PRODUCT MONOGRAPH

NAME OF DRUG

NOVO-TAMOXIFEN (tamoxifen citrate) Tablets BP

CONTRAINDICATIONS

Hypersensitivity to NOVO-TAMOXIFEN (tamoxifen citrate) or any of its components. Tamoxifen must not be given during pregnancy. There have been a small number of reports of spontaneous abortions, birth defects and fetal deaths after women have taken tamoxifen, although no causal relationship has been established. Reproductive toxicology studies in rats, rabbits and monkeys have shown no teratogenic potential. In rodent models of fetal reproductive tract development, tamoxifen was associated with changes similar to those caused by estradiol, ethynylestradiol, clomiphene and diethylstilboestrol (DES). Although the clinical relevance of these changes is unknown, some of them, especially vaginal adenosis, are similar to those seen in young women who were exposed to DES in utero and who have a 1 in 1000 risk of developing clear-cell carcinoma of the vagina or cervix. Only a small number of pregnant women have been exposed to tamoxifen. Such exposure has not been reported to cause subsequent vaginal adenosis or clear-cell carcinoma of the vagina or cervix in young women exposed in utero to tamoxifen. Women should be advised not to become pregnant while taking tamoxifen and should use barrier or other non-hormonal contraceptive methods if sexually active. Premenopausal patients must be carefully examined before treatment to exclude the possibility of pregnancy. Women should be appraised of the potential risks to the fetus, should they become pregnant while taking tamoxifen or within two months of cessation of therapy. When used in the prevention setting (an indication not approved in Canada), tamoxifen is contraindicated in patients with a history of stroke, deep vein thrombosis or pulmonary embolism, and in patients who are at increased risk of developing

WARNINGS

NOVO-TAMOXIFEN (tamoxifen citrate) should be used only for the conditions listed under the INDICATIONS section. In a proportion of pre-menopausal women receiving tamoxifen citrate for the treatment of breast cancer, there have been reports of menstrual function disorders including oligomenorrhea and amenorrhea. Available information has indicated that in those women who develop disturbances of menstrual function after receiving tamoxifen citrate for up to 2 years for the treatment of early breast cancer, a proportion return to normal cyclical bleeding on cessation of therapy. Hepatocellular carcinomas have been reported in a 2 year oncogenicity study in rats receiving tamoxifen (see TOXICOLOGY). In addition, gonadal tumors have been reported in mice receiving tamoxifen in long-term studies (see TOXICOLOGY). The clinical relevance of these cancer findings has not been established. Cataracts were also reported in the 2 year oncogenicity study in rats, and since then it has been established that treatment with tamoxifen has been associated with an increased incidence of cataracts. A number of second primary tumors, occurring at sites other than the endometrium and the opposite breast, have been reported in clinical trials, following the treatment of breast cancer patients with tamoxifen. No causal link has been established and the clinical significance of these observations remains unclear. An increased incidence of uterine malignancies has been reported in association with tamoxifen treatment. The underlying mechanism is unknown, but may be related to the estrogen-like effect of tamoxifen. Most uterine malignancies seen in association with tamoxifen are classified as adenocarcinoma of the endometrium. However, rare uterine sarcomas, including malignant mixed Mullerian tumours, have also been reported. Uterine sarcoma is generally associated with a higher FIGO stage (III/IV) at diagnosis, poorer prognosis, and shorter survival. Uterine sarcoma has been reported to occur more frequently among long-term users ($ 2 years) of tamoxifen than non-users. There is evidence of an increased incidence of thromboembolic events, including deep vein thrombosis and pulmonary embolism, during tamoxifen therapy. When tamoxifen is co-administered with chemotherapy, there may be a further increase in the incidence of thromboembolic effects. For treatment of breast cancer, the risks and benefits of tamoxifen should be carefully considered in women with a history of thromboembolic events. An increased risk of stroke has been found to be associated with tamoxifen therapy in high-risk patients being treated for the prevention of breast cancer. The use of tamoxifen for the prevention of breast cancer is not an approved indication in Canada. Incidence rates for the above events were estimated from a long-term clinical study called the National Surgical Adjuvant Breast and Bowel Project Breast Cancer Prevention (NSABP P-1) Trial. In this trial, high-risk patients were randomized to either tamoxifen therapy or placebo, for the prevention of breast cancer. Uterine malignancies were separated into cases of endometrial adenocarcinomas and uterine sarcomas. The relative risk of tamoxifen compared to placebo was 3.1 for endometrial cancer, 4.0 for uterine sarcomas, 1.6 for stroke, 3.0 for pulmonary embolism, and 1.6 for deep vein thrombosis.

PRECAUTIONS

NOVO-TAMOXIFEN (tamoxifen citrate) should be used cautiously in patients with existing leukopenia and/or thrombocytopenia since these effects have been reported on occasion with the use of tamoxifen citrate. Transient decreases in platelet counts, usually to 50,000-100,000/mm3, infrequently lower, have occurred in patients given tamoxifen citrate for breast cancer. No hemorrhagic tendency was recorded and platelet counts returned to normal levels even though treatment with tamoxifen citrate was continued. Transient decreases in leukocytes also have been observed occasionally during treatment. Although it was uncertain if these occasional incidences of leukopenia and thrombocytopenia were due to tamoxifen citrate therapy, complete blood counts, including platelet counts, should be obtained periodically. As with other additive hormonal therapy (estrogens and androgens), hypercalcemia has been reported in some breast cancer patients with bone metastases within a few weeks of starting treatment with tamoxifen citrate. Any symptoms suggestive of hypercalcemia should be promptly evaluated. Patients who have metastatic bone disease should have periodic serum calcium determinations during the first few weeks of NOVO-TAMOXIFEN therapy. If hypercalcemia occurs, appropriate measures should be taken and, if severe, NOVO-TAMOXIFEN should be discontinued. Following initiation of treatment, the first patient follow-up should be done within one month. Examinations may be performed at one to 2 month intervals, thereafter. In some patients, a reduction of dosage may control adverse reactions such as severe hot flashes, nausea or vomiting, without loss of effect on the disease. The use of analgesics may be required should bone pain occur. An increased incidence of endometrial cancer and uterine sarcoma (mostly malignant mixed Mullerian tumours) has been reported in association with tamoxifen treatment. The incidence and pattern of this increase suggest that the underlying mechanism may be related to estrogenic properties of tamoxifen. Any patient receiving tamoxifen or having previously received tamoxifen who report abnormal gynaecological symptoms, especially vaginal bleeding, should be promptly investigated. Two months has been the median duration of treatment before the onset of a definite objective response in clinical studies. However, 4 or more months of treatment was required before a definite objective response was recorded in approximately one-quarter of patients who eventually responded. The duration of treatment with NOVO-TAMOXIFEN will depend on the patient's response. As long as there is a favourable response, the drug should be continued. The drug should be discontinued with obvious disease progression. However, it is sometimes difficult to determine whether the patient's disease is progressing or whether it will stabilize or respond to continued therapy during the first few weeks of treatment because an occasional patient will have a local disease flare (see ADVERSE REACTIONS) or an increase in bone pain shortly after starting tamoxifen citrate. The data suggest that treatment should not be discontinued before a minimum of 3 to 4 weeks, if possible.

Drug Interactions:

Tamoxifen/Anticoagulants

: When NOVO-TAMOXIFEN is used in combination with coumarin-type anticoagulants (e.g. warfarin), a significant increase in anticoagulant effect may occur. Where such coadministration exists, careful monitoring of the patient's prothrombin time is recommended. Cases of life-threatening interactions have been reported.

Tamoxifen/Antineoplastic Drugs

: The known principal pathway for tamoxifen metabolism in humans is demethylation catalysed by CYP3A4 enzymes. A pharmacokinetic interaction with the CYP3A4 inducing agent rifampicin, involving a reduction in tamoxifen plasma levels has been reported in the literature. The relevance of this to clinical practice is not known.

Tamoxifen/Bromocriptine

: Serum tamoxifen concentrations reportedly were increased in patients receiving bromocriptine and tamoxifen concomitantly.

Use in Nursing Mothers

: NOVO-TAMOXIFEN should not be used during lactation since it is not known if tamoxifen is excreted in human milk. In deciding whether to discontinue nursing or discontinue NOVO-TAMOXIFEN, the importance of the drug to the mother should be considered.

PHARMACOLOGY

Pharmacokinetics and Metabolism: In women given radio-labelled tamoxifen, most of the radioactivity is slowly excreted in the feces while only small amounts appear in the urine. The drug is excreted mainly as conjugates; unchanged drug and hydroxylated metabolites account for 30% of the total. Blood levels of total radioactivity following single oral doses of approximately 0.3 mg/kg reached peak levels of 0.06 to 0.14 :g/mL at 3 to 7 hours after dosing, with only 20 to 30% of the drug present as tamoxifen. The initial half-life was 7 to 14 hours, with secondary peaks 4 or more days later. The prolongation of blood levels and fecal excretion is believed to be due to enterohepatic circulation.

Antiestrogenic Effect: In those species where tamoxifen is an estrogen antagonist, the antiestrogenic effect can be shown in various ways. In spayed rats, vaginal cornification in response to the daily subcutaneous injection of estradiol can be prevented by concomitant oral dosing with tamoxifen while in immature rats the uterotrophic effect of estrogen can be similarly inhibited. In rats, tamoxifen will also terminate early pregnancy by preventing implantation of the blastocysts. In this species, it is known that estrogen, secreted by the ovaries on day 4 of pregnancy, initiates implantation on day 5. There is evidence that, at the lowest dose needed to prevent implantation, tamoxifen acts by counteracting this estrogen. In normal female rats having regular estrous cycles, ovulation can be delayed by administration of a single dose of tamoxifen given on or before the day of diestrous. In the rat (and other spontaneously ovulating species), it appears that the ovulatory discharge of luteinizing hormone (LH) from the pituitary is "triggered" by the action of estrogen on the hypothalamus and/or pituitary. The secretion of estrogen from the ovaries reaches a peak before this LH discharge. Tamoxifen's inhibitory effect on ovulation is attributed to interference with the "feedback" action of estrogen at the hypothalamic and/or pituitary level. The activity of tamoxifen as an estrogen antagonist in the pig-tailed monkey (M. nemestrina) is shown by its effect on the response to estrogen of the perineal region ("sexual skin"). Mature females of this species menstruate regularly at intervals of about 28 days. An edematous swelling of the "sexual skin" develops during the follicular phase of the cycle and subsides more rapidly at about the presumed time of ovulation. The swelling is due to endogenous estrogen and is not seen in the ovariectomized animals unless estrogen is given. In an ovariectomized pig-tail, large daily doses of tamoxifen caused no swelling of the "sexual skin". On the other hand, the swelling induced by daily injection of estradiol was reduced almost to zero by small (oral) doses of tamoxifen given at the same time. Although the capacity of tamoxifen (demonstrated in spayed rats and monkeys) to inhibit the response to estrogen suffices to explain its effects, outlined above, in intact animals of these species, the possibility that it may also inhibit the endogenous production of estrogen cannot yet be excluded. In very large doses, tamoxifen has some degree of estrogenic activity since it causes a limited increase in uterine weight and incomplete vaginal cornification in spayed rats. In the mouse, tamoxifen behaves as an estrogen without demonstrable estrogen antagonistic activity at any dose.

TOXICOLOGY

Acute Toxicity: Tamoxifen citrate has a low acute toxicity in all species studied, including mice, rats, rabbits and marmosets. The acute oral LD50 is greater than 1 g/kg in all species treated. Chronic toxicity studies were conducted in rats, dogs and marmosets. In a 3 month rat study, daily doses of 2, 20 and 100 mg/kg tamoxifen citrate were administered as a mixture containing approximately 10% of the corresponding cis-isomer, an estrogen. The changes induced were reduced weights of ovaries, testes, seminal vesicles and ventral prostate in relation to body weight. Also noted were decreased numbers of corpora lutea and follicular cysts, as well as reduction in uterine size. A complete absence of glands was found in the endometrium of all dosed rats. The epithelium consisted of a single layer of columnar cells with small areas of flattening and occasional squamous metaplasia. The endometrial stroma was somewhat condensed giving it a more fibrous appearance. Cessation of maturation of spermatozoa was shown in high-dose male rats. Seminiferous epithelium showed scattered necrotic cells. A similar, but less severe change, was seen in males receiving the intermediate dose. In low dose rats, the testes showed reduced numbers of spermatocytes and occasional atrophic tubules. The height of the thyroid epithelium was marginally increased in a few treated rats, and a thin zone of adrenal cortical congestion and edema was shown in all treated rats. Tamoxifen was administered to rats in a 6 month study with oral doses of 0.05 mg, 0.8 mg, 2.4 mg, 4.8 mg and 9.6 mg/kg. Changes produced by tamoxifen were observed mainly in rats treated with the 3 highest doses. The reproductive organs showed severe atrophic changes increasing with dose from 2.4 to 9.6 mg/kg. Serum alkaline phosphatase and sodium levels were raised and alanine aminotransferase, aspartate aminotransferase and albumin levels were lowered. There were no significant histological findings observed in the liver. A 2 year study in the rat is presently in its 15th month. The rats are receiving 5, 20 and 35 mg/kg tamoxifen by gavage (all of which represent significant multiples of the recommended human dose of 20 - 40 mg/day). Thus far, atrophy of the reproductive organs has been reported together with hepatocellular carcinomata in the groups receiving 20 and 35 mg/kg. In addition, there appears to be a dose related increase in cataracts. Oral doses of 1, 10 and 50 mg/kg tamoxifen were administered to dogs for 3 months. The same cis-trans mixture was used as in the 3 month rat study. The treated males in all groups showed a decrease in weight of the testes and pituitary. The females showed an increase in weight of the uterus. Histological observations were as follows: All dosed dogs had atrophic testes. The seminiferous epithelium in most tubules comprised only a layer of spermatogonia and Sertoli cells. There was a considerable increase in the fibrous stroma around the tubules due to the condensation of the normal interstitial tissue as a result of atrophy. This change was attributed to the "estrogenic" effect of the cis-trans mixture. In treated females, reduced numbers of follicles, cessation of ovulation, and hyperplasia of the germinal epithelium was seen in the ovaries. This last change is an exaggeration of the physiological changes seen in metestrus. These changes were less marked in the dogs receiving the lower doses. In all treated females, there was squamous metaplasia of the uterine endometrium with severe endometritis. The myometrium showed separation of the muscle bundle by a markedly edematous connective tissue which resulted in an "attenuated" appearance of the muscle. However, it was unlikely that there was an alteration in the total bulk of the muscle. In the highest dose group, bile plugs in the bile canaliculi and pigment in the Kupffer cells were shown in the livers of three males and one female. Apart from slight thinning of the cell cords, the liver was normal. These findings are in keeping with the biochemical observation of serum alkaline phosphatase elevations. It should be remembered that, in this case, the dose is 500 times that required to prevent implantation in the dog. All other organs were within normal limits. Oral doses of 0.8, 4.0 and 8.0 mg/kg tamoxifen were administered to the marmoset in a 6 month chronic dosing study. The only treatment related, pathologically significant effect due to dosing was the formation of cystically enlarged follicles in the ovaries of the females treated at 8.0 mg/kg. An additional study of two months' duration was conducted in rats where the activity of tamoxifen was compared with that of pure cis-isomer and pure trans-isomer at an oral dose of 20 mg/kg. The reproductive tissue changes were similar to those listed above for all treatment groups, but the adrenal and thyroid lesions were seen only in those rats which received the cis-isomer. Oral doses of 0.5 and 2.0 mg/kg tamoxifen citrate were administered to female rats in a 3 month reversibility test. One-third of the animals were held without drug for an additional three months. After 3 months' dosing, the ovaries and uteri exhibited changes similar to those described above. In rats held an additional three months without tamoxifen citrate, these changes were not present. Tamoxifen citrate was compared with stilbestrol and clomiphene in a reversibility study conducted in female dogs. A dose of 0.1 mg/kg tamoxifen citrate was administered for three months with one animal out of four left untreated for an additional month to test for reversibility. In the uterus of dogs dosed with tamoxifen citrate, squamous metaplasia was not present. There was a diminution of collagen with fragmentation of the bundles in the myometrium. The muscle bundles were separated by edema. Withdrawal of tamoxifen citrate produced an effect similar to a mild estrogenic change with increased collagen in thick bundles. The ovaries showed cessation of ovulation and slight hyperplasia of the germinal epithelium. In studies comparing tamoxifen with conventional estrogens, it was shown that the estrogenic activity of tamoxifen in mice was responsible for gonadal tumors. Chronic studies in mice included an initial 15-month study where the cis-trans mixture described above was administered orally at doses of 5 and 50 mg/kg. This was followed by a 13 month study where the pure cis and trans forms were compared with the cis-trans mixture at a dose of 20 mg/kg and with stilbestrol and ethinyl estradiol. An additional study of 14 months was conducted using a dose of 0.1 mg/kg to investigate the effects of lower doses of the cis, trans, and cis-trans mixture of tamoxifen with stilbestrol and ethinyl estradiol. Interstitial cell tumors of the testes and granulosa cell tumors of the ovary were found and were compound related. After six months of treatment, the mice developed a spinal deformity with kyphosis. The lesion was characterized as elongation of vertebral bodies. In addition, there was increased opacity of long bone due to ossification of the medullary cavity. Some of these can be attributed to estrogenic activity; others were of unknown etiology and did not occur at lower doses. To evaluate the ocular toxicity of tamoxifen citrate as compared to compounds which caused ocular lesions and have a similar chemical structure, such as clomiphene and triparanol, a series of three tests were conducted. The first two tests consisted of female rats being mated and treated with tamoxifen citrate, clomiphene or clomiphene B on day 11 of pregnancy and killed on day 19 or 20. The eyes of the fetuses were examined histologically in addition to observations on the uterine and fetal changes. The females of the third experiment were given clomiphene on day 11 of pregnancy and the fetuses were delivered by cesarean section on day 22. They were immediately fostered to control animals and allowed to develop to weaning, at which time they were killed and examined for cataracts. There were no significant increases in embryonic or fetal deaths in any of the treatment groups of the first two studies. Hydramnios was observed in treated rats together with an increase in placental weight and a decrease in uterine weight. Fetal cataracts were observed with clomiphene and clomiphene B but not with tamoxifen citrate. Clomiphene was shown to induce cataracts in fostered neonates in the third test with an incidence of 9.5%.

Reproductive Studies: Teratogenic studies were conducted in rats and rabbits. Some difficulties were encountered in these studies since tamoxifen inhibits implantations. Oral doses (administered in the feed) ranged from 0.02 to 4.0 mg/kg in rats and from 0.01 to 2.0 mg/kg in rabbits. A reversible rib deformity in rats which, under certain conditions, had an incidence as high as 50% was the only drug-induced abnormality detected. Evidence is presented which suggests that the cause of the deformity is mechanical due to the failure of uterine growth caused by the antiestrogenic property of the compound.

Mutagenicity: Tamoxifen is not mutagenic in a range of in vitro and in vivo mutagenicity studies. Tamoxifen was genotoxic in some in vitro tests and in vivo genotoxicity tests in rodents.

ESTROGEN RECEPTOR ASSAY

Introduction: Recently, studies in estrogen-dependent tissues have led to the discovery of a cytoplasmic protein which binds estrogen with high affinity and specificity. Estrogen enters the cytoplasm of all cells whether or not they are estrogen-dependent. However, in the cytoplasm of estrogen-dependent cells are found specific protein molecules that are termed receptors. These receptor proteins bind estrogen biologically with great affinity and specificity. Following this initial binding step, the estrogen receptor complex undergoes an activation which allows the complex to enter the nucleus of the cell and bind to chromatin, the genetic information of the cell. Once bound to the chromatin, the interaction of the estrogen receptor complex with the genetic information of the cell leads to the elaboration of new species of messenger RNA. These molecules are then released into the cytoplasm where they can be translated on polysomes into new proteins. Antiestrogens are also able to enter the cytoplasm of the estrogen-dependent cell and bind biologically to the protein receptor with affinity and specificity, thus activating the complex to also translocate to the nucleus. However, the normal estrogen transcriptional processes are altered. Hence, antiestrogens interfere with estrogen- dependent tumor growth by competing with estrogens for the receptor site and by turning off the normal processes of the genetic information within the nucleus. Reports concerning the relationship between clinical responses of patients with breast cancer receiving endocrine therapy and the presence or absence of estrogen receptors have been compiled. In patients with tumors positive for estrogen receptors, the response rate to endocrine therapy was approximately 56%; and in patients with tumors negative for estrogen receptors, the response rate was about 10%. It was concluded that estrogen receptor assays are useful in predicting the results of endocrine therapy in patients with breast cancer.

Methods:

1. Dextran-Coated Charcoal Assay (DCC)

The Dextran-Coated Charcoal assay (DCC) involves the extraction of the highly labile estradiol receptor from a cytosol prepared from the tumor tissue. After incubating with tritiated estradiol, which interacts with the binding sites of receptors, the excess estradiol is separated from the incubate with dextran- coated charcoal. The amount of non-specific binding (e.g., albumin) is then determined and the quantity of estradiol receptors in the tissue is estimated from the difference in the total binding less non-specific binding per milligram of protein. Tumors which show binding capacity similar to benign tumors are designated ER-negative, while those with higher binding capacity are designated ER-positive.

Sucrose Gradient Method (SG)

The weighed tumor specimen is immersed in liquid nitrogen and shattered. The residual tissue powder is homogenized with efficient cooling in four volumes of buffer, using a tissue disintegrator with two or three homogenization periods, each followed by a cooling period. The homogenate is centrifuged to precipitate the particulate matter. Two portions of the cytosol fraction are removed and treated with either buffer alone or buffer containing an agonist. When equilibrium is reached, tritiated estradiol is added to each mixture. After mixing and standing in the cold, a portion of each mixture is layered on a 10 to 30% sucrose gradient containing buffer, and centrifuged. Successive fractions are collected, from which the radioactivity is counted. Receptor-positive tumor specimens exhibit 8S complex, whereas others show various amounts of specific binding in the 4S region as well. Radioactivity associated with the 8S form of estrophilin is estimated from the difference in the sedimentation curves, with and without inhibitor, from fraction 1 to the minimum observed around fractions 18 to 22, depending on the ultra-centrifugation. The 4S radioactivity is similarly calculated by difference of the curves between the minimum and the point where the curve with inhibitor crosses the curve without inhibitor.

Interpretation of Results: Laboratory results of the estrogen receptor assay should be interpreted by a qualified expert, as results may vary due to technique, handling and storage of the specimen, and the patient's menopausal status or recent drug therapy. Quantitative results vary among laboratories and methods. As a result of retrospective correlation made by various investigators based upon patients' responses to hormonal manipulation, a result of less than 3 fmol/mg of cytosol protein is considered ER-negative, 3 to 10 fmol/mg cytosol protein is equivocal and over 10 fmol/mg cytosol protein is considered ER- positive. For a more detailed description of the analytic techniques and interpretation of results, the following references may be consulted.

ESTROGEN RECEPTOR MONOCLONAL ANTIBODIES

The quantitative determination of estrogen and progesterone receptors in human breast cancers has served as a guide to therapeutic invention as well as prognosis. Analysis of the receptor content of the primary tumor at the time of mastectomy is able to predict response to endocrine therapy should the tumor recur as well as estimating the probability and rapidity of recurrence. However, current methods for determination of estrogen and progesterone receptors suffer from several deficiencies. They are costly in terms of laboratory time, they require a large sampling of tumor tissue, rapid receptor deterioration during specimen processing or storage can often lead to erroneous results and ligand-binding assays fail to detect receptor that is already complexed with non- radioactive hormone of endogenous or therapeutic origin. These limitations have led to investigation of improved techniques for a simple, accurate and inexpensive assay which will recognize the receptor whether or not it retains its ability to bind hormones. Most recently monoclonal antibody technology has been used to generate a number of monoclonal antibodies specific for antigenic determinants on or near the estrogen receptor site. A number of antibodies have been produced by Greene and Jensen at the Ben May Laboratory for Cancer Research, University of Chicago, Chicago, Illinois. These specific monoclonal antibodies recognize the extranuclear estrogen receptor of the MCF-7 human breast cancer cell line. These antibodies bind to nuclear and cytosolic estrogen receptors from a variety of tissues and are therefore unique and specific probes for examining the structure and function of the estrogen receptor. Three such antibodies (D58, D75, D547) have been described to recognize different antigenic determinants on the receptor molecule. A combination of two such antibodies can be used in a sandwich technique for the immunoradiometric (IRMA) or enzyme-linked immunosorbent (ELISA) determination of estrogen receptor. These three antibodies recognize estrogen receptors in human breast cancer specimens as well as estrogen receptor in uterine tissue from other species. Further studies with the D547 and D58 monoclonal antibodies have revealed that these antibodies can distinguish among various forms of the estradiol-estrogen receptor complex. The antigenic determinants recognized by these particular antibodies on breast tumor cytosolic receptors are not significantly altered by the binding of either estrogen or antiestrogen to the receptor. Studies such as this are able to demonstrate fundamental differences in the subcellular fate of the estrogen or antiestrogen-receptor complexes, and provide clues to the mechanism of action of estrogens and antiestrogens. Poulsen has used two monoclonal antibodies specific for MCF-7 estrogen receptor to stain human breast cancer tissue sections using an immunoperoxidase technique. The immunoperoxidase staining was predominantly located in the nucleus of the malignant epithelial cells. No relationship between tumor type or degree of differentiation of invasive ductal carcinomas and staining features was observed. Poulsen found a significant positive correlation between the number of positively stained cells and cytosol receptor content. Similarly, King has developed monoclonal antibody D-5, an IgG1 which binds to soluble estrogen receptor in a dose-dependent manner. Antibody D-5 is specific for human soluble estrogen receptor and will not react with other steroid- binding proteins or nuclear estrogen receptor. King found a highly significant correlation between estrogen receptor content and D-5 reactivity in human breast cancer sections. Kodama has used similar techniques to study the expression of estrogen receptors of human breast cancer clonal growth using the soft-agar cloning assay. He found that estrogen receptor expression increased with clonal growth of tumor cells to colonies and that estrogen receptor appeared to be expressed in the differentiation process. Finally, Dr. Edwards has developed a monoclonal antibody to the chicken oviduct progesterone receptor. This antibody also recognizes denatured human progesterone receptor as its antigen. Further applications of this monoclonal antibody are currently being examined. The development of specific monoclonal antibodies directed at antigenic determinants of the estrogen or progesterone receptor will make it possible to more accurately and precisely define levels of estrogen or progesterone receptors in human tumor tissue. This technology will allow such assays to be performed on much smaller amounts of tumor tissue than are currently needed for standard receptor assays. In addition the future availability of standardized kits for performing monoclonal antibody assays will help provide uniformity when results of receptor levels are described.

MONOCLONAL ANTIBODY ESTROGEN RECEPTOR REFERENCES

1. Greene GL, Fitch FW, Jensen EV. Monoclonal antibodies to oestrophilin: probes for the study of oestrogen receptors. Proceedings National Academy of Sciences, USA 1980; 77: 157-161.

2. Greene GL, Jensen EV. Monoclonal antibodies as probes for estrogen receptor detection and characterization. J Steroid Biochemistry 1982; 16: 349-353.

3. Jensen EV, Greene GL, Hospelhorn VD, DeSombre ER. Improved procedures for the determining of oestrogen receptors in breast cancers. Reviews on Endocrine Related Cancer 1981, Suppl 9: 13-21.

4. Tate AC, DeSombre ER, Greene GL, Jensen EV, Jordan VC. Interaction of (3H) estradiol-(3H) monohydroxytamoxifen-estrogen receptor complexes with a monoclonal antibody. Breast Cancer Research and Treatment 1983; 3: 267- 277.

5. Poulsen HS, Ozzello L, King WJ, Greene GL. The use of monoclonal antibodies to estrogen receptors for immunoperoxidase detection of ER in human breast cancer, Second International Symposium on Antihormones in Breast Cancer, Berlin, West Germany, October 21-24, 1984, page 25.

6. King RJB, Coffer AI, Louis K. Studies with monoclonal antibody raised against partially purified oestradiol receptor from human myometrium, Second International Symposium on Antihormones in Breast Cancer, Berlin, West Germany, October 21-24, 1984, page 24.

7. Kodama F, Salmon SE, Soehnlen B, Greene GL. Expression of estrogen receptor as a clonal marker of differentiation in MCF-7 cells. Am Assoc for Cancer Research 1984; 25: 218 (Abstract 863).

8. Edwards D, McGuire W. Presentation of monoclonal antibody to progesterone receptor, Workshop on Estrogen and Antiestrogen Action: Basic and Clinical Aspects, Wisconsin Clinical Cancer Center, Madison, WI, June 26-29, 1984.

ESTROGEN RECEPTOR REFERENCES

1. Bishop HM, Poole T, Blamey RW, Nichsolson RI, Griffiths K. The importance of rapid freezing of breast tumors for the accurate determination of cytoplasmic oestradiol receptor (ER) status in primary breast cancer. Clin Oncol 1982; 8: 86.

2. Chamness GC, McGuire WL. Scatchard plots: common errors in correction and interpretation. Steroids 1976; 26: 538-542.

3. Hawkins RA, Roberts MM, Forrest APM. Oestrogen receptors and breast cancer: current status. Brit J Surg 1980; 67: 153-169.

4. Horwitz KB, McGuire WL, Pearson OH, Segaloff A. Predicting response to endocrine therapy in human breast cancer: a hypothesis. Science 1975; 189: 726-727.

5. Layne E. Spectrophotometric and turbidimetric methods for measuring proteins.

Methods Enzymol 1957; 3: 337-454. Leung BS, Moseley HS, Davenport CE, Kippaehne WW, Fletcher WS. Estrogen receptor in prediction of clinical responses to endocrine ablation. In: WL McGuire, PP Carbone, and EP Vollmer (eds. ). Estrogen Receptors in Human Breast Cancer, pp. 107-129. New York: Raven Press, 1975. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 265-275. McGuire WL. Current status of estrogen receptors in human breast cancer. Cancer 1975; 36: 638-644. McGuire WL, De La Garza M. Improved sensitivity in the measurement of estrogen receptor in human breast cancer. J Clin Endocrinol Metab 1973; 37: 986-989. McGuire WL, Pearson OH, Segaloff A. Predicting hormone responsiveness in human breast cancer. In: WL McGuire, PP Carbone and EP Vollmer (eds. ). Estrogen Receptors in Human Breast Cancer, pp. 17-30, New York: Raven Press, 1975. McGuire WL. Therapeutic significance of hormonal receptor assays. In: Reviews on endocrine-related cancer, Proc. Sym. King's College, Cambridge, July 4-5, 1978: Suppl 3, pp. 41-49, 1979. Rice RH, Means GE. Radioactive labelling of proteins in vitro. J Biol Chem 1971; 246: 831-832. Scatchard G. The attraction of proteins for small molecules and ions. Ann NY Acad Sci 1949; 51: 660-672.

REFERENCES

1. Emmens CW. Inhibitors of implantation. J Reprod Fertil Suppl 1973; 18: 177- 184.

2. Jordan VC, Koerner S. Tamoxifen (ICI 46, 474) and the human carcinoma 8S oestrogen receptor. Eur J Cancer 1975; 11: 205-206.

3. Mortel R, Levy C, Wolff J-P, Nicolas J-C, Robel P, Baulieu E-E. Female sex steroid receptors in postmenopausal endometrial carcinoma and biochemical response to an antiestrogen. Cancer Res 1981; 41: 1140-1147.

4. Harper MJK, Walpole AL. A new derivative of triphenylethylene: effect on implantation and mode of action in rats. J Reprod Fertil 1967; 13: 101-119.

5. Lippman M, Bolan G, Huff K. The effects of estrogens and antiestrogens on hormone-responsive human breast cancer in long-term tissue culture. Cancer Res 1976; 36: 4595-4601.

6. Coezy E, Borgna J-L, Rochefort H. Tamoxifen and metabolites in MCF, cells: correlation between binding to estrogen receptor and inhibition of cell growth. Cancer Res 1982; 42: 317-323.

7. Eckert RL, Katzenellenbogen BS. Effects of estrogens and antiestrogens on estrogen receptor dynamics and the induction of progesterone receptor in MCF-7 human breast cancer cells. Cancer Res 1982; 42: 139-144.

8. Green MD, Whybourne AM, Taylor IW, Sutherland RL. Effects of antioestrogens on the growth and cell cycle kinetics of cultured human mammary carcinoma cells. In: RL Sutherland, VC Jordan (eds): Non-steroidal antioestrogens. Australia: Academic Press, 1981; 397-412.

9. Osborne CK, Boldt DH, Clark GM, Trent JM. Effects of tamoxifen on human breast cancer cell cycle kinetics: accumulation cells in early G phase. Cancer Res 1983; 43: 3583-3585.

10. Sutherland RL, Green MD, Hall RE, Reddel RR, Taylor IW. Tamoxifen induces accumulation of MCF-7 human mammary carcinoma cells in G0/G1 phase of the cell cycle. Eur J Cancer Clin Oncol 1983; 19: 615-621.

11. Golder MP, Phillips MEA, Fahmy DR, Preece PE, Jones V, Henk JM, et al. Plasma hormones in patients with advanced breast cancer treated with tamoxifen. Eur J Cancer 1976; 12: 719-723.

12. Groom GV, Griffiths K. Effect of the anti-oestrogen tamoxifen on plasma levels of luteinizing hormone, follicle-stimulating hormone, prolactin, oestradiol and progesterone in normal pre-menopausal women. J Endocrinol 1976; 70: 421- 428.

13. Kiang DT, Kennedy BJ. Tamoxifen (antiestrogen) therapy in advanced breast cancer. Ann Intern Med 1977; 87: 687-690.

14. Manni A, Trujillo J, Marshall JS, Pearson OH. Antiestrogen-induced remissions in Stage IV breast cancer. Cancer Treat Rep 1976; 60: 1445-1450.

15. Williamson JG, Ellis JD. The induction of ovulation by tamoxifen. J Obstet Gynecol Br Common 1973; 80: 844-847.

16. Adam HK, Douglas EJ, Kemp JV. The metabolism of tamoxifen in humans. Biochem Pharmacol 1979; 28: 145-147.

17. Daniel P, Gaskell SJ, Bishop H, Campbell C, Nicholson RI. Determination of tamoxifen and biologically active metabolites in human breast tumours and plasma. Eur J Cancer Clin Oncol 1981; 17: 1183-1189.

18. Fabian C, Sternson L, Barnett M. Clinical pharmacology of tamoxifen in patients with breast cancer: comparison of traditional and loading dose schedules. Cancer Treat Rep 1980; 64: 765-773.

19. Fromson JM, Pearson S, Bramah S. The metabolism of tamoxifen (ICI 46,474). Part II: in female patients. Xenobiotica 1973; 3: 711-714.

20. Wilkinson P, Ribeiro G, Adam H, Patterson J. Clinical pharmacology of tamoxifen and N-desmethyltamoxifen in patients with advanced breast cancer. Cancer Chemother Pharmacol 1980; 5: 109-111.

21. Fromson JM, Pearson S, Bramah S. The metabolism of tamoxifen (ICI 46,474). Part I: in laboratory animals. Xenobiotica 1973; 3: 693-709.

22. Fabian C, Tilzer L, Sternson L. Comparative binding affinities of tamoxifen, 4- hydroxytamoxifen, and desmethyltamoxifen for estrogen receptors isolated from human breast carcinoma: correlation with blood levels in patients with metastatic breast cancer. Biopharm Drug Dispos 1981; 2: 381-390.

23. Watanbee M, et al. Oral acute, subacute and chronic toxicity studies of tamoxifen in small animals. Jitchuken-Zenrinsho Kenkyuho 1980; 6: 1-36 (translated from Japanese).

24. Harper MJK, Walpole AL. A new derivative of triphenylethylene: effect on implantation and mode of action in rats. J Reprod Fert 1967; 13: 101-119.

25. Bloxham PA, et al. An effect of tamoxifen (ICI 46,474) on the surface coat of the late preimplantation mouse blastocyst. J Reprod Fert 1975; 45: 181-183.

26. Pugh DM, Sumano HS. The anti-implantation action of tamoxifen in mice. New Toxicology for Old Arch Toxicol Suppl 1982; 5: 209-213.

27. Furr BJ, et al. Tamoxifen. In: Goldberg Me, ed. Pharmacological and Biochemical Properties of Drug Substances. Vol. 2. Washington, DC: American Pharmaceutical Association 1979; 355-399.

28. Chamness GC, et al. Abnormal reproductive development in rats after neonatally administered antiestrogen (tamoxifen). Biol Reprod 1979; 21: 1087- 1090.

29. Clark JH, McCormack SA. The effect of clomid and other triphenylethylene derivatives during pregnancy and the neonatal period. J Steroid Biochem 1980; 12: 47-53.

30. Drenckhahn D, et al. Corneal lipidosis in rats treated with amphiphilic cationic drugs. Arzneim Forsch Drug Res 1983; 33: 827-831.

31. Bratherton DG, Brown CH, Buchanan R, Hall V, Kingsley Pillers EM, Wheeler TK, Williams CJ. A comparison of two doses of tamoxifen (Nolvadex(r)) in postmenopausal women with advanced breast cancer: 10 mg bd versus 20 mg bd. Br J Cancer 1984; 50: 199-205.

32. Braunsberg H. Factors influencing the estimation of oestrogen receptors in human malignant breast tumours. Eur J Cancer 1975; 11: 499-507.

33. Buchanan RB, Blamey RW, Durrant KR, Howell A, Paterson AG, Preece PE, Smith DC, Williams CJ, Wilson RG. A randomized comparison of tamoxifen with surgical oophorectomy in premenopausal patients with advanced breast cancer. J Clin Oncol 1986; 4: 1326-30.

34. Dorr RT, Fritz WL. Tamoxifen. Cancer Chemotherapy Handbook, Elsevier, New York, 1980; 641-51.

35. Fentiman IS, Powles TJ. Tamoxifen and benign breast problems. The Lancet 1987; 2:1070-1.

36. Goldberg ME. Tamoxifen. Pharmacological and biochemical properties of drug substances, Volume 2, American Pharmaceutical Association Academy of Pharmaceutical Sciences, Washington, D.C., 1979.

37. Hawkins RA, Roberts MM, Forrest APM. Oestrogen receptors and breast cancer: current status. Br J Surg 1980; 67: 153-69.

38. Heel RC, Brogden RN, Speight TM, Avery GS. Tamoxifen: a review of its pharmacological properties and therapeutic use in the treatment of breast cancer. Drugs 1978; 16: 1-24.

39. Ingle JN. Additive hormonal therapy in women with advance breast cancer. Cancer 1984; 53: 766-77.

40. Legha SS. Tamoxifen in the treatment of breast cancer. Ann Int Med 1988; 109: 219-28.

41. Ribeiro G, Swindell R. The Christie hospital adjuvant tamoxifen trial - status at 10 years. Br J Cancer 1988; 57: 601-3.

42. Riippa P, Kauppila A, Sundstrom H, Vihko R. Hepatic impairment during simultaneous administration of medroxyprogesterone acetate and tamoxifen in the treatment of endometrial and ovarian carcinoma. Anticancer Res 1984; 4: 109-12.

43. United States Food and Drug Administration Summary for Approval Documents for Nolvadex(r) (tamoxifen citrate), NDA 17-970 (Including Division Directors Review and Initial Pharmacologists Review).

44. AHFS Drug Information 1988. American Society of Hospital Pharmacists Inc., Bethesda, MD, USA, 1988; 550-2.

45. Comparative Bioavailability Studies of Tamoxifen Citrate Tablets in Normal Human Volunteers. July 20, 1984 and February 18, 1998. Data on File at Novopharm Limited.

46. Valavaara R, Kangas L. The significance of estrogen receptors in tamoxifen and toremifene therapy. Ann Clin Res 1988; 20: 380-8.

47. Walter JB, Israel MS. General Pathology, 6th Edition. Spread of malignant tumours (Chapter 28). Longman Group UK Limited 1987: 387-94.

48. Nolvadex(r) (tamoxifen citrate) Product Monograph, Zeneca Pharma (Canada) Inc., Mississauga, Ontario.