Pyrrolidino tamoxifen (at half the dose) gives levels of DNA adducts as high as tamoxifen in rat liver (White et al., 1992). Mice show interstitral hyperplasia in the ovaries and the expected decrease in uterine weights after 4 weeks of treatment with 25-50 mg/kg of daily idoxifene. A single dose study in mice at 100 mg/kg shows no mortality or behavioral changes (Chander et al., 1991). Tamoxifen produces similar actions in the mouse but the metabolic profile in the mouse differs from the rat (Robinson et al., 1991) and mice do not produce liver tumors in response to tamoxifen (Furr and Jordan, 1984). Idoxifene is designed to be an agent that is ‘metabolism restricted’ and as such requires careful evaluation for unusual or toxic routes of metabolism not previously noted with tamoxifen.

Clinical1Pharmacology and Endocrinology Preliminary studies in the laboratory with idoxifene demonstrate no metabolism of idoxifene over 48 hours of administration (Carnochan et al., 1994). The studies are being used to establish a data base for the evaluation of [lz41] idoxifene by PET imaging during clinical studies. Idoxifene can be measured by HPLC (Coombes et al., 1995), but although 4’-hydroxyidoxifene (Fig. 18) is the most likely metabolite it has not been reported in clinical studies. Idoxifene has an initial half-life of 15 hours and a terminal half-life of 23.3 days (e.g. three times greater than tamoxifen). Idoxifene causes a modest decrease in LH and FSH but no increase in SHBG.

214

V. C. Jordan and W. J. Gradishar

Clinical Evaluation Only one clinical trial in humans has been reported with idoxifene. Coombes et al. (1995) reported the results of a phase I clinical trial in which 20 patients with advanced breast cancer (ER-positive or unknown) were treated with one of four dose levels of idoxifene. The majority of patients previously received tamoxifen, secondline hormone therapy, and chemotherapy. Partial responses were observed in 14% of patients and additional 29% of patients had stable disease for 1.4-14 months (Table 7) Toxicity was mild and not dose related.

TAT-59 TAT-59 is a prodrug and is being developed in Japan for the treatment of advanced breast

by the Taiho cancer.

Pharmaceuticals

Co. Ltd.

3

NN

0

& 1:

,:I

1:

IDOXIFENE

I

3

NN

0

c

I

Jsi+ 1 ;

OH

3

4’-HYDROXYIDOXIFENE Fig. 18.

The possible metabolite

of the metabolically

resistant

antiestrogen

idoxifene.

Molecular

Mechanisms

and Future Uses of Antiestrogens

215

Table 7. Clinical trial involving idoxifene Study Coombes (1995)

et al.

Patient population

Dose/schedule

Response

Toxicity

20 patients

10, 20, 40, 60 mg po qd. (14/20 patients received idoxifene longer than 2 weeks.)

RR PR SD 14% 14% 29% SD duration 1.5-1.4 months

Mild, not dose related Nausea 15% Anorexia 15% Fatigue 20%

with metastatic breast cancer. lo/20 patients prior TAM rx. 16/20 patients prior second-line hormonal rx. ER+ or unknown. 13120 patients received prior chemotherapy

Abbreviations: RR, response disease; qd, daily.

rate;

TAM,

tamoxifen;

PR, partial

response;

po, oral; SD, stable

o\b,O

HO/ \oa

TAT-59

dephosphorylation

HO

DP-TAT-59 (active metabolite) Fig.

19.

The metabolic

activation

of the antiestrogen

TAT-59

to the 4-hydroxy

derivative.

V. C. Jordan and W. J. Gradishar

216

Laboratory Studies TAT-59 is active in inhibiting the growth of ER-positive, DMBA-induced rat carcinomas (Toko et al., 1990). The drug is converted to its mammary dephosphorylated metabolite (Toko et al., 1990) that has high binding affinity for the ER (Toko et al., 1992) (Fig. 19). The drug is active in inhibiting the growth of estrogen-stimulated, ER-positive breast cancer cells transplanted into athymic mice (Koh et al., 1992; Iino et al., 1994).

Clinical Evaluations Clinical studies been published.

using

TAT-59

for the treatment

of advanced

breast

cancer

have not

Chapter 7

Pure Antiestrogens

Pure antiestrogens were first described in the mid-1980s (Wakeling and Bowler, 1987). The compound ICI 182,780 (ZM.182780) is being developed by Zeneca Pharmaceuticals, UK. for the treatment of advanced breast cancer following the failure of long-term adjuvant tamoxifen therapy. Pure antiestrogens could also find application in gynecology and other non-malignant conditions.

Laboratory Studies ICI 182,780 is a competitive inhibitor of estrogen action by blocking estrogen binding to the ER (Wakeling et al., 1991) and causing a destruction of the ER (Dauvois et al., 1992; Gibson et al., 1991). The antiestrogen is a potent inhibitor of the growth of MCF-7 cells (10-9-10-7M) (Wakeling et al., 1991; Thompson et al., 1989) and causes a. more complete inhibition of growth compared to tamoxifen (Thompson et cell line, MDA-MB-231, is unaffected by pure al., 1989). The ER-negative antiestrogens. ICI 182,780 is a potent and complete antiestrogen when given orally or subcutaneously to immature rats. Furthermore ICI 182,780 can inhibit the partial estrogen-like effects of tamoxifen on the rat uterus (Wakeling et al., 1991). This may be important because tamoxifen can eventually encourage the growth of MCF-7 breast c,ancer cells implanted into athymic mice (Osborne et al., 1987; Gottardis et al., 1989a). Similarly tamoxifen-stimulated endometrial carcinoma has been reported to grow in athymic animals (Satyaswaroop et al., 1984; Gottardis et al., 1988). Pure antiestrogens will inhibit the growth of tamoxifen-stimulated breast and endometrial tumors in the laboratory (Gottardis et al., 1989b; Gottardis et al., 1990). The antiestrogen ICI 182,780 will control the growth of tamoxifen-stimulated tumors for prolonged periods, however growth eventually occurs. Preliminary studies (Osborne et al., 1995:) suggest that ICI 182,780 resistant tumors may be developed but further work is required to describe the model in detail. It is known in the laboratory that ER+ breast cancer cells will develop subclones that are ERif they are maintained in an estrogen-deprived state (Pink and Jordan, 1996). Whether this is true clinically remains to be determined. Rats with established DMBA-induced tumors show a more rapid decrease in tumor size and uterine weight with a combination of the luteinizing hormone-releasing hormone (LHRH) analog, goserelin, and ICI 164,384 compared

217

218

V. C. Jordan and W. J. Gradishar

with goserelin alone (Nicholson et al., 1990). The complete estrogen blockade that might eventually premenopausal patients with advanced disease.

combination be valuable

may provide a more for the treatment of

Toxicology There have been no reports of genotoxicity or carcinogenesis with ICI 182,780. Unlike estradiol or tamoxifen, the administration of ICI 164,384 to neonatal female rats did not accelerate the onset of puberty or lead to abnormalities in the development of the reproductive tract (Wakeling and Bowler, 1988). Although ZM 189,154, a pure nonsteroidal antiestrogen not intended for clinical application does not affect bone density in the rat (Dukes et al., 1994), ICI 182,780 reduces cancellous bone volumes in female rats (Gallagher et al., 1993).

Clinical Pharmacology and Endocrinology There are no reports about the metabolism of ICI 182,780 but injection of 18 mg/day produces blood levels of 25 ng/ml after 1 week of treatment. ICI 182,780 is determined by radioimmunoassay (DeFriend et al., 1994). Patients treated with ICI 182,780 for a few days have a significant decrease in the Ki67, PgR and ER in their breast tumors (DeFriend et al., 1994). ICI 182,780 has no effect on LH, FSH, or SHBG (DeFriend et al., 1994). The pharmacokinetics of ICI 182,780 were evaluated following 1 month and 6 month treatments with 250 mg/month. The AUC were 140.5 and 206.8 ng/day/ml on the first and sixth month of dosing, respectively, These data were consistent with drug accumulation after multiple doses and suggested that lower doses of the drug may be effective in maintaining therapeutic serum drug levels. A recent therapeutic study using depot ICI 182,780 showed rises in gonadotrophins but interestingly, no associated rise in circulating cholesterol. Howell et al. (1996) noted that all patients had previously been treated with tamoxifen, thus accounting but the fact that tamoxifen caused a decrease in for rises in gonadotropins, cholesterol, that was maintained during ICI 182,780 treatment is very intriguing. One is the possibility that ICI 182,780 is explanation that should be investigated, metabolized locally in the liver to an estrogen prior to biliary excretion. Serial endometrial ultrasound was performed in five responding patients. The endometrial thickness in all five patients was greater than that found in the normal postmenopausal uterus. However, this increased thickness was attributed to prior tamoxifen treatment. ICI 182,780 inhibits further endometrial proliferation but did not cause regression of pre-existing hypertrophied endometrium (Howell et al., 1996). in both ovariectomized (Dukes et al., 1992) and intact adult female (Dukes et demonstrate a complete blockade of the uterotrophic effects of estradiol by ICI 182,780. The pure antiestrogens could find use in the treatment of endometrial disorders and endometrial carcinoma. A clinical comparison with progestins might demonstrate fewer side-effects with a pure antiestrogen. Studies

al., 1993) monkeys

Molecular

Mechanisms

and Future Uses of Antiestrogens

Table 8. Clinical trials evaluating the pure antiestrogen

219

ICI 182,780

Study

Patient population

Dose/schedule

Response

Toxicity

DeFriend et al. (1994)

56 postmenopausal patients with histologically, confirmed primary breast cancer

In ER+ tumors, ER, PR and Ki 67 were significantly reduced after rx.

Minor/moderate headache 6

Howell et al. (1996)

19 postmenopausal, TAM-resistant, advanced breast cancer

Controls 10 patients 6mg,IMx7 days preop 21 patients 18mg,IMx7 days preop 16 patients 250 mg IM/ month

PR 37%

None

SD 32%

Soft tissue, bone, and visceral sites responded. Abbreviations:

TAM, tamoxifen;

IM, intramuscular.

Clinical Evaluations from clinical trials evaluating the activity of pure antiestrogens are limited (Table 8). DeFriend et al. (1994) assessed the tolerance, pharmacokinetics and shortterm biological activity of ICI 182,780 in 56 women with primary breast cancer. Patients were randomized to a control group (n = 19) in which no preoperative therapy was administered, or to one of two preoperative treatment groups (treatment treatment group 2: 18 mg IM x 7 days, group I : 6 mg IM x 7 days, n = 21 patients; n = 16 patients). ICI 182,780 was not associated with any significant toxicity.

Data

In a group of 19 tamoxifen-resistant, advanced breast cancer patients, Howell et al. (1996) reported a 37% partial response rate using ICI 182,780 (250 mg IM/month). Soft-tissue, bone and visceral sites of disease responded. In addition, 32% of patients maintained stable disease status. No significant toxicity was observed. The lack of cross-resistance with tamoxifen in 69% of patients suggest that ICI 182,780 may be useful as a first-line therapy in advanced disease or as a second-line therapy in advanced disease where tamoxifen has been previously used. The activity of pure antiestrogens in advanced disease suggest that they may also have efficacy in the adjuvant setting, although no data are available.

Chapter 8

Targeted Antiestrogens

Raloxifene (originally called keoxifene) is being developed by Eli Lilly Laboratories as a treatment for osteoporosis. The drug being referred to as a selective estrogen recept0.r modulator (SERM) which builds on the concept that targeted antiestrogens can be ifound to have estrogenic effects on the cardiovascular system and bone but an antiestrogenic action on the breast and uterus (Lerner and Jordan, 1990; Tonetti and Jordan, 1996). Raloxifene is also being evaluated as an antitumor agent in ER-positive advanced breast cancer patients.

Laboratory Studies Raloxifene has a binding affinity for the ER equivalent to that of estradiol (Black et al., 1983). The compound is a potent inhibitor of the growth of breast cancer cells in culture (Poulin et al., 1989). Raloxifene is an antiestrogen in the immature rat uterine weight ‘test but has little agonist action on the uterus well established that raloxifene can maintain bone (Jordan et al., 1987; Evans et al., 1994; Black et al., the drug also reduces circulating cholesterol (Black et Raloxifene has antitumor activity in induced (Gottardis and Jordan, Raloxifene-stimulated breast tumors inhibits the tamoxifen-stimulated EnCalOl, in athymic mice (Gottardis

when administered alone. It is density in the ovariectomized 1994; Sato et al., 1995) rat, but al., 1994).

the DMBA- (Clemens et al., 1983) and NMU1987) rat mammary carcinoma models. have not been described, but raloxifene partially growth of the human endometrial carcinoma, et al., 1990).

Toxicollogy No reports

of DNA adducts

or hepatocarcinogenesis

have appeared

with raloxifene.

Clinical Pharmacology and Endocrinology An analytical method to determine raloxifene and its metabolites has not been published. The drug causes a decrease in LDL cholesterol, but HDL cholesterol remains unchanged during treatment with 200 and 600 mg daily (Draper et al., 1993). 221

222

V. C. Jordan and W. J. Gradishar

Clinical Evaluation In the 1980s a series of phase I studies of raloxifene were carried out in healthy male subjects. A once-daily, oral dosing schedule was well tolerated, and acceptable blood levels were achieved. No clinically adverse events were detected, and there was evidence for an antiestrogenic effect (Draper et al., 1995). Physicians at the M.D. Anderson Hospital reported the results of a phase II trial of raloxifene in female patients with metastatic breast cancer who were refractory to tamoxifen therapy (Buzdar et al., 1988). Raloxifene, 200 mglday, was administered orally in accordance with the highest dose given in phase I studies. Fourteen patients received raloxifene daily for up to 8 months. The drug was well tolerated with no significant clinical or laboratory abnormalities detected, but no objective responses were observed (Buzdar et al., 1988). It is clear that raloxifene has no deleterious side-effects on breast cancer patients. As a result of the intense interest in raloxifene as a potential treatment for osteoporosis, a small international clinical trial is underway to evaluate the activity of raloxifene in hormone receptor-positive, postmenopausal patients with metastatic breast cancer who have not received prior hormonal therapy or chemotherapy for metastatic disease. The ability of raloxifene to maintain bone density in the rat (Jordan et al., 1987; Evans et al., 1994; Black et al., 1994; Sato et al., 1995) has encouraged the clinical testing of raloxifene as a treatment for osteoporosis. Large international clinical trials are currently underway to evaluate the effect of raloxifene on the progression of osteoporosis.

Chapter9

Comparison of New Antiestrogen and Conclusions

The successful- development of tamoxifen has created numerous new opportunities for the development of drugs that could be applied throughout medicine. In this review we have described the properties of several new agents being tested clinically but it is important to stress that the current development of these new agents is focused on several different therapeutic goals. Although this fact makes a direct comparison of compounds difficult, it is useful to evaluate the available data in the areas that have caused the most concern with tamoxifen. The principal areas of evaluation in the laboratory and clinic are shown in Table 9. Obviously additional information is available within the individual pharmaceutical companies to justify current clinical trials with regulatory authorities. However, it is possible to cite only published data in the medical literature. Nevertheless, compounds that are intended for prolonged usage must be evaluated clinically to the same high standards already achieved with tamoxifen to avoid misinterpretation of laboratory data that describes clinical. safety of new agent prematurely. Only carefully organised prospective clinical studies can provide the appropriate safety data. Additionally, for those agents that are intended to be used for the treatment of advanced breast cancer, it is essential to demonstrate the new drug is not cross

Table 9. A comparative

summary

DNA adducts Rat liver tumors Endometrial cancer (lab) Endometrial cancer (clinic) Cross-resistance with tamoxifen Advanced breast cancer Adjuvant therapy Cholesterol (+) (labor clinic) Preserves bones (+) (labor clinic)

of published reports about the toxicology antiestrogens Toremifene

Droloxifene

No No

No No

and application

of new

ICI 182,780 ICI 164,384

Raloxifene Keoxifene

Inhibit

Inhibit

Yes Yes

Yes

No Yes

Yes Yes

Yes

No

Yes Yes

a characteristic

was found

No, a report where a characteristic was not found; yes, a report where and a da.sh indicates no pubished studies. 223

224

V. C. Jordan and W. J. Gradishar

resistant with tamoxifen. All patients who present with stage I and II breast cancer that is ER-positive will be treated with adjuvant tamoxifen. There is clearly no point in treating disease that is resistant to tamoxifen with a cross resistant agent. This can be tested in the laboratory and must be addressed.

Overall, much progress has been made during the past decade. However, as illustrated in Table 9 there are still significant gaps in our knowledge about the efficacy and long-term safety of the new agents. A focused program of development in each of the targeted areas of therapeutics will result in the introduction of numerous new ‘antiestrogens’ (SERMS) into clinical practice by the end of the 1990s. Overall, the fascinating and multifaceted pharmacology of the nonsteroidal antiestrogens is converging to make new drugs available in diverse areas of medicine. This means that the knowledge of the value and side-effects of an anticancer drug tamoxifen, originally the exclusive domain of the oncologist, must be distributed to a wider constituency. As the original suggestions (Lerner and Jordan, 1990) that targeted antiestrogens should be sought to treat diseases of the menopause, and that as a beneficial side-effect the prevention of breast cancer becomes a reality, then issues of duration of treatment, endometrial cancer and agent-resistant breast cancer must become centre stage. It is known that long durations of treatment will be essential to maintain bone density and protect against coronary heart disease. But what of the growth of pre-existing endometrial cancer and the eventual development of agent resistant breast cancer? It is clear that, based on the experiences with tamoxifen and endometrial cancer and the epidemiology of breast cancer, these concerns will laboratory models exist to test the extrapolate into rare events. Nevertheless, hypothesis and provide guidance for prospective clinical trials and patient monitoring. Patient piece of mind and safety must be the goal of ethical pharmaceutical companies as we enter a new era of women’s health.

Acknowledgements We are especially grateful for Susan Tripoli typing and correcting our manuscript, Henry Muenzner for completing all the diagrams and to Ruth O’Regan, MD for her invaluable help with the references. We are deeply grateful to the Lynn Sage Foundation for their continuing support for our Breast Cancer Program. The program is also supported in part by a National Cancer Institute Breast Cancer Program Development Grant R21CA-65764.

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