US 20010047033A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2001/0047033 A1 Taylor et al. (43) Pub. Date: Nov. 29, 2001

(54) COMPOSITION FOR AND METHOD OF Publication Classification PREVENTING OR TREATING BREAST CANCER (51) Int. Cl." ...... A61K 31/35; A61K 31/138 (52) U.S. Cl...... 514/456; 514/651 (75) Inventors: Richard B. Taylor, Valley Park, MO (US); Edna C. Henley, Athens, GA (57) ABSTRACT (US) The present invention is a composition for preventing, Correspondence Address: minimizing, or reversing the development or growth of Richard B. Taylor breast cancer. The composition contains a combination of a Protein Technologies International, Inc. Selective estrogen receptor modulator Selected from at least P.O. BOX 88940 one of raloxifene, droloxifene, toremifene, 4'-iodotamox St. Louis, MO 63188 (US) ifen, and idoxifene and at least one isoflavone Selected from genistein, daidzein, biochanin A, formononetin, and their (73) Assignee: Protein Technologies International, respective naturally occurring glucosides and glucoside con Inc., St. Louis, MO jugates. The present invention also provides a method of (21) Appl. No.: 09/900,573 preventing, minimizing, or reversing the development or growth of breast cancer in which a Selective estrogen (22) Filed: Jul. 6, 2001 receptor modulator Selected from at least one of raloxifene, droloxifene, toremifene, 4'-iodotamoxifen, and idoxifene is Related U.S. Application Data co-administered with at least one isoflavone Selected from genistein, daidzein, biochanin A, formononetin, and their (62) Division of application No. 09/294,519, filed on Apr. naturally occuring glucosides and glucoside conjugates to a 20, 1999. woman having or predisposed to having breast cancer. Patent Application Publication Nov. 29, 2001 Sheet 1 of 4 US 2001/0047033A1

FIG. 1

O CH N-1N-1 3

HC N CH, Patent Application Publication Nov. 29, 2001 Sheet 2 of 4 US 2001/0047033A1

FG 2

Compound Droloxifene NMe2 Toremifene CH2Cl NMe2 4-Iodotamoxifen CH NMe2 Idoxifene CH pyrrolidino Patent Application Publication Nov. 29, 2001 Sheet 3 of 4 US 2001/0047033A1

FIG. 3 'N-1\ O

OH

HO

Compound Genistein Daidzein Glycitein Biochanin A Formononetin Patent Application Publication Nov. 29, 2001 Sheet 4 of 4 US 2001/0047033A1

Compound Genistin 6'-OMall genistin COCH2COH 6'-OAc genistin COCH, Daidzin H 6'-OMal daidzin COCHCO2H 6'-OAc daidzin COCH, Glycitin H 6'-OMal glycitin COCH US 2001/0047033 A1 Nov. 29, 2001

COMPOSITION FOR AND METHOD OF 0006 Tamoxifen, however, has an estrogenic effect on PREVENTING OR TREATING BREAST CANCER uterine tissues when endogenous estrogen levels are low, which occurs in postmenopausal women and Oopherectim FIELD OF THE INVENTION ized women. Uterine epithelial cell heights are significantly increased by the estrogenic effect of tamoxifen in postmeno 0001. The present invention relates to a composition pausal and oopherectimized women, leading to uterine containing a Selective estrogen receptor modulator and at hypertrophy. Tamoxifen also causes marked uterine eosino least one isoflavone, and a method of treating breast cancer philia. These effects have been associated with endometrial while inhibiting Selective estrogen receptor modulator carcinoma, and long term use of tamoxifen is linked to an induced uterotrophic effects. increased risk of endometrial cancer, up to a fivefold exceSS of risk relative to women not treated with tamoxifen therapy. BACKGROUND OF THE INVENTION Therefore, application of tamoxifen for long term breast 0002 Breast cancer is one of the leading causes of cancer cancer prevention and long term treatment of breast cancer mortality among Western women, and is predicted to has significant associated riskS. become a leading cause of cancer death in Oriental women 0007 Efforts have been made to develop new selective in countries Such as Japan in the near future. The American estrogen receptor modulators (“SERMS") which act in a Cancer Society estimates that 1 in 9 women face a lifetime mechanism similar to that of tamoxifen in breast tissue, risk of this disease, which will prove fatal for about one while avoiding the risks caused by the estrogenic effects of quarter of those afflicted with the disease. tamoxifen in uterine tissue. Several of these SERMS are 0003 Tamoxifen (FIG. 1), a synthetic nonsteroidal selec triphenylethylene tamoxifen analogs. As shown in FIG. 2, tive estrogen receptor modulator, has been used effectively droloxifene is a tamoxifen analog in which a 3'-hydroxyphe in the treatment of breast cancer for over twenty years. nyl moiety is Substituted in place of a phenyl moiety of Tamoxifen is one of the most widely prescribed antineoplas tamoxifen. Droloxifene has a binding affinity for the estro tic agents in the United States and Great Britain, and is one gen receptor which is ten times that of tamoxifen, has been of the initial hormonal treatments of choice in both pre shown to have antiestrogenic activity in breast tissue and to menopausal and postmenopausal women with estrogen be efficacious in treatment of advanced breast cancer, yet has receptor positive metastatic disease. Furthermore, adjuvant lower estrogenic effects in uterus tissue than tamoxifen. therapy Studies show a Substantial reduction of contralateral Droloxifene, a New Estrogen Antagonist/Agonist, Prevents primary breast carcinoma in tamoxifen treated women, Bone Loss in Ovariectomized Rats, Ke at al., Endocrinology which indicates that tamoxifen may be of use in breast 136:2435-2441 (1995). cancer prevention. 0008 Toremifene, shown in FIG. 2, is a tamoxifen 0004 Tamoxifen has tissue-specific estrogenic and anti analog having a 4-chloro Substituent. Pharmacologically estrogenic effects. Estrogen, an ovarian hormone, increases toremifene has quite similar effects as tamoxifen on breast the risk of breast and endometrial cancer by inducing an tissue, acting as potent antiestrogen. Toremifene also exhib estrogen receptor mediated increase in the frequency of its anti-tumor cytolytic effects at high doses which are breast and endometrial cell division. Cell division is essen independent of its antiestogenicity, effects which do not tial in the complex process of genesis of human cancer Since occur with high doses of tamoxifen. Antiestrogenic Potency it per Se increases the risk of genetic error-particularly of Toremifene and Tamoxifen in Postmenopausal Women, genetic errorS Such as inactivation of tumor Suppressor Homesley et al., Am. J. Clin. Onc., 16(2):117-122 (1993). geneS. 0009) 4-Iodotamoxifen, shown in FIG. 2, is another 0005 Tamoxifen has antiestrogenic effects in breast tis tamoxifen analog, having a 4'-iodophenyl Substituent in Sue. Tamoxifen's antiestrogenic effect in breast tissue is a place of a phenyl Substituent of tamoxifen. lodination of primary mechanism by which tamoxifen inhibits the prolif tamoxifen at the 4'-phenyl postion reduces estrogenic activ eration of breast cancer cells. Tamoxifen competes with ity, mimicking the high antiestrogenic activity of the tamox estrogen for binding to cytoplasmic estrogen receptors ifen metabolite 4'-hydroxytamoxifen, while giving the com (“ER”), with subsequent inhibition by the tamoxifen/ER pound a longer duration of action in Vivo by blocking complex of many of the activities of endogenous estrogen formation of the rapidly metabolized 4'-hydroxytamoxifen within tumor cells. Endogenous estrogen binds with ERS to metabolite. Pyrrolidino-4-iodotamoxifen and 4-Iodotamox promote cellular activities Such as estrogen/ER-mediated ifen, New Analogues of the AntieStrogen Tamoxifen for the gene transcription, DNA Synthesis, cancer cell growth, and Treatment of Breast Cancer, Chander et al., Cancer increases in autocrine polypeptides Such as transforming Research, 51:5851-5858 (Nov. 1, 1991); Idoxifene: Report growth factor-alpha, epidermal growth factor, insulin-like of a Phase I Study in Patients with Metastatic Breast Cancer, growth factor-II, and other growth factors that may be Coombes et al., Cancer Research, 55:1070-1074 (Mar. 1, involved in cell proliferation. Competitive inhibition of 1995). 4-Iodotamoxifen has been shown to have less estro estrogen binding to ERS by tamoxifen reduces or prevents genic agonist activity in uterine tissue than tamoxifen, and, Such cancer growth inducing cellular activities. As a result therefore, is less likely to cause endometrial cancer when of tamoxifen's antiestrogenic activity in breast tissue, administered over a long term. tamoxifen prevents the transition of breast cancer cells from the early G1 phase to the mid-G1 phase of the cell cycle and 0010 Idoxifene, also known as pyrrolidino-4-iodotamox exhibits a cytostatic effect on breast cancer cells. Tamoxifen ifen, shown in FIG. 2, is another tamoxifen analog, and is has been shown to reduce distant breast cancer metastasis as modeled on the 4'-iodotamoxifen analog. Idoxifene has the well as local-regional recurrence of Such cancers in both Same general molecular structure as 4'-iodotamoxifen, node-negative and node-positive women. except that the N,N-dimethylamino moiety of 4'-iodotamox US 2001/0047033 A1 Nov. 29, 2001 ifen is replaced with a pyrrolidino moiety. Substitution of the 0017 FIG. 3 is a molecular representation of the selec pyrrolidino group for the dimethylamino group reduces tive estrogen receptor modulator raloxifene. possible toxic side effects by inhibiting the metabolization of the compound by the liver to a desmethyl metabolite with 0018 FIG. 4 is a molecular representation of genistein, the concomitant release of potentially toxic formaldehyde. daidzein, biochanin A, and formononetin. Idoxifene has a 2.5 to 5 fold higher affinity for ERS than 0019 FIG. 5 is a molecular representation of the natu tamoxifen, and is 1.5-fold more efffective in inhibiting the rally occuring glucosides of genistein and daidZein. growth of MCF-7 breast cancer cells. Idoxifene also has less uterotrophic estogenic effects than tamoxifen and 4'-iodota DESCRIPTION OF THE PREFERRED moxifen, and produced uterotrophic effects comparable to EMBODIMENTS that of tamoxifen only at a dose which was ten times greater. Pyrrolidino-4-Iodotamoxifen and 4-Iodotamoxifen, New 0020. As used herein, the term “ER” refers to “estrogen Analogues of the Antiestrogen Tamoxifen for the Treatment receptor'. The term "breast cancer” means any cancer of Breast Cancer, Chander et al., Cancer Research, 51:5851 having its origin in breast cells, and includes metastatic and 5858 (November 1991); Idoxifene: Report of a Phase I local forms of breast cancer (node negative and node posi Study in Patients with Metastatic Breast Cancer, Coombes et tive), as well as ER positive and ER negative forms of breast al., Cancer Research, 55:1070-1074 (Mar. 1, 1995). cancer. The term “uterotrophic effect” means the prolifera tion of uterine epithelial cells, which frequently is a side 0011. Other SERMS which are not tamoxifen analogs effect of administration of Selective estrogen receptor modu have shown effectiveness in preventing or minimizing the lators to women, and which appears to be directly related to development of breast cancer. Raloxifene (FIG. 3), a ben development of endometrial cancer. As used herein “Mal” Zothiophene derivative, has shown potent antiestrogenic represents “malonyl and “Ac' represents “acetyl'. The term inhibition of estradiol binding to the ER and significantly “minimize', or a derivative thereof, includes a complete or inhibits estrogen dependent proliferation of MCF-7 cells partial inhibition of a specified biological effect (which is derived from human mammary tissue. Raloxifene, unlike apparent from the context in which the term minimize is tamoxifen and its analogs, exhibits an antiestrogenic effect used). The term “isoflavone” may mean both a single in uterine tissue, and provides a nearly complete blockade of isoflavone or plural isoflavones when the isoflavone is uterotrophic responses to estrogen as well as tamoxifen. defined as at least one of a Selected group of isoflavones. Selective Estrogen Receptor Modulators, Kauffman & Bry “SERM' means a selective estrogen receptor modulator and ant, DN&P 8(9) 531-539 (November 1995). its physiologically acceptable Salts, other than tamoxifen, 0012. It is desirable to utilize these SERMS to develop which is a compound which produces estrogen antagonist new compositions which may be used to improve the effects in one or more desired target tissues (e.g. breast tissue SERMS prevention or minimization of the development of and uterine tissue), while producing either estrogen agonist breast cancer while reducing their uterotrophic activity, if effects or minimal agonism in other non-target tissues. any. 0021. The present invention resides in the discovery that SUMMARY OF THE INVENTION the combination of selected SERMs with certain isoflavones can be used to treat or prevent breast cancer in a woman 0013 In one aspect, the present invention is a composi having or predisposed to breast cancer, and the isoflavones tion for preventing or minimizing the development or will augment the SERM induced prevention, minimization, growth of breast cancer. The composition comprises a or reversal of the development or growth of breast cancer, as combination of a Selective estrogen receptor modulator well as prevent or minimize uterotrophic effects associated Selected from at least one of raloxifene, droloxifene, with Some SERMs. The SERMs which are useful in the toremifene, 4-iodotamoxifen, and idoxifene, and at least one compositions and methods of the present invention are isoflavone Selected from genistein daidZein, biochanin A, droloxifene, toremifene, 4'-iodotamoxifen, idoxifene, and formononetin, or their respective naturally occuring gluco raloxifene. The isoflavones which are useful in the compo Sides or glucoside conjugates. Sitions and methods of the present invention are genistein, 0.014. In another aspect, the present invention is a method daidzein, glycitein, biochanin A, formononetin, their natu for preventing or minimizing the development or growth of rally occuring glycosides and their naturally occuring gly breast cancer in a human. A Selective estrogen receptor coside conjugates, shown in FIGS. 4 and 5. modulator and an isoflavone are co-administered to a human 0022 Materials to prevent or minimize the development or growth of breast cancer. The Selective estrogen receptor is Selected from at 0023 The selective estrogen receptor modulator com least one of raloxifene, droloxifene, toremifene, 4'-iodota pounds used in the compositions and methods of the present moxifen, and idoxifene. The isoflavone is Selected from at invention can be chemically Synthesized according to known least one of genistein, daidzein, biochanin A, formononetin, methods, and include the Salt forms of each of the com or their naturally occuring glucosides or glucoside conju pounds. Raloxifene, 6-hydroxy-2-(4-hydroxyphenyl)-3-4- gateS. (2-piperdinoethoxy)benzoylbenzobthiophene (FIG. 3), and its physiologically acceptable Salts may be produced BRIEF DESCRIPTION OF THE DRAWINGS according to the methods described in U.S. Pat. Nos. 4,418, 068 and 4,133,814, each of which is incorporated herein by 0.015 FIG. 1 is a molecular representation of tamoxifen. reference. Droloxifene, E-1-4'-(2-dimethylaminoethox 0016 FIG. 2 is a molecular representation of the selec y)phenyl)-1-(3'-hydroxyphenyl)-2-phenyl-1-butene (FIG. tive estrogen receptor modulators droloxifene, toremifene, 2), and its physiologically acceptable Salts may be produced 4'iodotamoxifen, and idoxifene. according to the methods described in U.S. Pat. No. 5,047, US 2001/0047033 A1 Nov. 29, 2001

431, which is incorporated herein by reference. Toremifene, prepared by a conventional Saponification of genistin with a 4-chloro-1,2-diphenyl-1-(4-2-(N,N-dimethylamino)et malonyl or an acetyl anhydride, respectively. hoxy)-phenyl-1-butene (FIG. 2), and its physiologically 0027 Biochanin A can be synthetically prepared by the acceptable Salts may be produced by the methods described method provided by Baker et al. (Nature 169:706 (1952)), in U.S. Pat. No. 4,696,949, which is incorporated herein by incorporated herein by reference. Biochanin A can also be reference. 4'-Iodotamoxifen, E-1-4-2-(dimethylamino)et separated from red clover by the method provided by Pope hoxyphenyl)-1-(4-iodophenyl)-2-phenyl-1-butene (FIG. et al. (Chem. & Ind. (London) p. 1092 (1953)), incorporated 2), and its physiologically acceptable salts may be produced herein by reference. Formononetin can be Synthetically according to combined methods described in Stereoselective prepared by the methods disclosed by Wessely et al. (Ber. Olefin Formation from the Dehydration of 1-(p-Alkoxyphe 66:685 (1933)) and Kagel et al. (Tetrahedron Letters, p. 593 nyl)-1,2-diphenylbutan-1-ols: Application to the Synthesis (1962)), both references of which are incorporated herein by of Tamoxifen, McCague, J. Chem. Soc. Perkin Trans., reference. Formononetin can be isolated from Soybean meal 1:1011-1015 (1987); and Derivatives of Tamoxifen. Depen by the method of Walz (Ann. 489:118 (1931)) or can be dence of Antiestrogenicity on the 4-Substituent, McCague et isolated from clover species by the method of Bradbury et al. al., J. Med. Chem., 32(12):2527-2533 (1989), each of which (J. Chem. Soc. p. 34.47 (1951)), both references of which are is incorporated herein by reference. Idoxifene, E-1-(4-io incorporated herein by reference. dophenyl)-1-4-(2-pyrrolidinoethoxy)phenyl)-2-phenyl-1- 0028. It is preferred to extract the isoflavones useful in butene (FIG. 2), may be produced according to combined the compositions and methods of the present invention from methods described in the references above that provide the plant materials in which they naturally occur. A preferred methods for producing 4'-iodotamoxifen. method of isolating the isoflavone compounds is to extract 0024. The isoflavone compounds used in the composi the plant materials with an alcohol, preferably methanol or tions and methods of the present invention are naturally ethanol, or an aqueous Solution, preferably an aqueous occurring Substances which may be found in plants Such as alkaline Solution, to remove the isoflavones from the plant legumes, clover, and the root of the kudzu vine (pueraria material. It is preferred to comminute the plant material root). Common legume Sources of these isoflavone com before extracting the isoflavone compounds to maximize pounds include Soy beans, chick peas, and various other recovery of the isoflavone compounds from the plant mate types of beans and peas. Clover Sources of these isoflavone rial. The isoflavone compounds can be isolated from the compounds include red clover and Subterranean clover. Soy extract by conventional Separation procedures Such as beans are a particularly preferred Source of the isoflavone reverse phase high performance liquid chromatography compounds (except biochanin A which is not present in Soy). (“HPLC). 0.025 The isoflavone compounds may be isolated from 0029. In a preferred embodiment, the isoflavone com the plant Sources in which they naturally occur, or may be pounds genistein, genistin, 6'-O-Mal genistin, 6'-O-Ac Synthetically prepared by processes known in the art. For genistin, daidzein, daidzin, 6'-O-Mal daidzin, 6'-O-Ac daid example, daidzein may be isolated from red clover as Zin, glycitein, glycitin, and 6'-O-Mal glycitin are isolated disclosed by Wong (J. Sci. Food Agri, Vol. 13, p. 304 (1962)) from a Soy material, preferably a commercially available Soy or may be isolated from the mold Micromonospora halo material. Soy materials from which the isoflavone com phytica as provided by Ganguly and Sarre (Chem. & Ind. pounds can be isolated include: Soy beans, dehulled Soy (London), p. 201 (1970)), both references of which are beans, Soy meal, Soy flour, Soy grits, Soy flakes (full fat and incorporated by reference herein. Daidzein may be Syntheti defatted), Soy cotyldeons, Soy molasses, Soy protein con cally prepared by the methods provided by Baker et al (J. centrate, Soy whey, Soy whey protein, and Soy protein Chem. Soc., p. 274 (1933)), Wesley et al. (Ber. Vol. 66, p. isolate. In one embodiment, the isoflavones are extracted 685 (1933)), Mahal et al. (J. Chem. Soc., p. 1769 (1934)), from Soybeans, dehulled Soybeans, Soy meal, Soy flour, Soy Baker et al. (J. Chem. Soc., p. 1852 (1953)), or Farkas (Ber. grits, Soy flakes, Soy protein concentrate, Soy whey protein, Vol. 90, p. 2940 (1957)), each reference of which is incor or Soy protein isolate, preferably Soy meal, Soy flour, Soy porated herein by reference. The isoflavone glucoside daid grits, or Soy flakes, with a low molecular weight organic Zin may be synthetically prepared by the method of Farkas extractant, preferably an alcohol, ethyl acetate, acetone, or et al. (Ber, Vol. 92, p. 819 (1959)), incorporated herein by ether, and most preferably aqueous ethyl alcohol or methyl reference. The daidZein isoflavone glucoside conjugates alcohol. Most preferably the extractant has a pH at about the 6'-O-Mal daidzin and 6'-O-Ac daidzin can be prepared by a isoelectric point of soy protein (about pH 4 to pH 5) to conventional Saponification of daidzin with a malonyl or an minimize the amount of Soy protein extracted by the extrac acetyl anhydride, respectively. tant. 0.026 Genistein may be synthetically prepared by the 0030 The extractant containing the isoflavones is sepa methods provided by Baker et al (J. Chem. Soc., p. 3115 rated from the insoluble soy materials to form an isoflavone (1928)); Narasimhachari et al. (J. Sci. Ind. Res., Vol. 12, p. enriched extract. If desired, an isoflavone enriched material 287 (1953)); Yoder et al., (Proc. Iowa Acad. Sci., Vol. 61, p. may be recovered by concentrating the extract to remove the 271 (1954); and Zemplen et al. (Acta. Chim. Acad. Sci. Solvent, thereby producing a Solid isoflavone enriched mate Hung, Vol. 19, p. 277 (1959)), each reference of which is rial. incorporated herein by reference. The isoflavone glucoside 0031. In a more preferred embodiment the isoflavone genistin may be Synthetically prepared by the method of compounds are further purified from other Soy materials Zemplen et al. (Ber, Vol 76B, p. 1110 (1943)), incorporated Soluble in the extract by contacting the extract with a herein by reference. The isoflavone glucoside conjugates of material which adsorbs the isoflavones in the extract, and genistein, 6'-O-Mal genistin and 6'-O-Ac genistin, can be eluting the adsorbed isoflavones out of the adsorbent mate US 2001/0047033 A1 Nov. 29, 2001

rial with a solvent which causes the isoflavones to be especially desirable in the compositions and methods of the differentially eluted from the adsorbent material. present invention Since, as noted above, they are believed to 0032. In a preferred embodiment, the isoflavones are be particularly active in inhibiting angiogenesis and inhib Separated from impurities in the extract by a conventional iting tyrosine kinase activity. reverse phase HPLC separation. After extraction of the 0037. The isoflavone glucoside conjugates 6"-O-Mal isoflavones from the Soy material and Separation of the genistin, 6"-O-Ac genistin, 6"-O-Mal daidzin, 6"-O-Ac extract from the insoluble Soy materials, the extract is daidzin, and 6"-O-Mal glycitin can be converted to their filtered to remove insoluble materials that could plug an respective glucosides genistin, daidzin, and glycitin by HPLC column. An HPLC column is prepared by packing a forming an aqueous alkaline Solution of the Substrate con conventional commercially available HPLC column with a taining the isoflavones having a pH of about 6 to about 13, particulate adsorbent material which will releasably bind the preferably about pH 9 to about pH 11, and treating the isoflavones and impurities in the extract in a compound aqueous alkaline Solution at a temperature of about 2 C. to Specific manner. The adsorbent material may be any reverse about 121°C., preferably about 25° C. to about 75° C., for phase HPLC packing material, however, a preferred packing a period of time Sufficient to effect the conversion, prefer material may be chosen by the criteria of load capacity, ably about 30 minutes to about 5 hours, more preferably Separation effectiveness, and cost. One Such preferred pack about 30 minutes to about 1.5 hours. The isoflavone gluco ing material is Kromasil C18 16 um 100A beads available Sides genistin, daidzin, and glycitin can be converted to their from Eka Nobel, Nobel Industries, Sweden. respective aglucone forms genistein, daidzein, and glycitein by contacting the isoflavone glucosides with an enzyme 0033. The filtered extract is passed through the packed capable of cleaving a 1,4-B-glucoside bond-preferably a HPLC column until all the binding sites of the column are commercially available beta-glucosidase enzyme, an alpha fully saturated with isoflavones, which is detected by the or beta-galactosidase enzyme, a pectinase enzyme, a lactase appearance of isoflavones in the effluent from the column. enzyme, or a glucoamylase enzyme-at a pH at which the The HPLC column may then be eluted with a solvent to effect the Separation. In a preferred embodiment, the eluent enzyme is active, typically from about pH 3 to about pH 9, is a polar Solvent Such as ethanol, methanol, ethyl acetate, or and at a temperature of about 25 C. to about 75 C., more acetonitrile, and preferably is an aqueous alcohol having an preferably about 45 C. to about 65 C., for a period of time alcohol content of between about 30% and about 90%, most sufficient to effect the conversion, typically about 1 hour to preferably about 50%, and most preferably the alcohol is about 24 hours, preferably about 1 hour to about 3 hours. ethanol. 0038. The aglucone isoflavones can be separated from the Substrate using conventional separation procedures. For 0034. The isoflavone compounds and impurities are sepa example, the aglucone isoflavones may be extracted from rately collected from the column effluent. The isoflavone the substrate with a low molecular weight alcohol. The fractions of the eluent may be identified from other eluent aglucone isoflavones may be separated from the extract by fractions in accordance with conventional HPLC and ana conventional recrystallization processes, or by HPLC. In a lytical chemistry techniques. In a preferred embodiment the particularly preferred embodiment, an isoflavone composi eluent fractions containing the aglucone isoflavones are tion isolated from a Soy SubStrate for formulation into a collected Separately since the aglucone isoflavones are composition for use in the method of the present invention believed to be particularly active tyrosine kinase inhibitors includes at least 40% genistein, at least 15% daidZein, and and anti-angiogenesis agents which inhibit the development at least 1% glycitein. In another particularly preferred or progression of breast cancer. Of the aglucone isoflavone embodiment of the invention, an isoflavone composition materials, the fraction of effluent containing daidzein elutes isolated from a Soy Substrate for formulation into a compo from the column first, followed by a glycitein fraction, Sition for use in the method of the present invention contains followed by the more polar genistein. at least 85% genistein, at least 5% daidzein, and at least 0035. The isoflavone fractions of the eluent may be 0.5% glycitein. In yet another preferred embodiment, each collected from the column, and the volatile content of the isoflavone is recovered Separately in pure form. Solvent (e.g. alcohol) can be removed by evaporation. The 0039. Several of the isoflavone compounds are commer isoflavone compounds can be recovered directly if all of the cially available, and may be purchased for formulation into Solvent is removed by evaporation, or may be recovered by compositions provided in the present invention, or used in chilling the remaining Solvent (e.g. water) to crystallize the the methods of the present invention. For example, isoflavones and centrifuging or filtering the remaining Sol genistein, daidzein, and glycitein are commercially available vent away from the crystallized isoflavones. and may be purchased, for example, from Indofine Chemical 0036). In a particularly preferred embodiment the Soy Company Inc., P.O. Box 473, Somerville, N.J. 08876, and isoflavone glucosides and isoflavone glucoside conjugates biochanin A is available from Aldrich Chemical Company, 6'-O-Mal genistin, 6'-O-Ac genistin, 6'-O-Mal daidzin, 6'-O- Inc., 940 West Saint Paul Avenue, Milwaukee, Wis. 53233. Ac daidzin, 6'-O-Mal glycitin, genistin, daidzin, and gly citin-are converted to their respective aglucone isoflavone 0040 Methods forms-genistein, daidzein, and glycitein. The conversion of 0041. In one aspect the present invention is a method for the isoflavone glucoside conjugates and the isoflavone glu preventing or minimizing the development or growth of cosides to the aglucone isoflavones can be effected in the breast cancer in a human by co-administering at least one Substrate from which the isoflavones are to be extracted SERM Selected from raloxifene, droloxifene, toremifene, prior to the extraction, or may be effected in the isoflavone 4-iodotamoxifen, and idoxifene, and at least one isoflavone enriched extract after Separation of the extract from the Selected from genistein, daidzein, biochanin A, formonon insoluble materials. The aglucone isoflavone compounds are etin, their respective glucosides, and their respective gluco US 2001/0047033 A1 Nov. 29, 2001

side conjugates. The SERM and isoflavone may be co mg to about 200 mg per day. The amount of isoflavone administered prophylactically to prevent the development of Sufficient to treat the development or growth of breast cancer breast cancer in Women Susceptible of developing breast to prevent, minimize, or reverse the development or growth cancer, or the SERM and isoflavone may be co-administered of the cancer is preferably at least 1 mg per day, more to treat breast cancer by preventing, minimizing, or revers preferably from about 1 mg to about 1000 mg per day, and ing the growth and development of the cancer. The SERM most preferably from about 50 mg to about 500 mg per day. may be obtained for use in accordance with the method the 0045. The isoflavones utilized in the method of the present invention as described above, or, preferably, may be present invention prevent, minimize, or reverse the growth provided in a composition of the present invention, as of breast cancer by Several mechanisms, which in combi described below. The isoflavone may be obtained for use in nation with the anti-estrogenic activity of the SERM in accordance with the method of the present invention as breast tissue, increase the relative anti-breast cancer activity described above, or, preferably, may be provided in a of each compound. First, the isoflavones are anti-estrogenic composition of the present invention, as described below. in breast tissue, and Serve to competitively inhibit estrogen 0042. The SERM and the isoflavone may be co-admin induced cancerous breast cell division by binding to the ER istered either concurrently or Sequentially within a Specified of the cell, where the isoflavone/ER complex inhibits cancer period of time, preferably daily, on a periodic basis. Most cell growth in much the same manner as tamoxifen and the preferably the SERM and the isoflavone are co-administered SERMs (e.g. daidzein halts cell growth in the G1 phase of concurrently in a composition of the present invention, as the cell cycle, and genistein halts cell growth in the G2 phase described below, on a periodic basis, preferably daily. Alter of the cell cycle). Second, Some of the isoflavones, particu natively, the SERM and the isoflavone are administered larly genistein and biochanin A, and to a lesser extent Sequentially as Separate components. “Sequentially' as used daidzein and formononetin, are tyrosine kinase inhibitors herein is intended to mean administration of desired which inhibit enzymatic tyrosine kinase activity. Tyrosine amounts of the SERM and isoflavone individually within a kinase activity is necessary for cancerous cells to produce Specified periodic period of time, for example daily, and is proteins required for cellular differentiation and growth. not intended to be limited to immediate consecutive admin Third, the isoflavones inhibit angiogenesis, thereby prevent istration of the SERM and isoflavone. ing a cancerous cell mass from developing the network of blood vessels necessary to Support the cell mass, limiting the 0043. The SERM is administered in an amount Sufficient Sustainable growth of the cell mass. Fourth, the isoflavones to prevent or treat the development or growth of breast decrease endogenous estrogen levels by interfering with cancer in combination with the isoflavone. The amount of pituitary and hypothalmus gland feedback mechanisms SERM sufficient to prevent or treat the development or which regulate the release of gonadotropins Such as estra growth of breast cancer in combination with the isoflavone diol. The effect of the combined mechanisms of action is to is dependent on the particular SERM utilized, the amount further prevent or minimize the development or growth of and activity of the isoflavone utilized, the size of the patient breast cancer when co-administered with a SERM effective to which the SERM is administered, whether the SERM is administered prophylatically or to treat breast cancer, and if to prevent or minimize the growth of breast cancer. used in treatment, the extent of the cancer. The amount of 0046. In a particularly preferred embodiment of the SERM sufficient to prevent the development of breast cancer method of the present invention, the isoflavone is co in a woman predisposed to breast cancer is preferably at administered with the SERM in an amount Sufficient to least 0.5 mg per day, more preferably from about 0.5 mg to prevent or minimize SERM induced uterotrophic effects. about 100 mg per day, and most preferably from about 5 mg Atlhough the SERMs utilized in the present invention are to about 50 mg per day. The amount of SERM sufficient to less uterotrophic than tamoxifen, each of the SERMs except treat the development or growth of breast cancer to prevent, raloxifene induces uterotrophic effects at relatively high minimize, or reverse the development or growth of the doses. The isoflavones utilized in the present method have cancer is preferably at least 0.5 mg per day, more preferably an antiestrogenic effect in uterine tissues when concenra from 0.5 mg to about 500 mg per day, and most preferably tions of estrogen or an estrogen agonist SERM are relatively from about 40 mg to about 400 mg per day. The SERM may high. One mechanism by which the isoflavones likely cause be administered in Several doses per day to achieve the daily an antiestrogenic effect in uterine tissue in the presence of amount of the SERM sufficient to prevent or treat breast uterine estrogen agonist SERMS is by binding to uterine cell cancer, however, it is preferred that the daily required ERS and competitively inhibiting the estrogen agonist amount of SERM be administered in one or two doses. SERMs from binding to the ERs. Unlike uterine tissue estrogen agonist SERMs, the isoflavones do not cause an 0044) The isoflavone is co-administered to the human in estrogenic response upon binding to uterine cell ERS, there an amount Sufficient to prevent or treat the development or fore, the isoflavones prevent, inhibit, or minimize the growth of breast cancer in combination with the SERM. The uterotrophic effects caused by uterine endothelial cell amount of isoflavone sufficient to prevent or treat the devel ER/SERM complexes. Preferably the isoflavone is co-ad opment or growth of breast cancer in combination with the ministered with the SERM to prevent or minimize SERM is dependent on the particular isoflavone utilized, the uterotrophic effects in a weight/weight ratio of isofla amount and activity of the co-administered SERM, the size of the patient, whether the isoflavone is administered pro vone:SERM of about 0.25:1 to about 100:1, and more phylatically or to treat breast cancer, and if used in treatment, preferably from about 0.5:1 to about 20:1. the extent of the cancer. The amount of isoflavone Sufficient 0047. In a particularly preferred embodiment of the to prevent the development of breast cancer in a woman method, co-administration of the isoflavone with a uterine predisposed to breast cancer in the present method is pref tissue estrogen agonist SERM in an amount Sufficient to erably at least 1 mg per day, more preferably from about 10 prevent or minimize uterotrophic effects is also effective to US 2001/0047033 A1 Nov. 29, 2001 prevent or minimize the development of endometrial cancer 0053 A composition in accordance with the present when the SERM is used to prevent or treat breast cancer. As invention containing a SERM and an isoflavone can be noted above, tamoxifen and uterine tissue estrogen agonist prepared by conventional procedures for blending and mix SERMs cause an increased risk of the development of ing compounds. Preferably, the composition also includes an endometrial cancer as a result of estrogen-like activity in excipient, most preferably a pharmacuetical excipient. Com uterine tissue and its uterotrophic effects. Co-administration positions containing an excipient and incorporating the of the isoflavone together with an uterine tissue estrogen SERM and isoflavone can be prepared by procedures known agonist SERM prevents or minimizes the development of in the art. For example, the SERM and the isoflavone can be endometrial cancer by preventing or minimizing SERM formulated into tablets, capsules, powders, Suspensions, induced uterotrophic effects. Solutions for parenteral administration including intrave 0048 Compositions nous, intramuscular, and Subcutaneous administration, and into Solutions for application onto patches for transdermal 0049. In another aspect, the present invention is a com application with common and conventional carriers, binders, position useful for preventing or minimizing the develop diluents, and excipients. ment or growth of breast cancer. The composition includes combination of a Selective estrogen receptor modulator 0054 Inert pharmaceutically acceptable carriers useful to Selected from at least one of raloxifene, droloxifene, form pharmaceutical compositions in accordance with the toremifene, 4-iodotamoxifen, and idoxifene, and at least one present invention include Starch, mannitol, calcium Sulfate, isoflavone Selected from genistein, daidzein, biochanin A, dicalcium phosphate, magnesium Stearate, Silicic deriva formononetin, their respective naturally occuring glucosides tives, and/or SugarS Such as Sucrose, lactose, and glucose. and glucoside conjugates. These SERM and isoflavone Binding agents include carboxymethyl cellulose and other materials necessary to form compositions in accordance cellulose derivatives, gelatin, natural and Synthetic gums with the present invention may be obtained as described including alginates Such as Sodium alginate, polyethylene above. The composition contains from about 1% to about glycol, waxes and the like. Diluents useful in the invention 99% SERM, by weight of biologically active ingredients, include a Suitable oil, Saline, Sugar Solutions Such as acqueous and from about 1% to about 99% isoflavone, by weight of dextrose or aqueous glucose, and glycols Such as polyeth biologically active ingredients. ylene or polypropylene glycol. Other excipients include 0050. The SERM is present in the composition in an lubricants Such as Sodium oleate, Sodium acetate, Sodium amount Sufficient to prevent, minimize, or reverse the devel Stearate, Sodium chloride, Sodium benzoate, talc, and mag opment or growth of breast cancer in a woman when nesium Stearate, and the like; disintegrating agents including co-administered with the isoflavone. Preferably at least 0.5 agar, calcium carbonate, Sodium bicarbonate, Starch, Xan mg of the SERM is present in the composition, more than gum, and the like; and adsorptive carrierS Such as preferably from about 0.5 mg to about 500 mg, and most bentonite and kaolin. Coloring and flavoring agents may preferably from about 5 mg to about 100 mg. Most prefer also be added to the pharmaceutical compositions. ably, the SERM is present in the composition in an amount 0055. The following non-limiting formulations illustrate Sufficient to prevent, minimize, or reverse the development pharmaceutical compositions of the present invention. or growth of breast cancer by itself. FORMULATIONS 0051 Preferably at least 1 mg of the isoflavone is present in the composition, more preferably from about 1 mg to 0056. The following Formulations 1-4 illustrate pharma about 1000 mg, and most preferably from about 10 mg to ceutical formulations including a SERM and an isoflavone. about 200 mg. In a preferred embodiment the isoflavone is Formulation 1 present in the composition in an amount Sufficient to aug ment the composition's SERM induced prevention or mini Gelatin Capsules mization of development or growth of breast cancer when 0057 Hard gelatin capsules are prepared using the fol the composition is administered to a woman. In a more lowing ingredients: SERM 0.5-100 mg/capsule; Isoflavone preferred embodiment, the isoflavone is present in the com 0.1-1000 mg/capsule; Starch, NF 0-600 mg/capsule; Starch position in an amount Sufficient to prevent, minimize, or flowable powder 0-600 mg/capsule; Silicone fluid 350 cen reverse the development or growth of breast cancer by itself. tistokes 0-20 mg/capsule. The ingredients are mixed, passed 0.052 In another preferred embodiment, the isoflavone is through a Sieve, and filled into capsules. present in the composition in an amount Sufficient to prevent Formulation 2 or minimize the composition's SERM induced uterotrophic effects when the composition is administered to a woman. Tablets The isoflavone should be present in a ratio of isofla 0058 Tablets are prepared using the following ingredi vone:SERM of from about 0.25:1 to about 100:1 by weight, ents: SERM 0.5-100 mg/tablet; Isoflavone 0.1-1000 mg/tab and more preferably from about 0.5:1 to about 50:1 by let; Microcrystalline cellulose 20-300 mg/tablet; Starch 0-50 weight, to be present in the composition in an amount mg/tablet, Magnesium Stearate or Stearate acid 0-15 mg/tab sufficient to prevent or minimize the composition's SERM let; Silicon dioxide, fumed 0-400 mg/tablet; silicon dioxide, induced uterotrophic effects. In a most preferred embodi colloidal 0-1 mg/tablet, and lactose 0-100 mg/tablet. The ment, the isoflavone is present in the composition in an ingredients are blended and compressed to form tablets. amount sufficient to augment the composition's SERM Formulation 3 induced prevention or minimization of the development or growth of breast cancer and to prevent or minimize the Suspensions composition's SERM induced uterotrophic effects when the 0059 Suspensions are prepared using the following composition is administered to a woman. ingredients: SERM 0.5-100 mg/5 ml; Isoflavone 0.1-1000 US 2001/0047033 A1 Nov. 29, 2001

mg/5 ml, Sodium carboxymethyl cellulose 50-700 mg/5 ml; 3. The method of claim 1 wherein co-administration of Sodium benzoate 0-10 mg/5 ml; Purified water 5 ml; and Said droloxifene and Said genistein is Sequential. flavor and color agents as needed. 4. The method of claim 1 wherein from about 0.5 mg to about 500 mg of said droloxifene is administered to said Formulation 4 woman per day. 5. The method of claim 4 wherein from about 5 mg to Parenteral Solutions about 100 mg of said droloxifene is administered to said 0060 A parenteral composition is prepared by stirring woman per day. 1.5% by weight of active ingredients (SERM and isoflavone 6. The method of claim 1 wherein from about 1 mg to wit/wt ratio of from 10:1 to 1:10) in 10% by volume about 1000 mg of Said genistein is administered to Said propylene glycol and water. The Solution is made isotonic woman per day. with sodium chloride and sterilized. 7. The method of claim 6 wherein from about 10 mg to about 200 mg of Said genistein is administered to Said 0061 The above description is intended to be illustrative woman per day. of the present invention, and is not intended to be limiting. 8. The method of claim 1 wherein said genistein is Other embodiments are within the claims. administered to Said woman in an amount Sufficient to What is claimed is: augment prevention, minimization, or reversal of the devel 1. A method for preventing, minimizing, or reversing the opment or growth of breast cancer provided by Said drolox development or growth of breast cancer in a woman com ifene. prising co-administering to Said woman droloxifene and 9. The method of claim 1 wherein said genistein is genistein, Said droloxifene being administered to Said administered to Said woman to prevent or minimize the woman in an amount effective to prevent, minimize, or development of endometrial cancer. reverse the development or growth of breast cancer, and Said 10. The method of claim 1 wherein said droloxifene and genistein being administered for the purpose of preventing Said genistein are administered in a pharmaceutical prepa or minimizing uterine hypertrophy induced by administra ration. tion of Said droloxifene wherein Said genistein is adminis 11. The method of claim 10 wherein said pharmaceutical tered in an amount effective to prevent or minimize uterine preparation is a tablet, capsule, powder, Suspension, or hypertrophy induced by administration of droloxifene. Solution. 2. The method of claim 1 wherein co-administration of Said droloxifene and Said genistein is concurrent.