Anti-estrogen and immune modulator combinations for treating breast cancer

Description of equivalent WO 03041654 A2

ANTI-ESTROGEN AND IMMUNE MODULATOR COMBINATIONS FOR TREATING BREAST CANCER

BACKGROUND OF THE INVENTION

0003Field of Invention The present invention generally relates to methods and compositions for the use of tamoxifen and other anti-estrogenic compounds in combination with immune modulator agents (immunoglobulin inhibitors of estrogen responsive cancer cell growth), to treat or prevent breast cancer. Description of Related Art In 1896, a British physician named Beatson reported that δophorectomy had palliative effects for breast cancer patients (1). In 1905, Lett confirmed this observation with a larger patient trial (2). Clearly ovarian products were either directly or indirectly significant in breast cancer growth. From these earliest clinical observations, chemical and endocrine research continued and culminated in the identification of the primary ovarian/follicular agents responsible. The active agents proved to be a class of cholesterol derived steroid hormones now designated estrogens. In 1929 and 1930, Doisy and colleagues crystallized estrogens including estrone [3-hydroxy-estra-l,3,5(10)-trien-17-one] (Ε_\) from human pregnancy urine (3,12). Estradiol-17j8 [estra-l,3,5(10)-triene-3, 17/3-diol] (E₂ ) was also isolated from sow follicular fluid (4). The remaining major estrogen, estriol [1,3,5- estratriene-3, 16 (X, 17 β-triol] (E₃) has also been defined.

0004The relative potency of these three hormones is known today to be E₂ > Ε_\ »» E₃ (5). With regard to breast cancer cell growth, E₂ and E₁ are in the main considered the most physiologically relevant (6-9). Estriol is most likely relevant during pregnancy when the maternal plasma level is significantly elevated (10). During pregnancy, maternal E₃ is formed primarily as a placental conversion product of a steroid produced by the fetal adrenals. Breast cancers are not uncommon during pregnancy (18,22-25). However, all three estrogens are increased in pregnancy (10). In pregnant women, breast cancer is often diagnosed at a later stage (18). It may be that the elevated hormones during this time cause growth of developing breast cancer cells in pregnant females (19). Clearly, however, pregnancy has opposing effects on breast cancer development. On the one hand the increase in hormones can promote cancer cell growth (35). On the other hand, pregnancy and high hormones induce tissue differentiation that ultimately protects the tissue (20,21). Apparently the elevated estrogen levels in pregnancy explain the transient increase in short-term risk of breast cancer following term pregnancy (19). The results of several studies indicate that all three of the estro genie steroid hormones (i.e. E , Ei and E₃) are important in breast cancer risk in humans (26-28).

0005The biosynthesis and metabolism of estrogens and estrogen-related steroid hormones has been reviewed (11). The majority of plasma E₂ and Ei is synthesized and secreted by cells of the ovarian follicle (29,30). The biochemical synthetic pathway begins with conversion of cholesterol to progesterone, followed by modification of the progestin to form androgens or androgen-like steroids. To form all three types of estrogen, the cholesterol origin "A" ring of "androgens" must be converted to a phenolic structure by the action of aromatases. These key enzymes in the biosynthesis of estrogens are located in the endoplasmic reticulum of ovarian cells.

0006Estrogens undergo a variety of metabolic transformations including hyroxylations, methylations and reduction. Also, the estrogens are converted to more water-soluble, biologically inactive, glucuronide and sulfate conjugates by the liver. The conjugates are excreted into urine and bile. Earlier studies indicated that estrogen conjugates (e.g. estrone sulfate) might serve as sources of free estrogen in breast cancer cells possessing the appropriate cleaving enzyme(s) to form free steroid (31). More recent work (32-34) indicates this is unlikely, based on tissue culture studies with eight different ER⁺ cell lines. Estrogen sulfates and glucuronides are cleaved by intestinal flora to regenerate free estrogens that again appear in the plasma and urine via the enterohepatic circulation (36). A high fiber-low fat diet tends to decrease this process. Other intestinal microbial processes also convert inactive estrogen metabolites to active steroid hormones (37). Thus, recycling of estrogens is entirely possible.

0007However, the sites of synthesis of estrogenic substances in the body are not limited to the ovary (13). While it is understood with premenopausal women that estrogens are primarily of ovarian origin, this is not the case in postmenopausal females (38-41). The question is "what is the origin(s) of estrogens in the postmenopausal female"? This is important because breast cancer rates are much higher in postmenopausal women (42) even though estrogen levels are declining Nonetheless, 80 or 90% of breast cancers in postmenopausal women are ER⁺ (43), implying they are estrogen growth promoted. This paradox can be explained in part by the suggestion that postmenopausal women with higher risk of developing breast cancer show relatively higher concentrations of endogenous estradiol (44). Also, it is now very clear that adrenal androgenic steroids can be converted to estrogens via the action of aromatases located in mammalian tissues (45). Its activity provides a significant portion of the plasma estrogens even in postmenopausal women (38- 41). Aromatase activity has a broad tissue distribution in mammals (45). However, in human women after menopause, adipose tissue is the primary source of endogenous estrogens (46,47). Indeed, obesity is positively correlated with breast cancer (48). Also, aromatase is present in breast tissue and cells and represents an "intracrine" source of stimulating steroid hormone (49). Because of the major role of aromatase in generating breast cancer promoting estrogens in postmenopausal women, a series of aromatase inhibitors has been developed and are now in use as pharmaceutical products or are in and clinical trials as breast cancer treatments (41).

0008The question of how estrogens regulate target tissue gene expression and growth is of great consequence to this discussion. In 1962, Jensen & Jacobsen (14) came to the conclusion that estrogens acted on sex steroid hormone target tissues via specific cellular receptors. By 1972 to 1974, this research was sufficiently advanced to outline the mechanisms of estrogen action as mediated by an intracellular receptor (15-17). For several years, intense study has proceeded and has been reported in nearly 20 thousand publications (PubMed literature search of "estrogen receptors"). In 1986, the molecular cloning of the original estrogen receptor, now designated ERα, was reported (50,51). This 64-kDalton protein is functionally and structurally related to other receptors and has been classified as a member of the steroid and thyroid hormone superfamily (52). Today, these similar receptors include those for androgens, corticosteroids, progestins, thyroid hormones, vitamin D and retinoic acid.

0009Although for several years ERα was acknowledged as the only estrogen receptor, variants of it were being identified (55,56). However, in 1995, another type of estrogen receptor, designated ER/3, was cloned from a rat prostate and ovary (57). This initiated a boom of new activity to define the function and properties of ER/3 (58,60,61). Indeed, the results suggest that the role of estrogens in male accessory organ function deserves renewed study (58). The characteristics and properties of ERc versus ER/3 have been reviewed (58,61,63). For the purposes of this disclosure, it should be noted that the binding affinities of both receptors are approximately equal (61). This was expected. However, one startling fact has surfaced. Mice gene knockout experiments for both ERα (62) and ER/3 (60) have confirmed developmental functions for both of these receptors, but have fallen short of providing conclusive evidence that either receptor regulates growth (58). In fact, transfection of ER^" cells with a functional ER led to an estrogen-induced inhibition of cell growth (59). There is a possibility that ERα is a receptor regulating expression of differentiated functions. It is well recognized that growth and differentiation are 'dprJαsirig¹ 'c ϊrMctio'ϊϊaT 'states?¹' Differentiated cells divide only slowly if at all. This issue has been reviewed in detail in recent U.S. Patent Application Nos. 09/852,547 and 09/852,958 and in International Patent Application Nos. PCT/US01/15171 (WO 01/86307) and PCT/USOl/15183 (WO 01/85210), also identified in the list of References, below, as items 53 and 54, and hereby incorporated herein by reference). This led to the proposal in those applications that there is another growth regulating estrogen receptor, tentatively designated ER7 (53,54).

0010The characteristics of ERγ are that it binds estrogens with 10 to 100-fold higher affinities than ERc or ER/3. Furthermore, it is proposed that this receptor is a new gene that is expressed in all estrogen growth responsive target tissues. Data obtained indicate that this receptor is present in eight well-known estrogen responsive tumor cell lines derived from four tissues and three species including human (32-34,53,54).

0011However, there exist potential alternatives regarding the identity of ERγ. Investigators have cloned two ERα-like "orphan receptors" with unknown functions (64,65). Other forms of estrogen receptors appear to arise as gene product splice variants (58,66). Those with major deletions of the hormone binding domain or the DNA binding domain may be expected to be inactive with respect to estrogen induced growth of breast cancer cells. The function of most of the other types of known variants remains to be established.

0012Another potentially significant variant has been identified. It is a point mutation that affects the border of the nge-hormone-binding domains (67). This mutation was found in 34% of a series of 59 specimens of premalignant hyperplasia. Transfection of this mutated ERα caused MCF-7 human breast cancer cells to respond to lower concentrations of estrogen in culture. The full implications of this mutation await more study, but it is clear from the results available at this time, and those presented in the above-identified patent applications (53,54) and other recent publications (32-34), that MCF-7 as well as T47D and ZR-75-1 ER⁺ breast cancer cells respond to very low concentrations of E₂ even without transfection of the mutated ERα. It may be possible that the hypersensitive mutated receptor (67) is present in all ER⁺ cell types including those from rat mammary and rat pituitary tumors as well as from estrogen-induced kidney tumor cells from Syrian hamster (32-34). This means that a specific mechanism must exist for formation of this receptor in target tissue cells, or that this receptor is derived from a new gene. The latter possibility implies that the response of ER⁺ cells to very low concentrations of E₂ involves the proposed new ERγ (53,54). The currently available knowledge about esfrogen'fufϊctioή and ^"estrogen receptόrs^"na led to one of the most common treatments for disseminated and/or local ER⁺ breast cancer, especially in postmenopausal women. Today, selective estrogen receptor modulators (SERMs) are the compounds of choice (68). The mechanism of action of these drugs is to block the growth promoting action of estrogens at the cellular/receptor level, no matter whether the sex steroid hormones are delivered systemically or formed locally in breast tissue via aromatase action on adrenal steroid precursors. Hence, these drugs are classified as anti- estrogens. As a general mechanism of action, anti-estrogens are thought to interfere with the binding of natural estrogens to the growth promoting estrogen receptor(s). The first potent anti-estrogen developed 1958 was MER-25 or ethamoxytriphetol

0013(76). It then was used to derive clomiphene (77) which is now used to treat amenorrhea. Clomiphene was then modified to give rise to tamoxifen (78). Although several anti- estrogens have been developed, only two are currently FDA approved for treatment of human breast cancer. These are tamoxifen and toremifene. These, and id xifene and droloxifene, are triphenylethylene derivatives. Notably, the toremifene structure differs from tamoxifen by only a single chlorine atom (69). Since its approval in 1977, tamoxifen has been the SERM of choice for treatment of ER⁺ breast cancer worldwide (70). Tamoxifen is classified as a "mixed" anti-estrogen because it displays both antagonistic properties (i.e. inhibits breast cancer cell growth) and agnostic properties (i.e. stimulates endometrial cell growth and tumor development) (71).

0014The action of the anti-estrogens is reversed by lower concentrations of the natural estrogens (53,54). The affinity of tamoxifen for the estrogen receptor is 10 to 100-fold less than that of E . This is commonly recognized throughout the endocrine cancer field. It is therefore useful to suppress natural estrogens along with application of tamoxifen treatment. This fact is often not recognized clinically. Postmenopausal women are not completely devoid of estrogens. Tamoxifen effectiveness is reduced by residual estrogenic steroid hormones. It is also reduced by the tamoxifen induced elevation of DHEA, E and Ei (81- 83). This is an unfortunate side effect of using this drug alone.

0015One of the commonly cited facts concerning tamoxifen is that it acts at cellular sites separate from the estrogen receptor. It is known to influence such cellular activities as protein kinase C as well as several other cellular mechanisms including those related to apoptosis (72). Although non-steroid hormone receptor directed actions are usually considered undesirable, certain very recent co-owned patent disclosures (53,54) describe targeting a non-steroid hormone receptor with new drug combinations whose actions are based on anti-estrogen augmentation/mimicking of the inhibition of growth of ER⁺ breast cancer cells by the immunoglobulins IgA and IgM of the natural secretory immune system. As described (53,54), the secretory immune system acts as a paracrine negative regulator of ER⁺ breast cancer cell growth. Employing new serum-free defined culture assay methods (53,54), tamoxifen was shown to mimic the inhibition caused by IgA or IgM in the complete absence of estrogens. This new tamoxifen function represents a clear departure from previous thought concerning how this "mixed function" anti-estrogen acts. Previously, other investigators had reported that tamoxifen inhibited growth factor dependent proliferation of human breast cancer cells in cultures devoid of estrogens and estrogen-like agents (73). However, there was no indication at that time that this anti-estrogen was capable of acting by mimicking the growth inhibitory effects of the natural secretory immune system immunoglobulins IgA, IgM and IgGl.

0016Another class of anti-estrogens is defined as "pure" because they only affect growth via interaction with estrogen receptors (71). The pure anti-estrogens were discovered about 15 years ago (74). Currently, five compounds are under intense investigation (71). They are abbreviated ICI 164384, ICI 182780, EM-800, RU 58688 and EM-139 (71). Two of these, ICI 164384 and ICI 182780 are in clinical trials. Because tamoxifen resistance develops with time (75), the pure anti-estrogens are thought to be useful as second-line therapies after tamoxifen failure (71). Furthermore, pure anti-estrogens are thought useful because they cause no increase in endometrial cancer (71).

0017However, the pure anti-estrogens have marked deleterious effects on the cardiovascular and skeletal systems (71), and their usefulness is yet to be established. There remains a need for effective anti-estrogens and for combination therapies of tamoxifen or tamoxifen-like drugs and the "pure" anti-estrogens that may be more effective than either class of drug alone.

0018SUMMARY OF PREFERRED EMBODIMENTS New compositions and methods are provided which advantageously employ compounds having a newly defined immune modulating function, or which have the ability to mimic that immune modulating function, or a combination of such compounds. For the purposes of the present disclosure, the terms "immune mimic," "immune modulating,"

0019"immune modulator," "immune modulation," "immune control," "immune inhibition,"

0020"immune suppressor," and the like, refer in most instances to the newly identified cancer cell growth (i.e., proliferation) inhibitory effect of the secretory immune system (i.e., dimeric/polymeric IgA and pentameric IgM) that is mediated by a newly identified Poly-Ig receptor or Poly-Ig-like receptor (also classified as an Fc-like receptor), and not to the usual antibody/antigen recognition based immune function of the immune system. In this context, the terms "immune modulation" or "immune enhancement" refer especially to the modulation or enhancement of these cell growth inhibitory immunoglobulins of the secretory immune system. The term "immune mimic" refers to a substance (e.g., tamoxifen) that can function in a similar manner to an irnmunoglobulin inhibitor of cell growth. In some instances, however, reference is also made herein to "natural immune inhibition," "immune enhancer," "immune modulator," "immune system," "immune therapy," and "immune response," and the like, in which the conventional meanings of those terms are intended and the context so indicates, especially when prior art methods, compounds and compositions are described. Hereinafter, an indication has been made in appropriate instances whether a conventional definition or the "new" meaning, or both, is intended.

0021In some aspects of the present invention, tamoxifen is used as a breast cancer treatment taking advantage of its newly identified function as an immune mimic instead of an anti-estrogen. That tamoxifen is a mixed anti-estrogen is well known. It not only binds to cellular estrogen receptors, but it also has other unrelated sites of cellular action. This new function for tamoxifen makes possible new combination therapies as well as new diagnostic methods to determine whether breast or other mucosal origin cancers are expected to be susceptible to these therapies. It is concluded that combination therapies of tamoxifen and the "pure" anti-estrogens may be more effective than either class of drug alone.

0022Tamoxifen treatment alone has several positive aspects as well as a number of negatives. The negatives can be overcome by placing this well known anti-estrogen in combinations with other compounds. The preferred combinations represent those that permit the mixture to act more effectively than the individual component alone. The combinations may include two or more breast cancer treatment drugs, some of which are classified as "pure" anti-estrogens while others are defined as immune modulators.

0023In accordance with certain embodiments of the present invention, a new tamoxifen- based therapeutic method is provided, in which tamoxifen acts as an immune inhibitor mimic ("immune mimic"). The method preferably includes employing a new diagnostic test to identify breast cancer cells expressing the inhibitor-mediating receptor (a Poly-Ig receptor or Poly-Ig like receptor), also classified as an Fc-like receptor, as an indication of sensitivity to cell growth inhibition by tamoxifen.

0024In accordance with another embodiment, the above-described tamoxifen therapy and diagnostic testing method is extended to mucosal cancers other than breast, including those of the prostate, colon, kidney, bladder, lung, pancreas, riasdphafynxV' ovai ehdo^'meMumJ vagina, and cervix.

0025In still other embodiments, combinations of tamoxifen and aromatase inhibitors are employed to treat breast and gynecologic cancers. In some embodiments, tamoxifen and a "pure" anti-estrogen compound are combined for treating breast and gynecologic cancers.

0026Some embodiments of the present invention provide compositions or therapeutic methods using chemically modified MER-25 to treat secretory immune system related cancers. In some embodiments, MER-25 or modified MER-25 is combined with progesterone or another hormone for treating breast cancer. Modified MER-25 or derivative compounds of MER-25 that may have satisfactory anti-estro genie or immune mimicking activity include methylated, alkylated, benzylated, halogenated, unsaturations, altered charge properties, and conformationally altered or stereoisomers of MER-25.

0027In certain embodiments, combinations of tamoxifen and levamisole are used as an immune mimic and immune modulator to treat breast and other mucosal cancers, including colon cancer. In certain embodiments, combinations of tamoxifen and imiquimod are used as an immune mimic and immune modulator to treat breast and other mucosal cancers. In certain embodiments, tamoxifen and OK-432 (picibanil) are used as an immune mimic and immune modulator to treat breast and other mucosal cancers.

0028Certain embodiments of the present invention provide compositions or therapeutic methods employing a combination of tamoxifen and DHEA (dehydroepiandrosterone) as an immune mimic and immune modulator to treat breast and other mucosal cancers. In certain embodiments, a therapeutic method is provided in which tamoxifen and an Fc-like receptor gene therapy are used together to treat breast and other mucosal cancers.

0029In still other embodiments of the invention, methods are provided for identifying anti- estrogenic compounds or for evaluating modified forms of existing compounds that might be more effective anti-estrogenic agents. These methods employ cell growth assays that, preferably, use certain serum-containing or serum-free media. In some embodiments, methods are provided for screening new compounds and for determining how combinations of compounds act on cells directly. These and other embodiments, features and advantages of the present invention will become apparent with reference to the following description. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

0030Example 1: Tamoxifen Therapy and New Diagnostic Test for Immune Modulation Applications with Breast Cancer.

0031Co-pending U.S. Patent Application Nos.09/852,958 and 09/852,547/PCT Published Application Nos. WO 01/86307 and WO 01/85210 establish that tamoxifen mimics the cell growth inhibitory actions of the secretory immune system immunoglobulins IgA and IgM

0032(53,54). The disclosures of those applications are hereby incorporated herein by reference.

0033The immunoglobulin action is mediated by a Poly-Ig receptor or a Poly-Ig-like receptor (also classified as an Fc-like receptor) that is identified by antibody raised against the extracellular five domains commonly called the "secretory component" (SC) (86). Those breast cancer cells expressing this Fc-like receptor are sensitive to inhibition by tamoxifen. Those cells not expressing the Fc-like receptor are not tamoxifen sensitive. Because the analysis can be done in completely serum-free defined medium (53,54) without estrogens, it is concluded that tamoxifen acts to mimic the Fc-like receptor mediated inhibition of cell growth by secretory immune system IgA and IgM. Using immunohistochemical analysis methods (88), breast cancer specimens will be examined for SC positive receptors. It has already been demonstrated that this methodology will identify breast cancers at the early stage when they are expressing an SC detectable receptor (87). These tumors are candidates for immune mimicking regulation. Those tumors that are Fc-like receptor positive are candidates for tamoxifen therapy or combined therapy with tamoxifen and the other agents described below. As a specific test is developed to detect the ERγ, the use of double label fluorescence will permit very accurate determination of patients that are strong candidates for the therapies described.

0034It is expected that tamoxifen inhibits breast cancer cell growth not by interaction with the commonly recognized ERα or ERβ but instead with the ERγ (53,54). The direct histochemical measurement of ERγ is expected to significantly increase the reliability of the decision to initiate anti-estrogen therapy. Further, the identification of ERγ will permit reanalysis of existing and new compounds for anti-ERγ activity. This approach can be expected to significantly advance how new SERMs are selected. Example 2: Tamoxifen Therapy and New Diagnostic Test for Immune Modulation Applications with other Mucosal Cancers including Prostate, Colon, Kidney, Bladder Lung, Pancreas, Nasopharynx, Ovarian, Endometrial, Vaginal and Cervical Cancer.

0035The analysis outlined in Example 1 will be used to determine the application of the tarnoxifen-based therapies to tumors arising from other mucosal tissues. Since the same secretory immune system is functional in all of the tissues (prostate, colon, kidney, bladder, lung, pancreas, nasopharynx, ovary, endometrium, vagina and cervix), the immunohistochemical analysis for SC detectable Fc-like receptor can be conducted. It has already been shown (89) that colon cancers progress through stages in which the SC is expressed (i.e. early differentiated tumors) to stages in which there is little or no detectable

0036SC (i.e. late malignancy stage). Tamoxifen and/or the combinations described will be used to treat Fc-like receptor positive (FcLR⁺) tumors by the new protocols. This new approach is expected to provide an expanded rationale for the use of tamoxifen to treat cancers not yet recognized as sensitive to this immune mimicking anti-estrogen. Further, as a test for ERγ is developed, it can be used to further refine the tumor types susceptible to the new modes of tamoxifen combination therapies.

0037Example 3: Combinations of Tamoxifen and Aromatase Inhibitors to Treat Breast and Gynecologic Cancers.

0038It is proposed that simultaneous treatment with aromatase inhibitors and tamoxifen will be more effective than either drug alone. The therapeutic potential of this new combination is all the more significant in view of the fact that aromatase inhibitors do not completely inhibit estrogen synthesis. An unfortunate side effect of tamoxifen treatment is that it increases the concentration of estrogens in the plasma, which will have a marked effect on reducing the potency of tamoxifen as an anti-estrogen. The simultaneous use of aromatase inhibitors is expected to suppress this estrogen inductive effect and therefore may help to resolve the problem of escape of tumors from tamoxifen inhibition. In conventional therapies, tamoxifen resistance is currently thought to be a major problem with longer-term tamoxifen treatment. As additional tumor types beyond breast are identified as tamoxifen sensitive (e.g. endometrial, ovarian, vaginal, cervical and possibly prostate), the combined tamoxifen-aromatase inhibitor therapy will have even broader application than is recognized today for either drug.

0039Example 4: Combinations of Tamoxifen and Pure Anti-estrogens to Treat Breast and Gynecologic Cancers.

0040The negative aspect of the current "pure" anti-estrogens discussed above is that they deplete the body of estrogen action so effectively that there are major cardiovascular and skeletal problems. However, by using the combination of tamoxifen and the "pure" anti- estrogens, the agonist action of tamoxifen can be expected to reduce this problem without supplying unwanted natural estrogens. Tamoxifen has positive effects on both the cardiovascular system and bone. Furthermore, it is expected that this combination will also reduce the problem of induction of endometrial cancers thought todly "t'd"^*be"^*a" problem^' wϊtrf the use of tamoxifen alone. Hence, the combination reduces major negative aspects seen with each drug alone. The balance of the two components can be varied to achieve specific end points. Example 5: Use of Chemically Modified MER-25 to Treat Secretory Immune System Related Cancers.

0041MER-25 is an anti-estrogen by virtue of its inhibitory effects on estrogen target tissues. It also has the benefit that it does not interact with the estrogen receptor to accomplish its action (79). The advantage of MER-25 (or its modified forms) is that systemic or locally produced estrogens will not interfere. Thus, it can be used with ER pre- and postmenopausal women without concern for suppression of endogenous estrogen levels. The results available support the present suggestion that MER-25 may mimic the immune activity of IgA and IgM even more strongly than tamoxifen.

0042Early anti-estrogens such as MER-25 were by-passed by previous investigators because of their potency and adverse side effects. Although MER-25 has many desirable properties as an anti-estrogen, it has been reported to be too toxic for use in humans (78). However, if MER-25 and related compounds can be modified to achieve high levels of immune modulation without the serious side effects, this will open additional new avenues of breast cancer therapy. Accordingly, the chemical structure will be modified particularly in the O-C-C-N segment of the side chain to change the conformation and to prevent hydrogen bonding with neighboring hydroxyl groups (80). Only limited modifications in MER-25 have been sought (84). Other chemical changes in the structure are expected to attenuate the side effects considered most severe. Computer based molecular modeling will be used to develop the chemical modifications. The modifications are expected to include, for example, methylation, halogenation, unsaturations, alterations on charged groups, changes in conformation and selection of stable stereoisomers of the MER-25 structure. The chemically modified forms will be evaluated for anti-estrogenic activity using both the serum-containing and serum-free assay methods described in co-owned U.S. and PCT patent applications (53,54), incorporated herein by reference. This is a rapid and effective method of determining when a derivative has been obtained that still retains the desired potency against estrogen target cells in culture but cannot be reversed by exogenous estrogens in culture. Those that are effective under the strict tests outlined (53,54) will be tested for in vivo anti- tumor activity using rat mammary and pituitary models as well^" as wιtE^' eri^'6^'graffs of uman breast cancer cell lines in athymic nude mice.

0043Example 6: Combinations of MER-25 and Modified MER-25 with Progesterone and other Hormones. The undesirable side effects of MER-25 and chemically modified forms may also be attenuated by simultaneous treatment with progesterone. In other studies, it has been shown that eating behavior and body weight regulation are affected by MER-25 (85). The administration of progesterone in rats corrected those side effects. Thus, at least some adverse properties in vivo may be due to altered hormonal influences. Administration of MER-25, and derivative compounds, will be evaluated for causation of endocrine changes. Any changes identified will be corrected by simultaneous application of the appropriate hormone(s). MER-25 or one of its derivatives may influence, for example, pituitary hormone secretion, thyroid hormones, adrenal hormones and/or neurogenic amines. Cytokines are also included in this group. Such changes are expected to yield the severe side effects reported (78). Accordingly, a hormone derived from pituitary, adrenals or thyroid, or a cytokine or a neurogenic hormone may be administered together with MER-25 or a modified form of

0044MER-25 to deter the occurrence of side effects from the drug.

0045Example 7: Combination Tamoxifen and Levamisole as Immune Mimic and Immune Modulator to Treat Breast and Other Mucosal Cancers including Colon. Levamisole is known to be immunoregulatory at multiple levels (90). It is known to enhance an impaired immune system (91). Levamisole is currently used to treat Stage III colon cancer, and is recognized to be an immunostimulant, in the conventional sense, to assist the natural immune system (92). Drawing from the inventor's prior observations that increased secretory immunoglobulins IgA and IgM are not only cytostatic for breast cancer cells, but also cytotoxic, therapies that enhance immune function, increasing the presence of these secretory immunoglobulins in particular, are thus expected to be beneficial. It has been shown by others that a general elevation of the immune system by levamisole can retard colon cancer, but is not completely effective. The addition of tamoxifen is expected to enhance cancer cell death via apoptosis mechanisms. The diagnostic test for SC outlined above can be used to decide which patients should receive a combined levamisole/tamoxifen therapy.

0046Together, the combination of levamisole and tamoxifen for breast cancer is expected to have effects beyond that achievable with each compound alone. Levamisole will enhance the natural immune inhibition of breast cancer growth while tamoxifen offers an additional direct cellular effect. This combination approaches therapy from two different aspects of regulation. The use of levamisole to treat breast cancer is a new application, particularly when placed in combination with tamoxifen. Other components of preferred therapeutic compositions include aromatase inhibitors and/or "pure" anti-estrogens. Example 8: Combination Tamoxifen and Imiquimod as Immune Mimic and Immune Modulator to Treat Breast and Other Mucosal Cancers.

0047Imiquimod is a conventional immune enhancer that is effective both as a topical preparation and when administered orally (93,94). The known use of this compound in breast cancer therapy is based on the action of interferon which is induced by imiquimod. The drug alone has only limited long term effects. Imiquimod therapy is expected to be highly effective in combination with an anti-estrogen such as tamoxifen or a new MER-25 derivative. The elevation of interferon affects the immune system as well as having potential effects directly on breast cancer cells. The addition of tamoxifen is expected to enhance any effects of interferon. This combination has three possible cytostatic/cytotoxic modes. First is the direct effect of the anti-estrogen. Second is an immune enhancing action of imiquimod, which is expected to include enhancement of the secretory immunoglobulin inhibitors of cancer cell growth. Third is the direct cytotoxic effect of interferon. This modality may be enhanced by measurement of the interferon receptor in breast specimens along with the Fc- like receptor. Example 9: Combination Tamoxifen and OK-432 (Picibanil) as Immune Mimic and Immune Modulator to Treat Breast and Other Mucosal Cancers.

0048Ok-432 (Picibanil) is a streptococcal preparation that has a strong immune modulating effect (95), employing the conventional meaning of "immune modulating," which generally refers to the antibody/antigen recognition function of the immune system. The active moiety of this preparation has not been identified. This preparation cannot be delivered orally. It has been used in breast cancer as intratumor injections (96). In those prior studies, the results were mixed but additional results from cell culture suggest that a combination with an anti- estrogen may have greater effect than OK-432 alone (97). Another route to administration of OK-432 is intrapleural administration, which was evaluated as a treatment for breast malignancy in pleural effusions (98). The results of the conventional immune therapy alone on disseminated breast cancer were encouraging. It is now proposed that the combination of OK-432 with tamoxifen or an aromatase inhibitor will provide additional benefits and have anti-cancer effects beyond those that could have previously been predicted for OK-432 and tamoxifen. The role of OK-432 may be direct on tumor cells, or may involve a critical conventional immune response that then suppresses tumor" cell <img file="WO03041654A2_D0001.tif" /> possible use of this preparation may be as an oral challenge to develop mucosal immunity as described (53,54). This route of administration and development of mucosal immunity represents an entirely new approach to the use of this immune modulator. Example 10: Combination Tamoxifen and DHEA (dehydroepiandrosterone) as Immune Mimic and Immune Modulator to Treat Breast and Other Mucosal Cancers.

0049One recent report (101) asks the question "is DHEA a panacea or snake oil"? The answer likely rests with proper experimental design. DHEA use must be critically evaluated to achieve meaningful results. For example, the use of DHEA as a conventional immune modulator has been evaluated in postmenopausal women (99). That report stated that evidence was clear that DHEA was a positive immune modulator in these females. Furthermore, it was considered useful that DHEA metabolism in breast yields androgens that likely act as inhibitors of breast cancer growth. However, the evidence with a combination of DHEA and the "pure" anti-estrogen EM-800 with ZR-75-1 breast cancer cell xenografts in athymic nude mice were not as encouraging (100). DHEA inhibited alone, and the "pure" anti-estrogen alone inhibited. These results are pointed out to demonstrate that the proposal of using the "mixed" anti-estrogen tamoxifen with DHEA has merit. Tamoxifen acts as a direct immune mimic, as described in Example 1, in addition to blocking the estrogen receptor. DHEA acts to stimulate the immune system and to deliver inhibitory androgens to breast cancer cells. It is believed that the conventional immune stimulatory action of DHEA will also serve to enhance the presence of the inhibitory secretory immunoglobulins. This multilevel approach is expected to be more effective than each of the compounds used alone. It is also expected to be more effective than use of a "pure" antiestrogen with only one mechanism of action. In addition, this combination may be even more effective when an aromatase inhibitor is added. Indeed, but applying the immunohistochemical classifications outlined above, along with determining the androgen receptor content, the combination therapy has a strong rational basis. Today androgen receptors are rarely measured in specimens of female breast cancer.

0050Example 11: Combination Tamoxifen and Fc-like Receptor Gene therapy to Treat Breast and Other Mucosal Cancers.

0051Because tamoxifen is effective only with cells that express the Poly-Ig (Fc) receptor or a Poly-Ig-like (Fc) receptor, introduction of this receptor into cells lacking immune control offers an entirely new approach to treatment of breast and other mucosal cancers. Viral vectors bearing the DNA coding for the full length functional Fc-like receptor can be used to transform disseminated cancer such that the tumor cells regain^" se£siϊi^i'ty^,to^,lϊamόxιfen. ^lTnis'¹' is a significant concept because it permits activation of killing over a long duration and with multiple exposures to the virus plus tamoxifen. Since tamoxifen can typically be used over a five-year period, and viral infections repeated, this new approach has considerable promise and is supported by the recognized fact that all cancer cells will not be killed after even the first few viral infections. The properties of the receptor to be used have been described

0052(53,54), and techniques for incorporating a desired DNA sequence into a suitable viral vector, and for transforming a population of cells are known and have been described in the literature. Example 12: Use of Serum-Containing and Serum-free Medium Assays to Define New Anti-estrogenic Compounds or to Modify Existing Compounds to More Effective Agents.

0053The above-identified co-owned U.S and PCT patent applications (53,54), hereby incorporated herein by reference, describe two different types of assays that will be used to characterize new anti-estrogenic compounds and derivatives. One assay is done with ER⁺ cell lines grown in medium supplemented with steroid hormone depleted serum. The serum is preferably prepared by either charcoal dextran extraction, or by XAD-4 resin treatment (53,54). Similar assays can be done under completely serum-free defined conditions. The results of the two assays can be compared directly. Several suitable cell lines for use in the assays are available from three different species and four different tissues (32-34), although another cell line that is capable of growing both in cell culture and when implanted into a compatible host could also be used. Use of steroid hormone depleted serum permits evaluation of its effect on the activity of the new compound. The presence of serum factors may alter activity and therefore indicate problems before initiation of time consuming and expensive animal testing.

REFERENCES

0055(1) Beatson GT (1896) On treatment of inoperable cases of carcinoma of the mamma: suggestions for a new method of treatment with illustrative cases. Lancet (Part 1) July 11:104 -107; (Part 2) July 18:162-165. (2) Lett H (1905) An analysis of 99 cases of inoperable carcinoma of the breast treated by δophorectomy. Lancet January 28:227-228.

0056(3) Doisy EA, Veler CD &amp; Thayer S (1929) Folliculin from urine of pregnant women.

0057Am J Physiol 90:329-330.

0058(4) MacCorquodale DW, Thayer SA &amp; Doisy EA (1936) The isolation of the principal estrogenic substance of liquor folliculi. J Biol Chem 115:435-448.

0059(5) Clark JH &amp; Markaverich BM (1983) The agonistic and antagonistic effects of short acting estrogens: a review. Pharm Ther 21 :429-453. (6) Lippman ME, Monaco ME &amp; Bolan G (1977) Effects ό¥ strOnt,'^l'estfaϊi "antf estriol on hormone responsive human breast cancer in long term tissue culture. Cancer Res 37:1901-1907.

0060(7) Jozan S, Moure C, Gillois M &amp; Bayard F (1979) Effects of estrone on cell proliferation of human breast cancer (MCF-7) in long term tissue culture. J

0061Steroid Biochem 10:341-342.

0062(8) Katzenellenbogen BS (1984) Biology and receptor interactions of estriol and estradiol derivatives in vitro and in vivo. J Steroid Biochem 20:1033-1037.

0063(9) Karey KP &amp; Sirbasku DA (1988) Differential responsiveness of the human breast cancer cell lines MCF-7 and T47-D to growth factors and 17/3-estradiol. Cancer

0064Res 48:4083-4092.

0065(10) Freinkel N &amp; Metzger BE (1992) Metabolic changes in pregnancy. In: Williams

0066Textbook of Endocrinology, 8^th Edition, Wilson JD &amp; Foster DW (eds), WB

0067Saunders Company, Philadelphia, pp 993-1005. (11) Gore-Langton &amp; Armstrong DT (1988) Folicular steroidogenesis and its control.

0068In: The Physiology of Reproduction, Knobile E &amp; Neill JD (Eds-in-chief),

0069Raven Press, New York, pp 331-385. (12) Doisy EA, Veler CD &amp; Thayer SA (1930) The preparation of the crystalline ovarian hormone from the urine of pregnant women. Am J Physiol 86:499-509. (13) Corner GW (1983) The sites of formation of estrogenic substances in the animal body. Physiol Rev 18:154-172.

0070(14) Jensen EV &amp; Jacobsen HI (1962) Basic guides to the mechanism of estrogen action. Recent Prog Horm Res 18:387-414.

0071(15) Jensen EV &amp; DeSombre ER (1972) Mechanism of action of the female sex hormones. Ann Rev Biochem 41:203-230.

0072(16) Jensen EV &amp; DeSombre ER (1973) Estrogen-receptor interaction. Estrogenic hormones effect transformation of specific receptor proteins to a biochemically functional form. Science (Wash DC) 182:126-134.

0073(17) O'Malley BW &amp; Means AR (1974) Female steroid hormones and target cell nuclei. Science (Wash DC) 183:610-620.

0074(18) Gemignani ML &amp; Petrek JA (2000) Breast cancer during pregnancy: diagnostic and therapeutic dilemmas. Adv Surg 34:273-286.

0075(19) Lambe M, Hsieh C-C, Trichopoulos D, Ekbom A, Pavia M &amp; Adami H-O (1994)

0076Transient increase in the risk of breast cancer after giving birth. N Eng J Med 331:5-9.

0077(20) Miller WR ( 1993) Hormonal factors and risk of breast cancer. Lancet 341:25-26.

0078(21) MacMahon B (1993) General Motors Cancer Research Prizewinners Laureates

0079Lectures. Charles S. Mott Prize. Reproduction and cancer of the breast. Cancer

008071:85-88. (22) Ibrahim EM, Ezzat AA, Baloush A, Hussain ZH &amp; Mohammed GH (2000)

0081Pregnancy-associated breast cancer: a case-control study in a young population with high fertility rate. Med Oncol 17:293-300. (23) Gwyn K &amp; Theriault R (2001) Breast cancer during pregnancy. Oncology

0082(Huntingt) 15:39-46; discussion 46. (24) Moore HC &amp; Foster RS Jr (2000) Semin Oncol 27:646-653.

0083(25) Gemignani ML, Petrek JA &amp; Borgen PI (1999) Breast cancer and pregnancy. Surg

0084Clin North Am 79:1157-1169.

0085(26) Key TJ (1999) Serum oestradiol and breast cancer risk. Endocrine-Related Cancer

00866:175-180. (27) Persson I (2000) Estrogens in the causation of breast, endometrial and ovarian^" cancers — evidence and hypotheses from epidemiological findings. J Steroid Biochem Mol Biol 74:357-364.

0087(28) Hulka BS &amp; Moorman PG (2001) Breast cancer: hormones and other risk factors. Maturitas 38:103-113; discussion 113-116.

0088(29) Ross GT &amp; Vande Wiele RL (1974) Chapter 7: The Ovaries. In: Textbook of

0089Endocrinology, 5^th Edition, Williams RH (ed), WB Saunders, Philadelphia, pp 368-422.

0090(30) Williams CL &amp; Stancel GM (1995) Chapter 57: Estrogens and Progestins. In: Goodman &amp; Gilman's The Pharmacological Basis of Therapeutics, 9^th Edition,

0091Hardman JG &amp; Limbird LE (Eds-in-Chief), McGraw-Hill, New York, pp 1411- 1440.

0092(31) Vignon F, Terqui M, Westley B, Derocq D &amp; Rochefort H (1980) Effects of plasma estrogen sulfates in mammary cancer cells. Endocrinology 106:1079- 1086.

0093(32) Moreno-Cuevas JE &amp; Sirbasku DA (2000) Estrogen mitogenic action. I.

0094Demonstration of estrogen-dependent MTW9/P12 carcinogen-induced rat mammary tumor cell growth in serum-supplemented culture and technical implications. In Vitro Cell Dev Biol 36:410-427. (33) Sirbasku DA &amp; Moreno-Cuevas JE (2000) Estrogen mitogenic action. II. Negative regulation of the steroid hormone-responsive growth of cell lines derived from human and rodent target tissue tumors and conceptual implications, hi Vitro Cell Dev Biol 36:428-446.

0095(34) Moreno-Cuevas JE &amp; Sirbasku DA (2000) Estrogen mitogenic action. III. Is phenol red a "red herring"? hi Vitro Cell Dev Biol 36:447-464.

0096(35) Petrek JA (1994) Breast cancer and pregnancy. J Natl Cancer List Monograph

009716:113-121.

0098(36) Gorbach SL (1984) Estrogens, breast cancer, and intestinal flora. Rev Infect Dis

0099Suppl S85-S90. (37) Axelson M &amp; Sjovall J (1983) Formation of catechol estrogens by intestinal bacterial demethylation of 2-methoxyestrone. Biochim Biophys Acta 751:162-

0100165. (38) Simpson ER (2000) Role of aromatase in sex steroid action. J Mol Endocrinol

010125:149-156. (39) Longcope C (2001) Endocrine function of the postmenopausal ovary. J Soc

0102Gynecol Investig 8 (supρl):S67-S68.

0103(40) Van Zonneveld P, Scheffer GJ, Broekmans FJM &amp; te Velde ER (2001) Hormones and reproductive aging. Maturitas 38:83-94.

0104(41) Kuerer HM, Buzdar AU &amp; Singletary SE (2001) Biologic basis and evolving role of armoatase inhibitors in the management of invasive carcinoma of the breast. J

0105Surg Oncol 77:139-147.

0106(42) Ries LAG, Kosary CL, Hankey BF et al (1999) "SEER Cancer Statistics Review,

01071973-1996". Bethesda, Maryland: National Cancer Institute.

0108(43) Diab SG, Elledge RM &amp; Clark GM (2000) Tumor characteristics and clinical outcome of elderly women with breast cancer. J Natl Cancer Inst 92:550-556.

0109(44) Thomas HV, Reeves GK &amp; Key TJ (1997) Endogenous estrogen and postmenopausal breast cancer: a quantitative review. Cancer Causes Control 8:922-928.

0110(45) Conley A &amp; Hinshelwood M (2001) Mammalian aromatases. 121:685-695. (46 Siiteri PK &amp; MacDonald PC (1973) Role of extraglandular oestrogen in human endocrinology. In: Handbook of Physiology, Volume 2, Greep Ro &amp; Astwood

0111EB (eds) American Physiology Society, Washington DC, pp 619-629.

0112(47 Simpson ER, Zhao Y, Agarwal VR, Michael MD et al (1997) Aromatase expression in health and disease. Recent Prog Horm Res 52:185-213.

0113(48; Huang Z, Hankinson SE, Colditz GA, Stampfer MJ et al (1997) Dual effects of weight and weight gain on breast cancer risk. JAMA 278 : 1407-1411.

0114(49; Labrie F, Belanger A, Cusan L &amp; Candas B (1997) J Clin Endocrinol Metab

011582:2403-2409.

0116(50) Green S, Walter P, Kumar V Krust A et al (1986) Human oestrogen receptor cDNA: sequence, expression and homology to verb-A. Nature 320:134-139.

0117(51 Greene GL, Gilna P, Waterfield M, Baker A, Hort Y &amp; Shine J (1986) Sequence and expression of the estrogen receptor complimentary DNA. Science (Wash

0118DC) 231:1150-1154.

0119(52 Evans RM (1988) The steroid and thyroid hormone receptor superfamily. Science

0120(Wash DC) 240:889-895. (53 Sirbasku, David A. "Compositions and Methods for the Diagnosis, Treatment and

0121Prevention of Steroid Hormone Responsive Cancers" U.S. Patent Application

0122No. 09/852,547 (U.S. Published Application No. 20020006630) and corresponding PCT Published Application No. WO 01/86307.

0123(54; Sirbasku, David A. "Compositions and Methods for Demonstrating Secretory

0124Immune System Regulation of Steroid Hormone Responsive Cancer Cell

0125Growth" U.S. Patent No. 09/852,958 (U.S. Published Application No.

012620020012954 and corresponding PCT Published Application No. WO 01/85210.

0127(55 Fuqua SAW , Fitzgerald SD, Chamness GC, Tandon AK et al (1991) A variant human breast tumor estrogen receptor with constitutive transcriptional activity.

0128Cancer Res 51:105-109. (56 Zhang Q-X, Borg A, Wolf DM, Oesterreich S &amp; Fuqua SAW (1997) An estrogen receptor mutant with strong hormone-independent activity from metastatic breast cancer. Cancer Res 57:1244-1249.

0129(57 Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S &amp; Gustafsson J-A (1996)

0130Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad

0131Sci USA 93:5925-5930.

0132(58; Nilsson S, Makela S, Treuter E, Tujaque M et al (2001) Mechanisms of estrogen action. Physiol Rev 81:1535-1565. (59; Zajchowski DA, Sager R &amp; Webster L (1993) Estrogen inhibits the growth of estrogen receptor-negative, but not estrogen receptor-positive, human mammary epithelial cells expressing a recombinant estrogen receptor. Cancer Res

013353:5004-5011.

0134(6o; Gustafsson J-A &amp; Warner M (2000) Estrogen receptor β in the breast: role in estrogen responsiveness and development of breast cancer. J Steroid Biochem

0135Mol Biol 74:245-248.

0136(61 Dickson RB &amp; Stancel GM (1999) Chapter 8: Estrogen receptor-mediated processes in normal and cancer cells. J Natl Cancer Inst Monographs No.

013727:135-145.

0138(62 Couse JF &amp; Korach KS (1999) Estrogen receptor null mice: what have we learned and where will they lead us? Endocr Rev 20:358-417. (63 Couse JF, Curtis-Hewitt S &amp; Korach KS (2000) Receptor null mice reveal contrasting roles for estrogen receptor α and β in reproductive tissues. J Steroid

0139Biochem Mol Biol 74:287-296. (64) Giguere V, Yang N, Seui V &amp; Evans RM (198^)^'Η htifιfcέtiύn'W"a^l''neW^!"5ϊ^'a&amp;s-^:b ^lt steroid hormone receptors. Nature 331 :91-94.

0140(65) Beato M &amp; Klug J (2000) Steroid hormone receptors: an update. Human

0141Reproduction Update 6:225-236. (66) Hopp T &amp; Fuqua S (1998) Estrogen receptor variants. J Mammary Gland Biol

0142Neoplasia 3:73-83. (67) Fuqua SA, Wiltschke C, Zhang QX, Borg A et al (2000) A hypersensitive estrogen receptor alpha mutation in premalignant breast lesions. Cancer Res 60:4026- 4029. (68) Goldstein SR, Siddhanti S, Ciaccia AV &amp; Plouffe L Jr (2000) A pharmacological review of selective oestrogen receptor modulators. Human Reprod Update 6:212-224. (69) Buzdar AU &amp; Hortobagyi GN (1998) Tamoxifen and toremifene in breast cancer: comparison of safety and efficacy. J Clin Oncol 16:348-353. (70) Buzdar AU &amp; Hortobagyi G (1998) Update on endocrine therapy for breast cancer.

0143Clin Cancer Res 4:527-534.

0144(71) Hermenegildo C &amp; Cano A (2000) Pure anti-estrogens. Human Reproduction

0145Update 6:237-243.

0146(72) Mandlekar S &amp; Kong AN (2001) Mechanism of tamoxifen-induced apoptosis. Apoptosis 6:469-477.

0147(73) Vignon F, Bouton M-M &amp; Rochefort H (1987) Antiestrogens inhibit the mitogenic effect of growth factors on breast cancer cells in the total absence of estrogens. Biochem Biophys Res Commun 146:1502-1508.

0148(74) Wakeling AE &amp; Bowler J (1987) Steroidal pure antiestrogens. J Endocrinol 112:R7-R10.

0149(75) Jordan VC (1993) A current view of tamoxifen for the treatment and prevention of breast cancer. Br J Pharmacol 110:507-517.

0150(76) Lerner LJ, Holthaus FG &amp; Thompson CR (1958) The non-steroidal estrogen antagonist (MER-25). Endocrinology 63:295-318. (77) Greenblatt RB, Roy S, Mahesh VB et al (1962) Induction of ovulation. Am J

0151Obstetrics Gynecology 84:900-909.

0152(78) Lerner LJ &amp; Jordan VC (1990) Development of antiestrogens and their use in breast cancer. Cancer Res 50:4177-4189.

0153(79) Lyman SD &amp; Jordan VC (1985) Possible mechanisms for the agonist actions of tamoxifen and the antagonist actions of MER-25 (ethamoxytriphetol) in mouse uterus. Biochem Pharmacol 34:2795-2806.

0154(80) Hossain MB, Symersky J, Neely SC, van der Helm D &amp; Magarian RA (1993)

0155Structure of 1 -(4-[2-(diethylamino)ethoxy]phenyl)-2-(4-methoxyphenyl)-l - phenylethan-1-ol, the non-steroidal antiestrogen MER-25. Acta Crystallogr 49:500-504.

0156(81) Lonning Pe, Johannessen DC, Lien EA et at (1995) Influence of tamoxifen on sex hormones, gonadotrophins and sex hormone binding globulin in postmenopausal breast cancer patients. J Steroid Biochem Mol Biol 52:491-496.

0157(82) Geisler J, Haarstad H, Gunderson S et al (1995) Influence of treatment with the antiestrogen 3-hydroxytamoxifen (droloxifene) on plasma sex hormone levels in postmenopausal patients with breast cancer. J Endocrinol 146:359-363.

0158(83) Lum SS, Woltering EA, Fletcher WS et al (1997) Changes in serum estrogen levels in women during tamoxifen therapy. Am J Surg 173:399-402. (84) Clark ER &amp; Jordan VC (1976) Oestrogenic, anti-oesfro^'genϊc and fertility effects of some triphenylethanes and triphenylethylenes related to ethamoxytriphetol (MER-25). Br J Pharmacol 57:487-493.

0159(85) Roy EJ &amp; Uade GN (1976) Estrogenic effects of an antiestrogen, MER-25, on eating and body weight in rats. J Comp Physiol Psychol 90:156-166.

0160(86) Krajci P, Kvale D, Tasken K &amp; Brandtzaeg P (1992) Molecular cloning and exon- intron mapping of the gene encoding human transmembrane secretory component (the Poly-Ig receptor). Eur J Immunol 22:2309-2315.

0161(87) Stern JE, Underdown BJ, Crichlow RW &amp; Wira CR (1985) Secretory component in breast cancer. Analysis of the levels in primary and metastatic disease.

0162Cancer Immunol Immunother 19:226-230.

0163(88) Brandtzaeg P &amp; Rognum TO (1984) Evaluation of nine different fixatives. 1.

0164Preservation of immunoglobulin isotypes, J chain, and secretory component in human tissues. Path Res Pract 179:250-266. (89) Krajci P, Meling GI, Andersen SN et al (1996) Secretory component mRNA and protein expression in colorectal adenomas and carcinomas. Br J Cancer 73:1503-1510.

0165(90) Goldstein G (1978) Mode of action of levamisole. J Rheumatol Suppl 4:143-148.

0166(91) Prakash MS, Rao VM &amp; Reddy V (1998) Effect of levamisole on the immune status of malnourished children. J Trop Pediatrics 44:165-166.

0167(92) Holcombe RF, Li A &amp; Stewart RM (1998) Levamisole and interleukin-2 for advanced malignancy. Biotherapy 11.255-258.

0168(93) Savage P, Horton V, Moore J et al (1996) A phase I trial clinical trial of imiquimod, an oral interferon inducer, administered daily. Br J Cancer 74:1482- 1486.

0169(94) Witt PL, Ritch PS, Reding D et al (1993) Phase I trial of an oral immunomodularor and interferon inducer in cancer patients. Cancer Res 53:5176-5180.

0170(95) Chirigos MA (1992) Immunomodulators: current and future development and application. Thymus 19 (suppl) :S7-S20. (96) Takeda T, Kobayashi T, Monden T et al (1993) The effect of local immunotherapy for breast cancer using a mixture of OK-432 and fibrinogen supplemented with activated macrophages. Biotherapy 7:47-53.

0171(97) Aoyagi H, lino Y, Takeo T et al (1997) Effects of OK-432 (picibanil) on estrogen receptors of MCF-7 cells and potentiation of antiproliferative effects of tamoxifen in combination with OK-432. Oncology 54:414-423.

0172(98) Kan N, Kodama H, Hori T et al (1993) Intrapleural adaptive immunotherapy for breast cancer patients with cytologically-confirmed malignant pleural effusions: an analysis of 67 patients in Kyoto and Shiga Prefecture, Japan. Breast Cancer Res Treat 27:203-210. (99) Casson PR, Andersen RN, Herrod HG et al (1993) Oral dehydroepiandrosterone in physiologic doses modulates immune function in postmenopausal women. Am J Obstet Gynecol 169:1536-1539.

0173(100) Couillard S, Labrie C, Belanger A et al (1998) Effect of dehydroepiandrosterone and the antiestrogen EM-800 on growth of human ZR-75-1 breast cancer xenografts. J Natl Cancer Inst 90:772-778.

0174(101) Sirrs SM &amp; Bebb RA (1999) DHEA: panacea or snake oil? Can Fam Physician

017545:1723-1728.

0176While the preferred embodiments of the invention have been shown and described, modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the invention. The embodiments described herein are exemplary only, and are not intended to be limiting. Many variations and modifications of the invention disclosed herein are possible and are within the scope of the invention. For example, the foregoing descriptions primarily focus on the treatment and prevention of breast cancer in high-risk individuals, however the same or similar approaches can be employed to with respect to other types of cancers of mucosal tissues, including prostate, ovary, endometrium, cervix, vagina, colon, kidney, lung, pancreas and nasopharynx. Cancers of those tissues, together with breast cancer, account for 80% of all human cancer. The disclosures of all patents, patent applications and publications cited hereinabove are hereby incorporated herein by reference. The discussion of certain references in the Description of Related Art, above, is not an admission that they are prior art to the present invention, especially any references that may have a publication date after the priority date of this application.