US 2003OO72760A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0072760 A1 Sirbasku (43) Pub. Date: Apr. 17, 2003

(54) ANT- AND IMMUNE Publication Classification MODULATOR COMBINATIONS FOR TREATING 51) Int.nt. Cl.C.7 ...... A61K 39/395; A61K 31/56 A61K 31/202; A61K 31/137 (76) (52) U.S. Cl...... 424/155.1; 514/170; 514/560; Inventor: David A. Sirbasku, Austin, TX (US) 514/649 Correspondence Address: CONLEY ROSE, PC. (57) ABSTRACT P. O. BOX 3267 Compositions for treating cancers of mucosal tissues includ HOUSTON, TX 77.253-3267 (US) ing breast, prostate, ovary, colon are disclosed which include various combinations of new or conventional anti-estrogen (21) Appl. No.: 10/293,439 compounds, aromatase inhibitors, immune modulators, Filed: Nov. 13, 2002 immune inhibitors, immune inhibitor mimicking com (22) pounds and or thyroid hormones. Methods of pre Related U.S. Application Data dicting Susceptibility of a cancer of mucosal origin to treatment with a composition containing an immune inhibi (63) Continuation-in-part of application No. 09/852,958, tor or an immune inhibitor mimicking compound are also filed on May 10, 2001. Continuation-in-part of appli disclosed. Preferred methods include identifying in a speci cation No. 09/852,547, filed on May 10, 2001. men of cancer cells the presence of a Poly-Ig (Fc) receptor or Poly-Ig-like (Fc) receptor capable of binding to an immune inhibitor or an immune inhibitor mimicking com (60) Provisional application No. 60/332,801, filed on Nov. pound and of mediating immune inhibition of cancer cell 14, 2001. growth. US 2003/0072760 A1 Apr. 17, 2003

ANT-ESTROGEN AND IMMUNE MODULATOR (19). Clearly, however, pregnancy has opposing effects on COMBINATIONS FOR TREATING BREAST breast cancer development. On the one hand the increase in CANCER hormones can promote cancer cell growth (35). On the other hand, pregnancy and high hormones induce tissue differen CROSS-REFERENCE TO RELATED tiation that ultimately protects the tissue (20.21). Apparently APPLICATIONS the elevated estrogen levels in pregnancy explain the tran 0001) This application claims the benefit under 35 U.S.C. Sient increase in short-term risk of breast cancer following S 119(e) of U.S. Provisional Patent Application No. 60/332, term pregnancy (19). The results of several Studies indicate 801 filed Nov. 14, 2001, and is a continuation-in-part of U.S. that all three of the estrogenic Steroid hormones (i.e. E., E. patent application Nos. 09/852,958 and 09/852,547, both and E) are important in breast cancer risk in humans filed May 10, 2001, the disclosure of each of which is hereby (26-28). incorporated herein by reference. 0008. The biosynthesis and metabolism of and estrogen-related Steroid hormones has been reviewed (11). STATEMENT REGARDING FEDERALLY The majority of plasma E and E is Synthesized and SPONSORED RESEARCH OR DEVELOPMENT secreted by cells of the ovarian follicle (29.30). The bio chemical Synthetic pathway begins with conversion of cho 0002 Research leading to the present invention was lesterol to progesterone, followed by modification of the Supported in part by the federal government under Grant progestin to form androgens or androgen-like . To Nos. DAMD 17-94-J-4473, DAMD 17-98-1-8337 and form all three types of estrogen, the origin “A” DAMD17-99-19405 awarded by the Defense Department ring of "androgens' must be converted to a phenolic Struc through the US Army Medical Research and Materiel Com ture by the action of aromatases. These key enzymes in the mand, Breast Cancer Research Program. The United States biosynthesis of estrogens are located in the endoplasmic government may have certain rights in the invention. reticulum of ovarian cells. BACKGROUND OF THE INVENTION 0009 Estrogens undergo a variety of metabolic transfor mations including hyroxylations, methylations and reduc 0003) 1. Field of Invention tion. Also, the estrogens are converted to more water 0004. The present invention generally relates to methods Soluble, biologically inactive, glucuronide and Sulfate and compositions for the use of and other anti conjugates by the liver. The conjugates are excreted into estrogenic compounds in combination with immune modu urine and bile. Earlier Studies indicated that estrogen con lator agents (immunoglobulin inhibitors of estrogen respon jugates (e.g. Sulfate) might serve as Sources of free Sive cancer cell growth), to treat or prevent breast cancer. estrogen in breast cancer cells possessing the appropriate cleaving enzyme(s) to form free steroid (31). More recent 0005 2. Description of Related Art work (32-34) indicates this is unlikely, based on tissue 0006. In 1896, a British physician named Beatson culture studies with eight different ER" cell lines. Estrogen reported that Öophorectomy had palliative effects for breast Sulfates and glucuronides are cleaved by intestinal flora to cancer patients (1). In 1905, Lett confirmed this observation regenerate free estrogens that again appear in the plasma and with a larger patient trial (2). Clearly Ovarian products were urine via the enterohepatic circulation (36). A high fiber-low either directly or indirectly significant in breast cancer fat diet tends to decrease this process. Other intestinal growth. From these earliest clinical observations, chemical microbial processes also convert inactive estrogen metabo and endocrine research continued and culminated in the lites to active steroid hormones (37). Thus, recycling of identification of the primary ovarian/follicular agents estrogens is entirely possible. responsible. The active agents proved to be a class of 0010. However, the sites of synthesis of estrogenic Sub cholesterol derived Steroid hormones now designated estro stances in the body are not limited to the ovary (13). While gens. In 1929 and 1930, Doisy and colleagues crystallized it is understood with premenopausal women that estrogens estrogens including estrone 3-hydroxy-estra-1,3,5(10)- are primarily of Ovarian origin, this is not the case in trien-17-one (E) from human pregnancy urine (3,12). postmenopausal females (38-41). The question is “what is -17Bestra-1,3,5(10)-triene-3, 17B-diol (E) was the origin(s) of estrogens in the postmenopausal female'? also isolated from Sow follicular fluid (4). The remaining This is important because breast cancer rates are much major estrogen, 1,3,5-estratriene-3, 16 C, 17 ?-triol higher in postmenopausal women (42) even though estrogen (E) has also been defined. levels are declining Nonetheless, 80 or 90% of breast 0007. The relative potency of these three hormones is cancers in postmenopausal women are ER" (43), implying known today to be E>E>>>>E (5). With regard to breast they are estrogen growth promoted. This paradox can be cancer cell growth, E and E are in the main considered the explained in part by the Suggestion that postmenopausal most physiologically relevant (6-9). Estriol is most likely Women with higher risk of developing breast cancer Show relevant during pregnancy when the maternal plasma level is relatively higher concentrations of endogenous estradiol Significantly elevated (10). During pregnancy, maternal E is (44). Also, it is now very clear that adrenal androgenic formed primarily as a placental conversion product of a Steroids can be converted to estrogens via the action of Steroid produced by the fetal adrenals. Breast cancers are not aromatases located in mammalian tissues (45). Its activity uncommon during pregnancy (18,22-25). However, all three provides a Significant portion of the plasma estrogens even estrogens are increased in pregnancy (10). In pregnant in postmenopausal women (38-41). Aromatase activity has women, breast cancer is often diagnosed at a later stage (18). a broad tissue distribution in mammals (45). However, in It may be that the elevated hormones during this time cause human women after menopause, adipose tissue is the pri growth of developing breast cancer cells in pregnant females mary Source of endogenous estrogens (46,47). Indeed, obe US 2003/0072760 A1 Apr. 17, 2003 sity is positively correlated with breast cancer (48). Also, eight well-known estrogen responsive tumor cell lines aromatase is present in breast tissue and cells and represents derived from four tissues and three species including human an “intracrine” source of stimulating steroid hormone (49). (32-34,53,54). Because of the major role of aromatase in generating breast 0014. However, there exist potential alternatives regard cancer promoting estrogens in postmenopausal women, a ing the identity of ERY. Investigators have cloned two Series of aromatase inhibitorS has been developed and are ERC-like “orphan receptors” with unknown functions (64. now in use as pharmaceutical products or are in and clinical 65). Other forms of estrogen receptors appear to arise as trials as breast cancer treatments (41). gene product splice variants (58.66). Those with major 0.011 The question of how estrogens regulate target tis deletions of the hormone binding domain or the DNA Sue gene expression and growth is of great consequence to binding domain may be expected to be inactive with respect this discussion. In 1962, Jensen & Jacobsen (14) came to the to estrogen induced growth of breast cancer cells. The conclusion that estrogens acted on Sex Steroid hormone function of most of the other types of known variants target tissues via Specific cellular receptors. By 1972 to remains to be established. 1974, this research was sufficiently advanced to outline the 0015. Another potentially significant variant has been mechanisms of estrogen action as mediated by an intracel identified. It is a point mutation that affects the border of the lular receptor (15-17). For several years, intense study has hinge-hormone-binding domains (67). This mutation was proceeded and has been reported in nearly 20 thousand found in 34% of a series of 59 specimens of premalignant publications (PubMed literature search of “estrogen recep hyperplasia. Transfection of this mutated ERC. caused tors”). In 1986, the molecular cloning of the original estro MCF-7 human breast cancer cells to respond to lower gen receptor, now designated ERC, was reported (50.51). concentrations of estrogen in culture. The full implications This 64-kDalton protein is functionally and structurally of this mutation await more Study, but it is clear from the related to other receptors and has been classified as a results available at this time, and those presented in the member of the steroid and thyroid hormone Superfamily above-identified patent applications (53,54) and other recent (52). Today, these similar receptors include those for andro publications (32-34), that MCF-7 as well as T47D and gens, corticosteroids, progestins, thyroid hormones, Vitamin ZR-75-1 ER" breast cancer cells respond to very low con D and retinoic acid. centrations of E. even without transfection of the mutated ERC. It may be possible that the hypersensitive mutated 0012 Although for several years ERC. was acknowledged receptor (67) is present in all ER" cell types including those as the only , variants of it were being from rat mammary and rat pituitary tumors as well as from identified (55,56). However, in 1995, another type of estro estrogen-induced kidney tumor cells from Syrian hamster gen receptor, designated ERB, was cloned from a rat prostate (32-34). This means that a specific mechanism must exist for and ovary (57). This initiated a boom of new activity to formation of this receptor in target tissue cells, or that this define the function and properties of ERB (58,60,61). receptor is derived from a new gene. The latter possibility Indeed, the results Suggest that the role of estrogens in male accessory organ function deserves renewed study (58). The implies that the response of ER" cells to very low concen characteristics and properties of ERC. versus ERB have been trations of E involves the proposed new ERY (53.54). reviewed (58,61,63). For the purposes of this disclosure, it 0016. The currently available knowledge about estrogen should be noted that the binding affinities of both receptors function and estrogen receptorS has led to one of the most are approximately equal (61). This was expected. However, common treatments for disseminated and/or local ER" one startling fact has Surfaced. Mice gene knockout experi breast cancer, especially in postmenopausal women. Today, ments for both ERC (62) and ERB (60) have confirmed selective estrogen receptor modulators (SERMs) are the developmental functions for both of these receptors, but compounds of choice (68). The mechanism of action of have fallen Short of providing conclusive evidence that these drugs is to block the growth promoting action of either receptor regulates growth (58). In fact, transfection of estrogens at the cellular/receptor level, no matter whether ERT cells with a functional ERC. led to an estrogen-induced the Sex Steroid hormones are delivered Systemically or inhibition of cell growth (59). There is a possibility that ERC. formed locally in breast tissue via aromatase action on is a receptor regulating expression of differentiated func adrenal Steroid precursors. Hence, these drugs are classified tions. It is well recognized that growth and differentiation as anti-estrogens. As a general mechanism of action, anti are opposing cell functional States. Differentiated cells estrogens are thought to interfere with the binding of natural divide only slowly if at all. This issue has been reviewed in estrogens to the growth promoting estrogen receptor(s). detail in recent U.S. patent application Nos. 09/852,547 and 09/852,958 and in International Patent Application Nos. 0017. The first potent anti-estrogen developed 1958 was PCT/US01/15171 (WO 01/86307) and PCT/US01/15183 MER-25 or ethamoxytriphetol (76). It then was used to (WO 01/85210), also identified in the list of References, derive clomiphene (77) which is now used to treat amenor below, as items 53 and 54, and hereby incorporated herein rhea. Clomiphene was then modified to give rise to tamox by reference). This led to the proposal in those applications ifen (78). Although several anti-estrogens have been devel that there is another growth regulating estrogen receptor, oped, only two are currently FDA approved for treatment of tentatively designated ERY (53.54). human breast cancer. These are tamoxifen and . These, and and , are 0013 The characteristics of ERY are that it binds estro derivatives. Notably, the toremifene structure differs from gens with 10 to 100-fold higher affinities than ERC. or ERB. tamoxifen by only a single chlorine atom (69). Since its Furthermore, it is proposed that this receptor is a new gene approval in 1977, tamoxifen has been the SERM of choice that is expressed in all estrogen growth responsive target for treatment of ER" breast cancer worldwide (70). Tamox tissues. Data obtained indicate that this receptor is present in ifen is classified as a “mixed' anti-estrogen because it US 2003/0072760 A1 Apr. 17, 2003

displays both antagonistic properties (i.e. inhibits breast SUMMARY OF PREFERRED EMBODIMENTS cancer cell growth) and agnostic properties (i.e. Stimulates 0022 New compositions and methods are provided endometrial cell growth and tumor development) (71). which advantageously employ compounds having a newly defined immune modulating function, or which have the 0.018. The action of the anti-estrogens is reversed by ability to mimic that immune modulating function, or a lower concentrations of the natural estrogens (53,54). The combination of Such compounds. For the purposes of the affinity of tamoxifen for the estrogen receptor is 10 to present disclosure, the terms “immune mimic,”“immune 100-fold less than that of E. This is commonly recognized modulating,”“immune modulator,”“immune modulation, throughout the endocrine cancer field. It is therefore useful “immune control,”“immune inhibition,”“immune suppres to Suppress natural estrogens along with application of Sor,” and the like, refer in most instances to the newly tamoxifen treatment. This fact is often not recognized clini identified cancer cell growth (i.e., proliferation) inhibitory cally. Postmenopausal women are not completely devoid of effect of the Secretory immune System (i.e., dimeric/poly estrogens. Tamoxifen effectiveness is reduced by residual meric IgA and pentameric IgM) that is mediated by a newly estrogenic Steroid hormones. It is also reduced by the identified Poly-Ig receptor or Poly-Ig-like receptor (also tamoxifen induced elevation of DHEA, E and E (81-83). classified as an Fc-like receptor), and not to the usual This is an unfortunate side effect of using this drug alone. antibody/antigen recognition based immune function of the immune System. In this context, the terms “immune modu 0019. One of the commonly cited facts concerning lation” or “immune enhancement” refer especially to the tamoxifen is that it acts at cellular Sites Separate from the modulation or enhancement of these cell growth inhibitory estrogen receptor. It is known to influence Such cellular immunoglobulins of the Secretory immune System. The term activities as protein kinase C as well as Several other cellular “immune mimic' refers to a Substance (e.g., tamoxifen) that mechanisms including those related to apoptosis (72). can function in a similar manner to an immunoglobulin Although non-Steroid hormone receptor directed actions are inhibitor of cell growth. In Some instances, however, refer usually considered undesirable, certain very recent ence is also made herein to “natural immune inhibition, co-owned patent disclosures (53,54) describe targeting a immune enhancer,”“immune modulator,”“immune Sys non-Steroid hormone receptor with new drug combinations tem,”“immune therapy,” and “immune response,” and the whose actions are based on anti-estrogen augmentation/ like, in which the conventional meanings of those terms are mimicking of the inhibition of growth of ER" breast cancer intended and the context So indicates, especially when prior cells by the immunoglobulins IgA and IgM of the natural art methods, compounds and compositions are described. Secretory immune System. As described (53.54), the Secre Hereinafter, an indication has been made in appropriate tory immune System acts as a paracrine negative regulator of instances whether a conventional definition or the “new” ER" breast cancer cell growth. Employing new serum-free meaning, or both, is intended. defined culture assay methods (53.54), tamoxifen was 0023. In some aspects of the present invention, tamoxifen shown to mimic the inhibition caused by IgA or IgM in the is used as a breast cancer treatment taking advantage of its complete absence of estrogens. This new tamoxifen function newly identified function as an immune mimic instead of an represents a clear departure from previous thought concern anti-estrogen. That tamoxifen is a mixed anti-estrogen is ing how this “mixed function' anti-estrogen acts. Previ well known. It not only binds to cellular estrogen receptors, ously, other investigators had reported that tamoxifen inhib but it also has other unrelated sites of cellular action. This ited growth factor dependent proliferation of human breast new function for tamoxifen makes possible new combina cancer cells in cultures devoid of estrogens and estrogen-like tion therapies as well as new diagnostic methods to deter agents (73). However, there was no indication at that time mine whether breast or other mucosal origin cancers are that this anti-estrogen was capable of acting by mimicking expected to be Susceptible to these therapies. It is concluded the growth inhibitory effects of the natural secretory immune that combination therapies of tamoxifen and the “pure” System immunoglobulins IgA, IgM and IgG1. anti-estrogens may be more effective than either class of 0020. Another class of anti-estrogens is defined as “pure” drug alone. because they only affect growth via interaction with estrogen 0024 Tamoxifen treatment alone has several positive receptors (71). The pure anti-estrogens were discovered aspects as well as a number of negatives. The negatives can about 15 years ago (74). Currently, five compounds are be overcome by placing this well known anti-estrogen in under intense investigation (71). They are abbreviated ICI combinations with other compounds. The preferred combi 164384, ICI 182780, EM-800, RU 58688 and EM-139 (71). nations represent those that permit the mixture to act more Two of these, ICI 164384 and ICI 182780 are in clinical effectively than the individual component alone. The com trials. Because tamoxifen resistance develops with time (75), binations may include two or more breast cancer treatment the pure anti-estrogens are thought to be useful as Second drugs, Some of which are classified as "pure' anti-estrogens line therapies after tamoxifen failure (71). Furthermore, pure while others are defined as immune modulators. anti-estrogens are thought useful because they cause no 0025. In accordance with certain embodiments of the increase in (71). present invention, a new tamoxifen-based therapeutic 0021 However, the pure anti-estrogens have marked method is provided, in which tamoxifen acts as an immune deleterious effects on the cardiovascular and Skeletal Sys inhibitor mimic (“immune mimic"). The method preferably tems (71), and their usefulness is yet to be established. There includes employing a new diagnostic test to identify breast remains a need for effective anti-estrogens and for combi cancer cells expressing the inhibitor-mediating receptor (a nation therapies of tamoxifen or tamoxifen-like drugs and Poly-Ig receptor or Poly-Ig like receptor), also classified as the "pure' anti-estrogens that may be more effective than an Fc-like receptor, as an indication of Sensitivity to cell either class of drug alone. growth inhibition by tamoxifen. US 2003/0072760 A1 Apr. 17, 2003

0026. In accordance with another embodiment, the O1/863.07 and WO 01/852.10 establish that tamoxifen mim above-described tamoxifen therapy and diagnostic testing ics the cell growth inhibitory actions of the Secretory method is extended to mucosal cancers other than breast, immune system immunoglobulins IgA and IgM (53.54). The including those of the prostate, colon, kidney, bladder, lung, disclosures of those applications are hereby incorporated pancreas, nasopharynx, Ovary, endometrium, Vagina, and herein by reference. The immunoglobulin action is mediated cervix. by a Poly-Ig receptor or a Poly-Ig-like receptor (also clas sified as an Fc-like receptor) that is identified by antibody 0027. In still other embodiments, combinations of tamox raised against the extracellular five domains commonly ifen and aromatase inhibitors are employed to treat breast called the “secretory component” (SC) (86). Those breast and gynecologic cancers. In Some embodiments, tamoxifen cancer cells expressing this Fc-like receptor are Sensitive to and a "pure' anti-estrogen compound are combined for inhibition by tamoxifen. Those cells not expressing the treating breast and gynecologic cancers. Fc-like receptor are not tamoxifen Sensitive. Because the 0028. Some embodiments of the present invention pro analysis can be done in completely Serum-free defined vide compositions or therapeutic methods using chemically medium (53,54) without estrogens, it is concluded that modified MER-25 to treat secretory immune system related tamoxifen acts to mimic the Fc-like receptor mediated cancers. In some embodiments, MER-25 or modified MER inhibition of cell growth by Secretory immune System IgA 25 is combined with progesterone or another hormone for and IgM. Using immunohistochemical analysis methods treating breast cancer. Modified MER-25 or derivative com (88), breast cancer specimens will be examined for SC pounds of MER-25 that may have satisfactory anti-estro positive receptors. It has already been demonstrated that this genic or immune mimicking activity include methylated, methodology will identify breast cancers at the early Stage alkylated, benzylated, halogenated, unsaturations, altered when they are expressing an SC detectable receptor (87). charge properties, and conformationally altered or Stereoi These tumors are candidates for immune mimicking regu Somers of MER-25. lation. Those tumors that are Fc-like receptor positive are 0029. In certain embodiments, combinations of tamox candidates for tamoxifen therapy or combined therapy with ifen and levamisole are used as an immune mimic and tamoxifen and the other agents described below. AS a immune modulator to treat breast and other mucosal can specific test is developed to detect the ERY, the use of double cers, including colon cancer. In certain embodiments, com label fluorescence will permit very accurate determination of binations of tamoxifen and imiquimod are used as an patients that are Strong candidates for the therapies immune mimic and immune modulator to treat breast and described. other mucosal cancers. In certain embodiments, tamoxifen 0033. It is expected that tamoxifen inhibits breast cancer and OK-432 (picibanil) are used as an immune mimic and cell growth not by interaction with the commonly recog immune modulator to treat breast and other mucosal can nized ERC. or ERB but instead with the ERY (53,54). The CCS. direct histochemical measurement of ERY is expected to 0030 Certain embodiments of the present invention pro Significantly increase the reliability of the decision to initiate vide compositions or therapeutic methods employing a anti-estrogen therapy. Further, the identification of ERY will combination of tamoxifen and DHEA (dehydroepiandros permit reanalysis of existing and new compounds for anti terone) as an immune mimic and immune modulator to treat ERY activity. This approach can be expected to significantly breast and other mucosal cancers. In certain embodiments, a advance how new SERMs are selected. therapeutic method is provided in which tamoxifen and an Fc-like receptor gene therapy are used together to treat Example 2 breast and other mucosal cancers. Tamoxifen Therapy and New Diagnostic Test for 0031. In still other embodiments of the invention, meth Immune Modulation Applications with other ods are provided for identifying anti-estrogenic compounds Mucosal Cancers including ProState, Colon, or for evaluating modified forms of existing compounds that Kidney, Bladder Lung, Pancreas, Nasopharynx, might be more effective anti-estrogenic agents. These meth ods employ cell growth assays that, preferably, use certain Ovarian, Endometrial, Vaginal and Cervical Cancer. Serum-containing or Serum-free media. In Some embodi 0034. The analysis outlined in Example 1 will be used to ments, methods are provided for Screening new compounds determine the application of the tamoxifen-based therapies and for determining how combinations of compounds act on to tumors arising from other mucosal tissues. Since the same cells directly. These and other embodiments, features and Secretory immune System is functional in all of the tissues advantages of the present invention will become apparent (prostate, colon, kidney, bladder, lung, pancreas, nasophar with reference to the following description. ynx, ovary, endometrium, vagina and cervix), the immuno histochemical analysis for SC detectable Fc-like receptor DETAILED DESCRIPTION OF PREFERRED can be conducted. It has already been shown (89) that colon EMBODIMENTS cancers progreSS through Stages in which the SC is expressed (i.e. early differentiated tumors) to stages in which Example 1 there is little or no detectable SC (i.e. late malignancy stage). Tamoxifen and/or the combinations described will be used Tamoxifen Therapy and New Diagnostic Test for to treat Fc-like receptor positive (FcLR") tumors by the new Immune Modulation Applications with Breast protocols. This new approach is expected to provide an Cancer. expanded rationale for the use of tamoxifen to treat cancers 0.032 Co-pending U.S. patent application Nos. 09/852, not yet recognized as Sensitive to this immune mimicking 958 and 09/852,547/PCT Published Application Nos. WO anti-estrogen. Further, as a test for ERY is developed, it can US 2003/0072760 A1 Apr. 17, 2003 be used to further refine the tumor types susceptible to the 0038 Early anti-estrogens such as MER-25 were by new modes of tamoxifen combination therapies. passed by previous investigators because of their potency and adverse side effects. Although MER-25 has many desir Example 3 able properties as an anti-estrogen, it has been reported to be too toxic for use in humans (78). However, if MER-25 and Combinations of Tamoxifen and Aromatase related compounds can be modified to achieve high levels of Inhibitors to Treat Breast and Gynecologic Cancers. immune modulation without the serious side effects, this will 0035) It is proposed that simultaneous treatment with open additional new avenues of breast cancer therapy. aromatase inhibitors and tamoxifen will be more effective 0039. Accordingly, the chemical structure will be modi than either drug alone. The therapeutic potential of this new fied particularly in the O-C-C-N segment of the side combination is all the more Significant in View of the fact chain to change the conformation and to prevent hydrogen that aromatase inhibitors do not completely inhibit estrogen bonding with neighboring hydroxyl groups (80). Only lim Synthesis. An unfortunate side effect of tamoxifen treatment ited modifications in MER-25 have been sought (84). Other is that it increases the concentration of estrogens in the chemical changes in the Structure are expected to attenuate plasma, which will have a marked effect on reducing the the Side effects considered most Severe. Computer based potency of tamoxifen as an anti-estrogen. The Simultaneous molecular modeling will be used to develop the chemical use of aromatase inhibitors is expected to SuppreSS this modifications. The modifications are expected to include, for estrogen inductive effect and therefore may help to resolve example, methylation, halogenation, unsaturations, alter the problem of escape of tumors from tamoxifen inhibition. ations on charged groups, changes in conformation and In conventional therapies, tamoxifen resistance is currently Selection of stable stereoisomers of the MER-25 structure. thought to be a major problem with longer-term tamoxifen The chemically modified forms will be evaluated for anti treatment. AS additional tumor types beyond breast are estrogenic activity using both the Serum-containing and identified as tamoxifen Sensitive (e.g. endometrial, ovarian, Serum-free assay methods described in co-owned U.S. and vaginal, cervical and possibly prostate), the combined PCT patent applications (53.54), incorporated herein by tamoxifen-aromatase inhibitor therapy will have even reference. This is a rapid and effective method of determin broader application than is recognized today for either drug. ing when a derivative has been obtained that Still retains the desired potency against estrogen target cells in culture but Example 4 cannot be reversed by exogenous estrogens in culture. Those that are effective under the strict tests outlined (53.54) will Combinations of Tamoxifen and Pure be tested for in Vivo anti-tumor activity using rat mammary Anti-estrogens to Treat Breast and Gynecologic and pituitary models as well as with Xenografts of human CancerS. breast cancer cell lines in athymic nude mice. 0.036 The negative aspect of the current “pure' anti Example 6 estrogens discussed above is that they deplete the body of Combinations of MER-25 and Modified MER-25 estrogen action So effectively that there are major cardio with Progesterone and other Hormones. vascular and skeletal problems. However, by using the 0040. The undesirable side effects of MER-25 and chemi combination of tamoxifen and the "pure' anti-estrogens, the cally modified forms may also be attenuated by Simulta agonist action of tamoxifen can be expected to reduce this neous treatment with progesterone. In other Studies, it has problem without Supplying unwanted natural estrogens. been shown that eating behavior and body weight regulation Tamoxifen has positive effects on both the cardiovascular are affected by MER-25 (85). The administration of proges System and bone. Furthermore, it is expected that this terone in rats corrected those side effects. Thus, at least Some combination will also reduce the problem of induction of adverse properties in Vivo may be due to altered hormonal endometrial cancers thought today to be a problem with the influences. Administration of MER-25, and derivative com use of tamoxifen alone. Hence, the combination reduces pounds, will be evaluated for causation of endocrine major negative aspects Seen with each drug alone. The changes. Any changes identified will be corrected by Simul balance of the two components can be varied to achieve taneous application of the appropriate hormone(s). MER-25 Specific end points. or one of its derivatives may influence, for example, pitu itary hormone Secretion, thyroid hormones, adrenal hor Example 5 mones and/or neurogenic amines. Cytokines are also included in this group. Such changes are expected to yield Use of Chemically Modified MER-25 to Treat the Severe side effects reported (78). Accordingly, a hormone Secretory Immune System Related Cancers. derived from pituitary, adrenals or thyroid, or a cytokine or 0037 MER-25 is an anti-estrogen by virtue of its inhibi a neurogenic hormone may be administered together with tory effects on estrogen target tissues. It also has the benefit MER-25 or a modified form of MER-25 to deter the occur that it does not interact with the estrogen receptor to accom rence of Side effects from the drug. plish its action (79). The advantage of MER-25 (or its Example 7 modified forms) is that Systemic or locally produced estro gens will not interfere. Thus, it can be used with ER" pre Combination Tamoxifen and Levamisole as and postmenopausal women without concern for Suppres Immune Mimic and Immune Modulator to Treat Sion of endogenous estrogen levels. The results available Breast and Other Mucosal Cancers including Support the present Suggestion that MER-25 may mimic the Colon. immune activity of IgA and IgM even more Strongly than 0041 Levamisole is known to be immunoregulatory at tamoxifen. multiple levels (90). It is known to enhance an impaired US 2003/0072760 A1 Apr. 17, 2003 immune System (91). Levamisole is currently used to treat has not been identified. This preparation cannot be delivered Stage III colon cancer, and is recognized to be an immuno orally. It has been used in breast cancer as intratumor Stimulant, in the conventional Sense, to assist the natural injections (96). In those prior studies, the results were mixed immune system (92). Drawing from the inventor's prior but additional results from cell culture Suggest that a com observations that increased Secretory immunoglobulins IgA bination with an anti-estrogen may have greater effect than and IgM are not only cytostatic for breast cancer cells, but OK-432 alone (97). Another route to administration of also cytotoxic, therapies that enhance immune function, OK-432 is intrapleural administration, which was evaluated increasing the presence of these Secretory immunoglobulins as a treatment for breast malignancy in pleural effusions in particular, are thus expected to be beneficial. It has been shown by others that a general elevation of the immune (98). The results of the conventional immune therapy alone System by levamisole can retard colon cancer, but is not on disseminated breast cancer were encouraging. It is now completely effective. The addition of tamoxifen is expected proposed that the combination of OK-432 with tamoxifen or to enhance cancer cell death Via apoptosis mechanisms. The an aromatase inhibitor will provide additional benefits and diagnostic test for SC outlined above can be used to decide have anti-cancer effects beyond those that could have pre which patients should receive a combined levamisole/ viously been predicted for OK-432 and tamoxifen. The role tamoxifen therapy. of OK-432 may be direct on tumor cells, or may involve a critical conventional immune response that then Suppresses 0042. Together, the combination of levamisole and tumor cell growth. An additional possible use of this prepa tamoxifen for breast cancer is expected to have effects ration may be as an oral challenge to develop mucosal beyond that achievable with each compound alone. Levami immunity as described (53,54). This route of administration Sole will enhance the natural immune inhibition of breast and development of mucosal immunity represents an cancer growth while tamoxifen offers an additional direct cellular effect. This combination approaches therapy from entirely new approach to the use of this immune modulator. two different aspects of regulation. The use of levamisole to treat breast cancer is a new application, particularly when Example 10 placed in combination with tamoxifen. Other components of preferred therapeutic compositions include aromatase Combination Tamoxifen and DHEA inhibitors and/or "pure' anti-estrogens. () as Immune Mimic and Immune Modulator to Treat Breast and Other Example 8 Mucosal Cancers. Combination Tamoxifen and Imiquimod as Immune 0045 One recent report (101) asks the question “is Mimic and Immune Modulator to Treat Breast and DHEAa panacea or snake oil'? The answer likely rests with Other Mucosal Cancers. proper experimental design. DHEA use must be critically evaluated to achieve meaningful results. For example, the 0043. Imiquimod is a conventional immune enhancer that use of DHEA as a conventional immune modulator has been is effective both as a topical preparation and when admin istered orally (93.94). The known use of this compound in evaluated in postmenopausal women (99). That report stated breast cancer therapy is based on the action of interferon that evidence was clear that DHEA was a positive immune which is induced by imiquimod. The drug alone has only modulator in these females. Furthermore, it was considered limited long term effects. Imiquimod therapy is expected to useful that DHEA metabolism in breast yields androgens be highly effective in combination with an anti-estrogen that likely act as inhibitors of breast cancer growth. How Such as tamoxifen or a new MER-25 derivative. The eleva ever, the evidence with a combination of DHEA and the tion of interferon affects the immune System as well as “pure' anti-estrogen EM-800 with ZR-75-1 breast cancer having potential effects directly on breast cancer cells. The cell Xenografts in athymic nude mice were not as encour addition of tamoxifen is expected to enhance any effects of aging (100). DHEA inhibited alone, and the “pure” anti interferon. This combination has three possible cytostatic/ estrogen alone inhibited. These results are pointed out to cytotoxic modes. First is the direct effect of the anti demonstrate that the proposal of using the “mixed' anti estrogen. Second is an immune enhancing action of imiqui estrogen tamoxifen with DHEA has merit. Tamoxifen acts as mod, which is expected to include enhancement of the a direct immune mimic, as described in Example 1, in Secretory immunoglobulin inhibitors of cancer cell growth. addition to blocking the estrogen receptor. DHEA acts to stimulate the immune system and to deliver inhibitory Third is the direct cytotoxic effect of interferon. This modal androgens to breast cancer cells. It is believed that the ity may be enhanced by measurement of the interferon conventional immune stimulatory action of DHEA will also receptor in breast Specimens along with the Fc-like receptor. Serve to enhance the presence of the inhibitory Secretory Example 9 immunoglobulins. This multilevel approach is expected to be more effective than each of the compounds used alone. It Combination Tamoxifen and OK-432 (Picibanil) as is also expected to be more effective than use of a “pure' Immune Mimic and Immune Modulator to Treat anti-estrogen with only one mechanism of action. In addi Breast and Other Mucosal Cancers. tion, this combination may be even more effective when an aromatase inhibitor is added. Indeed, but applying the 0044) Ok-432 (Picibanil) is a streptococcal preparation immunohistochemical classifications outlined above, along that has a strong immune modulating effect (95), employing with determining the androgen receptor content, the com the conventional meaning of “immune modulating,” which bination therapy has a strong rational basis. Today androgen generally refers to the antibody/antigen recognition function receptors are rarely measured in Specimens of female breast of the immune System. The active moiety of this preparation CCC. US 2003/0072760 A1 Apr. 17, 2003

Example 11 0051 (4) MacCorquodale DW, Thayer SA & Doisy E A (1936) The isolation of the principal estrogenic Combination Tamoxifen and Fc-like Receptor Gene Substance of liquor folliculi. J Biol Chem 115:435-448. therapy to Treat Breast and Other Mucosal CancerS. 0052 (5) Clark J H & Markaverich B M (1983) The agonistic and antagonistic effects of Short acting estro 0.046 Because tamoxifen is effective only with cells that express the Poly-Ig (Fc) receptor or a Poly-Ig-like (Fc) gens: a review. Pharm Ther 21:429–453. receptor, introduction of this receptor into cells lacking 0053 (6) Lippman M E, Monaco M E & Bolan G immune control offers an entirely new approach to treatment (1977) Effects of estrone, estradiol, and estriol on of breast and other mucosal cancers. Viral vectors bearing hormone responsive human breast cancer in long term the DNA coding for the full length functional Fc-like tissue culture. Cancer Res 37:1901-1907. receptor can be used to transform disseminated cancer Such 0054 (7) Jozan S, Moure C, Gillois M & Bayard F that the tumor cells regain Sensitivity to tamoxifen. This is (1979) Effects of estrone on cell proliferation of human a significant concept because it permits activation of killing breast cancer (MCF-7) in long term tissue culture. J over a long duration and with multiple exposures to the virus Steroid Biochem 10:341-342. plus tamoxifen. Since tamoxifen can typically be used over a five-year period, and Viral infections repeated, this new 0055 (8) Katzenellenbogen BS (1984) Biology and approach has considerable promise and is Supported by the receptor interactions of estriol and estradiol derivatives recognized fact that all cancer cells will not be killed after in vitro and in vivo. J Steroid Biochem 20:1033-1037. even the first few viral infections. The properties of the 0056 (9) Karey K P & Sirbasku D A (1988) Differ receptor to be used have been described (53,54), and tech ential responsiveness of the human breast cancer cell niques for incorporating a desired DNA sequence into a lines MCF-7 and T47-D to growth factors and 17B Suitable viral vector, and for transforming a population of estradiol. Cancer Res 48:4083-4092. cells are known and have been descibed in the literature. 0057 (10) Freinkel N & Metzger B E (1992) Meta Example 12 bolic changes in pregnancy. In: Williams Textbook of Endocrinology, 8" Edition, Wilson J D & Foster DW Use of Serum-Containing and Serum-free Medium (eds), WB Saunders Company, Philadelphia, pp. 993 ASSays to Define New Anti-estrogenic Compounds 1005. or to Modify Existing Compounds to More 0058 (11) Gore-Langton & Armstrong DT (1988) Effective Agents: Folicular steroidogenesis and its control. In: The Physi 0047. The above-identified co-owned U.S and PCT ology of Reproduction, Knobile E & Neill J D (Eds patent applications (53.54), hereby incorporated herein by in-chief), Raven Press, New York, pp 331-385. reference, describe two different types of assays that will be used to characterize new anti-estrogenic compounds and 0059 (12) Doisy E A, Veler CD & Thayer SA (1930) derivatives. One assay is done with ER" cell lines grown in The preparation of the crystalline ovarian hormone medium Supplemented with Steroid hormone depleted from the urine of pregnant women. Am J Physiol Serum. The Serum is preferably prepared by either charcoal 86:499-509. dextran extraction, or by XAD-4 resin treatment (53.54). 0060 (13) Comer G W (1983) The sites of formation Similar assays can be done under completely Serum-free of in the animal body. Physiol defined conditions. The results of the two assays can be Rev 18:154-172. compared directly. 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0129 (82) Geisler J, Haarstad H, Gunderson S et al cancer using a mixture of OK-432 and fibrinogen (1995) Influence of treatment with the anti-estrogen Supplemented with activated macrophages. Biotherapy 3-hydroxytamoxifen (droloxifene) on plasma Sex hor 7:47-53. mone levels in postmenopausal patients with breast 0144) (97) Aoyagi H, Eino Y, Takeo T et al (1997) cancer. J. Endocrinol 146:359-363. Effects of OK-432 (picibanil) on estrogen receptors of 0130 (83) Lum SS, Woltering EA, Fletcher W S et al MCF-7 cells and potentiation of antiproliferative (1997) Changes in serum estrogen levels in women effects of tamoxifen in combination with OK-432. during tamoxifen therapy. Am J Surg 173:399-402. 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Preservation of Spirit and teachings of the invention. The embodiments immunoglobulin isotypes, J chain, and Secretory com described herein are exemplary only, and are not intended to ponent in human tissues. Path Res Pract 179:250-266. be limiting. Many variations and modifications of the inven tion disclosed herein are possible and are within the Scope of 0.136 (89) Krajci P. Meling GI, Andersen S N et al the invention. For example, the foregoing descriptions pri (1996) Secretory component mRNA and protein marily focus on the treatment and prevention of breast expression in colorectal adenomas and carcinomas. Br cancer in high-risk individuals, however the Same or similar J. Cancer 73:1503-1510. approaches can be employed to with respect to other types 0137 (90) Goldstein G (1978) Mode of action of of cancers of mucosal tissues, including prostate, ovary, levamisole. J Rheumatol Suppl 4:143-148. endometrium, cervix, Vagina, colon, kidney, lung, pancreas and nasopharynx. Cancers of those tissues, together with 0138 (91) Prakash MS, Rao V M & Reddy V (1998) breast cancer, account for 80% of all human cancer. The Effect of levamisole on the immune status of malnour disclosures of all patents, patent applications and publica ished children. J Trop Pediatrics 44.165-166. tions cited hereinabove are hereby incorporated herein by reference. The discussion of certain references in the 0139 (92) Holcombe R F, Li A & Stewart R M (1998) Description of Related Art, above, is not an admission that Levamisole and interleukin-2 for advanced malig they are prior art to the present invention, especially any nancy. Biotherapy 11:255-258. references that may have a publication date after the priority O140 93) Savage9. P, Horton V, Moore J et al (1996) A date of this application. phase I trial clinical trial of imiquimod, an oral inter feron inducer, administered daily. BrJ Cancer 74: 1482 What is claimed is: 1486. 1. A method of predicting Susceptibility of a cancer of mucosal origin to treatment with an immunoglobulin inhibi 0141 (94) Witt P L, Ritch PS, Reding D et al (1993) tor mimicking compound, the method comprising: identify Phase I trial of an oral immunomodularor and inter ing in a Specimen of Said cancer cells expression of a Poly-g feron inducer in cancer patients. Cancer Res 53:5176 (Fc) receptor or a Poly-Ig-like (Fc) receptor that is capable 518O. of binding to Said immunoglobulin inhibitor mimicking 0142 (95) Chirigos MA (1992) Immunomodulators: compound and of mediating immunoglobulin inhibition of current and future development and application. Thy cancer cell growth in cancer cells of mucosal origin. mus 19 (suppl):S7-S20. 2. The method of claim 1 wherein the Fc region of said identified Poly-Ig (Fc) receptor or Poly-Ig-like (Fc) receptor 0143 (96) Takeda T, Kobayashi T, Monden T et al is capable of binding to Said immunoglobulin inhibitor (1993) The effect of local immunotherapy for breast mimicking compound. US 2003/0072760 A1 Apr. 17, 2003

3. The method of claim 1 comprising determining whether 22. A pharmaceutical composition for treating a cancer of Said cancer cell is at least partially hormone responsive with mucosal origin comprising: respect to Stimulation of cell growth by Said hormone. 4. The method of claim 1 wherein said immunoglobulin an immune inhibitor of Steroid hormone reversible cancer inhibitor mimicking compound is tamoxifen. cell growth or an immune inhibitor mimicking com 5. The method of claim 1 wherein said immunoglobulin pound; and at least one at least one component chosen inhibitor mimicking compound is MER-25 or a chemically from the group consisting of: modified form thereof. an anti-estrogen compound, 6. The method of claim 1 wherein said cancer of mucosal origin is from breast, prostate, colon, kidney, bladder, lung, an aromatase inhibitor, pancreas, nasopharynx, ovarian, endometrial, vaginal or an agent that enhances immunoglobulin production, cervical. and 7. The method of claim 1 further comprising detecting a hormone, expression of Secretory component in Said cancer cells. 8. The method of claim 1 further comprising detecting the together with a pharmaceutically acceptable carrier. presence of ERY in Said cancer cells. 23. The composition of claim 22 wherein Said agent that 9. A method of treating breast cancer in an individual in enhances immunoglobulin production comprises a com need of Such treatment, the method comprising: pound chosen from the group consisting of Levimisole, Imiquimod, Picibanil and DHEA. in a specimen of breast cancer cells from Said individual, 24. The composition of claim 22 wherein said anti determining Susceptibility of at least a portion of Said estrogenic compound or Said immunoglobulin inhibitor cells to cell growth inhibition by an immunoglobulin mimicking compound comprises MER-25 or a chemically inhibitor mimicking compound by a method compris modified form thereof. ing identifying in at least a portion of Said cells the 25. The composition of claim 21 wherein said anti expression of a Poly-Ig receptor or Poly-Ig-like recep estrogenic compound comprises a compound chosen from tor, or the Fc receptor region thereof, that is capable of the group consisting of tamoxifen, toremifene, ICI 16384, binding to Said immunoglobulin inhibitor mimicking ICI 182780, EM-800, RU 58688 and EM-139. compound and mediating immunoglobulin inhibition 26. The composition of claim 21 wherein said hormone is of breast cancer cell growth; and chosen from the group consisting of pituitary hormones, administering Said immunoglobulin inhibitor mimicking adrenal hormones, thyroid hormones, cytokines and neuro compound to said individual. genic hormones. 10. The method of claim 9 further comprising adminis 27. The composition of claim 26 wherein said hormone tering an aromatase inhibitor to Said individual. comprises progesterone. 11. The method of claim 9 further comprising adminis 28. A method of Screening a compound for anti-estrogenic tering a pure anti-estrogen compound to Said individual. activity comprising: 12. The method of claim 9 further comprising adminis culturing a predetermined population of estrogen respon tering to Said individual an enhancer of immunoglobulin Sive cancer cells in a nutrient medium comprising a production. quantity of immunoglobulin inhibitor or immunoglo 13. The method of claim 12 wherein said enhancer bulin inhibitor mimicking compound Sufficient to comprises levimisole. inhibit cell growth in the absence of an inhibition 14. The method of claim 12 wherein said enhancer reversing amount of estrogen; comprises imiquimod. 15. The method of claim 12 wherein said enhancer adding a defined amount of the Substance of interest to the comprises picibanil. cell culture medium; 16. The method of claim 12 wherein said enhancer adding a defined amount of estrogen to Said cell culture comprises DHEA. medium, Said amount being Sufficient to Stimulate cell 17. The method of claim 9 further comprising transferring growth in the presence of said inhibitor or inhibitor a functional Poly-Ig receptor gene or Poly-Ig-like receptor mimicking agent under the same cell culture conditions gene to breast cancer cells in Said individual. but in the absence of Said Substance of interest, to 18. The method of claim 9 further comprising transferring provide a test culture; a DNA sequence encoding the Fc portion of a Poly-Ig receptor or Poly-Ig-like receptor to breast cancer calls in incubating Said test culture for a predetermined period of said individual. time under growth promoting conditions, 19. The method of claim 9 comprising administering testing Said Substance of interest for cytotoxic effects on MER-25 or a chemically modified form thereof, and at least Said cells, one hormone to Said individual. determining the cell population of the test culture after 20. The method of claim 18 wherein said at least one Said predetermined period of time, wherein a lack of hormone is chosen from the group consisting of pituitary increase in the cell population, not attributable to hormones, adrenal hormones, thyroid hormones, cytokines cytotoxic effects of Said Substance of interest, indicates and neurogenic hormones. an anti-estrogenic effect by Said Substance of interest. 21. The method of claim 20 wherein said at least one hormone comprises progesterone. k k k k k