TAMOXIFEN: A MOST UNLIKELY PIONEERING MEDICINE
V. Craig Jordan
For more than 25 years, tamoxifen has been the gold standard for the endocrine treatment of all stages of oestrogen-receptor-positive breast cancer, and the World Health Organization lists tamoxifen as an essential drug for the treatment of breast cancer. It is estimated that more than 400,000 women are alive today as a result of tamoxifen therapy, and millions more have benefited from palliation and extended disease-free survival. Interestingly, tamoxifen also became the first cancer chemopreventive approved by the Food and Drug Administration (FDA) for the reduction of breast-cancer incidence in both pre- and post-menopausal women at high risk. However, 40 years ago, it was hard to imagine that a non-toxic targeted treatment for breast cancer could be developed at all.

The Robert H. Lurie Comprehensive Cancer Center, Northwestern University Medical School, 303 East Chicago Avenue, Olson Pavilion 8258,
Chicago, Illinois 60611, USA.
e-mail: [email protected] doi:10.1038/nrd1031

History is lived forward, but is written in retrospect. “We know the end before we consider the beginning and we can never wholly recapture what it was to know the beginning only” (C. V. Wedgewood, William the Silent). That is, unless one has lived through the evolving applications of tamoxifen.
In the early years of the twentieth century, Paul Ehrlich proposed the now widely accepted model for drug development1. In this approach, a broad synthetic organic-chemistry programme is followed by testing of compounds in an appropriate laboratory model for the disease to be treated; compounds with low toxicity are then selected to be tested in prospective clinical trials. Although tamoxifen is a successful drug for the treatment of breast cancer, it was initially identified in the early 1960s in a programme designed to develop a contraceptive2–4. The Ehrlich strategy of focusing on translational models of relevance to the disease to be cured ultimately proved to be an appropriate formula in the 1970s for devising a successful strategy for the treatment and prevention of breast cancer.
It is fair to say that throughout the 1970s, the clinical evaluation of tamoxifen in advanced breast cancer was not hailed as a breakthrough by the clinical community. The drug only produced responses equivalent to other endocrine approaches, although with fewer side effects5–7.

By contrast, there was enormous enthusiasm for the discovery and application of cytotoxic chemotherapy. Some 30 years later, enthusiasm for the nonspecific approach of chemotherapy has waned and has been replaced by the anticipation of a new generation of targeted therapies. Tamoxifen, an agent kept on life support for the first 20 years of its existence (1962–1982), evolved into the first targeted medicine for breast cancer. This article describes the twists and turns in the discovery and development of tamoxifen, from its origins 40 years ago as a potential contraceptive to its current position as the most widely used anticancer drug (see TIMELINE).

Anti-oestrogens
Oestrogens are hormones that regulate the menstrual cycle, and are essential for successful reproduction. In addition, oestrogen has been implicated in the reduced risk of coronary heart disease (CHD) in premenopausal women compared with men, and it also helps to maintain bone density. After the menopause, at around 50 years of age, when oestrogen is no longer synthesized in the ovary, women have an increased risk of CHD and a reduction in bone density, which can lead to osteoporosis. However, the perceived beneficial effects of endogenous oestrogen on women’s physiology must also be balanced against the role of oestrogen in carcinogenesis.

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The link between ovarian function and the develop-ment of breast cancer has been known for more than a century8,9. The initial clinical observations were sub-sequently complemented by the laboratory finding that early ovariectomy can reduce the incidence of mammary cancer in high-incidence strains of mice10. The finding that oestrogens from ovarian extracts enhanced mammary tumorgenesis led Antoine Lacassagne11 to speculate in 1936 that if increased sensitivity to oestrogen was responsible for the hereditary susceptibility to breast cancer, then perhaps an antagonist of oestrogen accumulation could prevent the disease.
The discovery of non-steroidal anti-oestrogens is a good example of scientific serendipity. The compound ethamoxytriphetol (MER25) (FIG. 1), which was being tested in the cardiovascular program at the Merrell Company in Cincinnati, USA, was requested to be tested in an endocrinology program evaluating syn-thetic oestrogens12. The reason for the request was that ethamoxytriphetol had a structural similarity to the known oestrogen trianisylchlorethylene. Remarkably, ethamoxytriphetol had no oestrogenic activity in any species, but was, rather, anti-oestrogenic.
In 1958, Leonard Lerner reported the anti-oestrogenic properties of the first non-steroidal anti-oestrogen ethamoxytriphetol13 (FIG. 1), and subsequently clomiphene16 (FIG. 1). Remarkably, both compounds were effective post-coital contraceptives in laboratory animals, so one possible application was in fertility control. This was an extremely lucrative prospect for the pharmaceutical industry, as the steroidal oral contraceptive was already widely accepted during the 1950s and 1960s. Ethamoxytriphetol proved too toxic for clinical use, and, ironically, in women clomiphene had exactly the opposite antifertility action noted in rats16, and induced ovulation and enhanced fertility in subfertile women17,18. Despite the very small market at the time,

by the mid-1960s Clomid (clomiphene citrate mixed isomers) had become established as a standard therapy for the induction of ovulation19.
Although the scientists at Merrell envisaged many clinical applications for anti-oestrogens, such as regulation of menstrual disorders, regulation of blood lipids, atherosclerosis, modification of behaviour and as antitumour agents12, these were difficult to realize throughout the 1960s. Although there was a known link between ovarian hormones and the development of breast cancer, the application of ethamoxytriphetol, clomiphene20 and nafoxidine14,15 (discovered by Upjohn Co.) as experimental treatments for advanced breast cancer were all discontinued because of extensive side effects15,21 or concerns about potential unforeseen toxicity with the possibility of acute cataract formation (BOX 1)22 — difficult obstacles that had to be overcome in the development of tamoxifen.

Discovery of tamoxifen
The discovery and development of tamoxifen is, initially, a story of interpersonal relationships, rather than a pre-planned effort by ICI Pharmaceuticals Division — the manufacturer of tamoxifen — to establish themselves as a major player in oncology. The fact that this is what ultimately happened is testimony of how truly successful tamoxifen has been. However, in the 1960s, this advance was not anticipated.
ICI Pharmaceuticals (now known as AstraZeneca) had an interest in the pharmacology of synthetic derivatives of triphenylethylene in the 1940s and 1950s, and ICI chemists had synthesized several nonsteroidal oestrogens that were used at high doses to treat breast cancer27,28. However, the market for the treatment of advanced breast cancer was extremely modest, with at most a few thousand patients who might respond for about a year.

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By contrast, the market for contraceptives was already established. The failure of Merrell to market non-steroidal anti-oestrogens as contraceptives attracted the attention of Arthur Walpole and his col-leagues Michael J. K. Harper — a reproductive endocri-nologist — and Dora M. Richardson — a synthetic organic chemist — at ICI Pharmaceuticals Division. The goal was to synthesize a potent non-steroidal antioestrogen with a reduced ability to increase circulating desmosterol, which was linked to the serious toxicity of triparanol (BOX 1).
As the programme was tasked to find antifertility agents, the group used models of reproduction in rats to identify suitable contraceptive agents2–4,29–34. These studies culminated in the identification of ICI46,474 (FIG. 1), the trans isomer of a triphenylethylene — subsequently named tamoxifen — as a potentially safer medicine for clinical applications3.
The fact that tamoxifen had paradoxical full oestrogen-like actions in mice2,3, but not in rats, created some uncertainty in the late 1960s about the action of tamoxifen in humans. Fortunately, Walpole had a specific interest in cancer therapy28,35 and included coverage of the “control of hormone-dependent tumours” in the patent on tamoxifen, which primarily focused on “the management of the sexual cycle” (BOX 2). In 1972, numerous applications for tamoxifen — ranging from

the induction of ovulation36,37 to the treatment of breast cancer5,6 and menometrorrhagia38 — were reviewed at ICI Pharmaceutical headquarters in Alderly Park, but by all accounts the development programme was on the verge of termination. The potential applications as an inducer of ovulation, various gynaecological conditions or the treatment of advanced breast cancer in post-menopausal women did not seem to have the possibility of broad market exploitations or the prospects for enhancing revenues to offset investment in clinical research. Nevertheless, Walpole succeeded in convincing ICI Pharmaceuticals to market tamoxifen in the UK as a breast-cancer treatment (1973) and as an inducer of ovulation (1975).
Although it is impossible to recapture fully the general clinical indifference to the development of a new endocrine agent for the treatment of advanced breast cancer, the views of opinion leaders can be recalled by examining their published review articles. Emmens41 focused on the potential of anti-oestrogens as post-coital contraceptives. And the first major review of the basic and clinical uses of anti-oestrogens, by Lunan and Klopper42 in 1975, devotes only fourteen lines and three references to applications in breast cancer in an article totalling fifteen pages with 180 references. This review article focuses on the developing body of work on the clinical usefulness of anti-oestrogens in reproductive

Figure 1 | Getting the right formula. The withdrawal of triparanol, a cholesterol-lowering drug, focused concerns on any product that might potentially cause cataracts. The first non-steroidal anti-oestrogen, ethamoxytriphetol, was examined as a hormonal agent by Lerner13 because of its structural similarity to the clinically available oestrogen trianisylchlorethylene (TACE). However, the finding that the drug was only a weakly active anti-oestrogen and too toxic initiated a search for a more potent agent. Clomiphene is a mixture of geometric isomers conceived by placing the anti-oestrogenic alkylaminoethoxyside chain on a known oestrogenic triphenylethylene. The report of nafoxidine as an antifertility agent14 resolved the issue of which isomer of a mixture of triphenylethylenes would be responsible for anti-oestrogenic and antitumour activity (the anti-oestrogenic and antitumour effects are attributable to the trans isomer); however, nafoxidine failed testing as an anti-breast-cancer drug because of toxicity15.
Tamoxifen (ICI46,474) lowered cholesterol in animals, but did not increase desmosterol levels3. The anticancer properties and profertility properties resulted in clinical development in the 1970s, but the low toxicity of tamoxifen enhanced the prospects for expanded clinical applications as a long-term adjuvant therapy and ultimately led to it being tested as a chemopreventive.

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endocrinology, not breast cancer. The review closes with a look to the future: “Relatively little has been done to apply the results of animal experiments to humans but there is now enough evidence to suggest that antioestrogens have a great potential application to human therapy. A promising approach is to tailor make particular antioestrogens for specific indications – for example –ovulation induction or antifertility or anticancer”.
It is clear that although tamoxifen was approved for clinical use in the UK in 1973, much was still to be done to convert an essentially orphan drug with economic prospects of a few hundred thousands of pounds Sterling into a pioneering medicine with sales in the billions of pounds Sterling. This was to be realized by applying Ehrlich’s principles of using a laboratory test system to target the relevant disease. A drug discovered in a fertility-control program was re-invented as a pioneering therapy for breast cancer through the dimethylbenzathracene (DMBA)-induced rat mammary carcinoma model43. The key role of the unique patenting situation with tamoxifen in allowing these scientific principles to be validated in prospective clinical trials is described in BOX 2.

Development of tamoxifen in breast cancer
I first met Walpole in 1967 when I was a summer student at ICI Pharmaceuticals Division, and met him for the second time in 1972 when he examined my Ph.D. thesis from Leeds University, entitled,‘The oestrogenic and anti-oestrogenic properties of some substituted tri-phenylethylenes and triphenylethanes’. I was invited by Michael Harper (who by that time had moved to the Worcester Foundation, a research institute in Massa-chusetts credited with the discovery of the oral contra-ceptive) to work on the contraceptive properties of prostaglandins. However, before my arrival, Harper had

moved to the World Health Organization. On arrival at the Worcester Foundation in 1972, I was told to select a topic for my research project as an independent scientist. I proposed to develop anti-oestrogens as anti-breast-cancer drugs, and a transatlantic telephone call secured support from Walpole and funds from the newly appointed drug monitor of ICI46,474, Lois Trench at Stuart Pharmaceuticals in Wilmington, Delaware (acquired by ICI Pharmaceuticals Division). I was also fortunate to meet Elwood V. Jensen, the director of the Ben May Laboratory, in Chicago in November 1972, who was at the time a member of the scientific advisory board for the Worcester Foundation. Jensen had discovered the target of oestrogen action — the oestrogen receptor — in oestrogen target tissues44 and there was great excitement about the possibility of using the oestrogen-receptor assay to predict the clinical response of advanced breast cancer to endocrine therapy45. Jensen generously agreed to teach me oestrogen-receptor assays and the DMBA model so that a systematic study of the antitumour actions of tamoxifen could start at the Worcester Foundation (1972–1974).
By 1974, the oestrogen-receptor assay was successfully evaluated as a predictive test for the endocrine responsiveness to endocrine ablation (TABLE 1); however, the situation with anti-oestrogens was less clear, because the oestrogen-receptor assay did not initially prove to be useful in selecting patients for treatment (TABLE 1). Additionally, tamoxifen was shown to have a very low affinity for the oestrogen receptor in vitro46, despite demonstrating potent antifertility and anti-oestrogen action in the uterus3. The apparent anomaly was resolved with the finding that tamoxifen was a prodrug that accumulated and was then converted to 4-hydroxytamoxifen, an anti-oestrogenic metabolite with high affinity for the oestrogen receptor47,48. This knowledge advanced the

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study of structure–activity relationships of anti-oestro-gens, which resulted in the synthesis of droloxifene (3-hydroxytamoxifen), raloxifene, the pure anti-oestro-gens ICI164,384 and ICI182,780, and nearly all current selective oestrogen-receptor modulators (SERMs).
Evidence to support a targeted role for tamoxifen at the oestrogen receptor accumulated in the laboratory through the use of the DMBA-induced rat mammary carcinogen model. Tamoxifen inhibited oestradiol binding to breast and mammary tumour oestrogen receptors both in vivo and in vitro49–52, and oestrogen-receptor assays could be used to predict the response to tamoxifen in the DMBA model53 and, subsequently, in advanced breast cancer54. Paradoxically, in contrast to this evidence, the early publication of the Nolvadex Adjuvant Trial Organisation (NATO) trial data of adjuvant tamoxifen found no evidence to support the idea that a patient with an oestrogen-receptor-positive tumour was more likely to benefit from tamoxifen than an oestrogen-receptor-negative patient55,56. This lack of difference concluded from the NATO trial data was subsequently shown to be incorrect in the overview of the world-wide clinical trials conducted at Oxford every five years57. Nevertheless, the result of the NATO trial created some uncertainty for clinical trialists during the 1980s, and resulted in a prolonged laboratory examination of alternate mechanisms for tamoxifen action in oestrogen-receptor-negative patients (summarized in REF. 58). Unfortunately, these laboratory data have not translated to clinical advances.
Four years after tamoxifen had been approved for the treatment of breast cancer in the UK, the medicine was approved for the treatment of advanced breast cancer

in post-menopausal women in the United States in December, 1977. However, there was still little prospect for commercial success with yet another hormonal treatment for advanced disease. Nevertheless, a new strategy was in place to target tamoxifen towards the correct clinical application.
In the 1970s, adjuvant therapy following breast-cancer surgery was evaluated to determine whether cytotoxic chemotherapy would destroy all micrometas-tases and cure most breast cancer patients. Tamoxifen
— a relatively non-toxic palliative treatment — was also evaluated for its effectiveness as an adjuvant. Most trials chose to use one year of tamoxifen in unselected patient populations — that is, a mixture of oestrogen-receptor-positive, oestrogen-receptor-negative and oestrogen-receptor-unknown patients were recruited. The reason for recruiting a broad patient population was really because there were no standardized oestrogen-receptor assays generally available to clinical-trial organizations outside of the United States, and the choice of a one-year course of tamoxifen derived from the limited experience in advanced disease. Tamoxifen was effective for about one year in treating advanced disease7 and there was concern that a longer treatment regimen would result in premature drug resistance.
In the laboratory, high concentrations of tamoxifen had been shown to be cytotoxic to breast cancer cells in culture59, but studies in the DMBA-rat mammary carcinoma model showed that short-term therapy, even with large daily doses of tamoxifen, delayed — rather than completely prevented — the development of tumours60,61. By contrast, continuous therapy, with even small daily doses, resulted in 80% of the animals

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Table 1 | Objective breast tumour regressions with different therapies*

Therapy ER+ ER–
Adrenalectomy 32/66 4/33
Oophorectomy 25/33 4/53
Hypophysectomy 2/8 0/8
Total 59/107 = 55% 8/94 = 8%
Androgen 12/26 2/24
Oestrogen 37/57 5/58
Glucocorticoid 2/2 -
Total 51/85 = 60% 7/82 = 8%
Anti-oestrogen 8/20 5/27

*According to oestrogen receptor (ER) assay and type of therapy as judged by extramural review. Adapted from REF. 96.

remaining tumour free62,63. In other words, the treat-ment of rats for one month (equivalent to one year in humans), starting one month after DMBA, was unable to destroy all the microfoci of deranged mammary epithelial cells, but treatment for five months (equiva-lent to five years in humans) was extremely effective at controlling tumourigenesis. So, longer rather than shorter adjuvant tamoxifen was predicted to be more beneficial in the oestrogen-receptor-positive patient population, and this has indeed subsequently been found to be the case (BOX 3).
The fact that tamoxifen was classified as an anti-oestrogen raised concerns that long-term therapy would enhance the prospect of women developing osteoporo-sis or increase the risk of CHD. However, tamoxifen is a SERM: it is anti-oestrogenic in breast and mammary tissue52, but acts as an oestrogen-like compound in bone68 and also lowers circulating cholesterol, whereas it has mixed oestrogenic and anti-oestrogenic actions in

the uterus3. The discovery of the principle of selective oestrogen-receptor modulation68–70, and its translation to patients71,72, permitted the safe evaluation of tamoxifen in women only at risk for breast cancer. Tamoxifen prevents mammary carcinogenesis in laboratory models52,73,74; this observation, coupled with the key clinical observation that tamoxifen reduced contralateral breast cancer during adjuvant therapy75, and also, at that time, was perceived to have few side effects, provided the scientific rationale to test whether tamoxifen reduced the incidence of breast cancer in high-risk women76,77.
Tamoxifen was first tested in a pilot study of ~3,000 women at high risk of developing breast cancer by Trevor Powles to evaluate, initially, compliance and toxicological issues78. Compliance was generally good, but the study was under-powered to show a significant decrease in primary breast cancer with tamoxifen treatment79. By contrast, the National Surgical Adjunctive Breast and Bowel Project (NSABP) chemoprevention study in the United States, organized and led by Bernard Fisher, randomized 13,388 high-risk (as determined by the Gail model80) pre- and post-menopausal women to placebo or tamoxifen for five years. The results showed a 50% reduction in invasive breast cancer and ductal carcinoma in situ76 and a nonsignificant decrease in fractures in volunteers treated with tamoxifen. These data were recently confirmed by the International Breast Cancer Intervention Study77. Overall, menopausal symptoms, a modest increase in blood clots and endometrial cancer in post-menopausal volunteers are the most important side effects noted in all studies with tamoxifen.

Concerns about side effects
Although tamoxifen was the first medicine approved in the United States as an agent for the reduction of the risk of breast cancer in high-risk women, this pioneering

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Figure 2 | Tamoxifen and beyond. The clinical impact of tamoxifen and an understanding of its pharmacology has facilitated the clinical development of selective oestrogen-receptor modulators (SERMs) and the introduction of third-generation aromatase inhibitors (anastrozole, letrozole and exemestane) and a new pure anti-oestrogen (fulvestrant). Most importantly, the SERM principle — exemplified by the clinical usefulness of raloxifene to prevent osteoporosis, but with breast and endometrial safety — is being applied to other members of the steroid hormone superfamily of receptors.

advance was not without major concerns. In the mid-1980s, tamoxifen was found to enhance the growth of endometrial cancer in the laboratory81,82 and was pre-dicted to increase the risk of endometrial cancer in women82. There is a fourfold increase in endometrial cancer in post-menopausal women treated with tamoxifen83,84. In other words, the incidence increases from one to four endometrial cancers per 1,000 post-menopausal women per year. The fact that tamoxifen was tested in 1990 and found to produce rat liver tumours85 was a surprise — to say the least — as the medicine had been used clinically for twenty years! Fortunately, although a link between tamoxifen and a modest increase in endometrial cancer was noted, no significant increase in human liver cancer has been reported. Nevertheless, the unexpected scientific results did teach lessons. It is fair to say that if rat liver tumours had been noted in the early 1970s, drug development in this area would have stopped, as there was no successor to tamoxifen86. The molecule was a single shot at success, and a pipeline of other success-ful agents was only initiated in the late 1970s and early 1980s, that is, nearly 20 years after the initial United Kingdom patent application in 1962. This illustrates the change in fortunes for a drug with initially no clear application at the outset, which subsequently evolved into a pioneering targeted anti-breast-cancer drug during the 1970s. The validation of future applications for antihormonal therapy or SERMs now seems to be limited only by the cost of investment in clinical trials.

The end of the beginning
Tamoxifen was not the first non-steroidal anti-oestro-gen to be developed for clinical use, but it proved to be a pioneering medicine with ubiquitous applications as an endocrine therapy for breast cancer. Remarkably, the 40-year history of tamoxifen opened the door to the development of raloxifene for the treatment and

prevention of osteoporosis87 and the current testing of raloxifene as a breast-cancer chemopreventive — it is now known to reduce the risk of breast cancer88, and is being compared with tamoxifen in a large randomized clinical trial called the Study of Tamoxifen And Raloxifene (STAR), which involves 22,000 high-risk post-menopausal volunteers. Remarkably, raloxifene was originally intended as a therapy for breast cancer — developed under the name keoxifene — but the programme was closed down in the late 1980s because the drug was ineffective and cross-resistant with tamoxifen89. However, the finding of endometrial cancer associated with tamoxifen and the identification of the bone-preserving or bone-building qualities of non-steroidal anti-oestrogens68 changed that view, and resurrected raloxifene as a SERM. Now a whole new drug group — the SERMs — has been established on the basis of the paradoxical pharmacology of tamoxifen in different tissues and species70. Indeed, the need for new SERMs or designer oestrogens90 to enhance the positive effects of oestrogen selectivity is becoming urgent. Prospective studies of hormone-replacement therapy in post-menopausal women generally show benefits for reducing osteoporosis and colon cancer, but increased risk of CHD, blood clots and breast cancer91. Clearly, there are opportunities for targeted drug discovery.
The increasing clinical acceptance of tamoxifen as the endocrine treatment of choice for breast cancer therapy during the 1980s encouraged research to improve and refine the effectiveness of endocrine therapy, and aided the discovery and development of a new generation of aromatase inhibitors and pure anti-oestrogens92 (FIG. 2). The aromatase inhibitors and pure anti-oestrogens are directed against the same target — the oestrogen receptor in breast tumours. Aromatase inhibitors block oestrogen synthesis in post-menopausal women, whereas pure anti-oestrogens destroy the oestrogen receptor in tissues around the body. These new targeted agents are proving to be effective anticancer agents and seem to be superior to tamoxifen93–95. However, the ‘no oestrogen’ approach, rather than the balanced SERM approach, might have long-term physiological consequences on bone, CHD and the central nervous system if the new agents are developed for use as chemopreven-tives in women without disease.
The therapeutic advance with tamoxifen for the targeted treatment and prevention of breast cancer, and the laboratory data that translated to the clinic, has prompted the application of the principle of selective oestrogen-receptor modulation to other members of the superfamily of steroid receptors and related molecules. Imagine how advantageous it would be to be able to reduce inflammation with glucocorticoids without the deleterious effects on bone density, or to design androgens that are anabolic but free from deleterious effects on other androgen target tissues. All the opportunities for selective drug discovery with the androgen receptor, glucocorticoid receptor, progesterone receptor or the PPAR receptor are now possible because of the pathfinding drug tamoxifen.

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