US 20080293.683A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2008/0293683 A1 Zhu (43) Pub. Date: Nov. 27, 2008

(54) HORMONE REPLACEMENT THERAPY Related U.S. Application Data (75) Inventor: Bao Ting Zhu, Lexington, SC (US) (60) giga application No. 60/931,586, filed on May Correspondence Address: Publication Classification STINSON MORRISON HECKER LLP ATTN PATENT GROUP (51) Int. Cl. 1201 WALNUT STREET, SUITE2800 A6II 3/56 (2006.01) KANSAS CITY, MO 64106-2150 (US) A6IP 5/30 (2006.01) (52) U.S. Cl...... 514/178; 514/169; 514/182 (73) Assignee: University of Kansas Medical 57 ABSTRACT Center, Kansas City, KS (US) (57) A hormone replacement therapy formulation and method (21) Appl. No.: 12/111,328 comprising selective estrogenic compounds which preferen tially stimulate the receptor alpha over the estrogen (22) Filed: Apr. 29, 2008 receptor beta.

() 1 ()() 1OOO } O1 10 OO 1000 Concentrations (nM) ( ERO E. ERB F. 2) 15 e O TB O O TB v NSB o W NSB O 12 e SB SB o1 4. A. o - O

E 12 Zir o A---- 9 0, 70 nM - 0.75 nM N. 8 6 r 9 4 W3 s 6 W / Y O v^ w (L 97-of W - W - WW W1 (2- v.-v-V- v-V O O.O2 OO9 (39 156 6.25 25 (O2 O.O9 O.39 156 6.25 25 Patent Application Publication Nov. 27, 2008 Sheet 1 of 7 US 2008/0293683 A1

cro Y st e

iss aC E S. E se S E & Y E r E

O O. 1 1 0 1 OO 1000 O O. 1 1 1 0 1 OO 1000 Concentrations (nM) ( ERO E. ERB F. 2O 15 s o TB O O TB y W. NSB o W NSB 16 O SB / O SB O o 12 E /oolo - O - - ) 12 K = Y K = / 9 9 DT D 2 0.70 nM Ns 0.75 nM N. 8 6 r

W 4 W /* 4 W 1 / O W V OL (9 of- VW-VW W1 2V-V-V-V-d v-V O O.O2 O.O9 O.39 156 6.25 25 O.O2 O. O9 O.39 156 6.25 25

F.G. Patent Application Publication Nov. 27, 2008 Sheet 2 of 7 US 2008/0293683 A1

5 CO 75 5 25

25 O) 75 St. 5

25

5.

2-OH-3-MeO-E, 4-MeO-E, O - l . O O () O OC OOC Concentrations (nM) o ERC e ERB FG. Patent Application Publication Nov. 27, 2008 Sheet 3 of 7 US 2008/0293683 A1

-o-o-o-os--

2,3-dimeO-E,

O O. 10 OO OOO O O. O 1 OO OOO 25 OO 0 ERO e ERB

O. C 100 OOC Concentrations (nM) FG, 3 Patent Application Publication Nov. 27, 2008 Sheet 4 of 7 US 2008/0293683 A1

--- l

5 H OC-E isa =u. 8-0-8s sa.--CCo -- a-- o-o-o 75 - Y 50 - 25 - 11 a-OH-E, ll B-O-E,

O O O OO IOOC) O (). O 100 OOO s M. 10-iss, ERO. 5 - S. • ERB 50 N. : 25 - 11 3-MeO-EE, N. 0 - - oNS O () ) (OO) Concentrations (nv1) FIG. 4 Patent Application Publication Nov. 27, 2008 Sheet 5 of 7 US 2008/0293683 A1

7-Dehydro-E

OO & res= -2- N.Y. N.V. - 9(11)-Dehydro-E,NN NS -3s

5

SO 173-Dihydro NN. -- 7-Dehydro-E-17a. N O OO 100 ( ). O CO COO Concentrations (nM) o ERO o ERB F.G. 5 Patent Application Publication Nov. 27, 2008 Sheet 6 of 7 US 2008/0293683 A1

25

O)

f 5

SO 16B-O-E, 5 (16-) .

16a-OH-E-17a. "N 16B-Oli-E-17a.

(17-Epies triol) s (16,17-Epies triol) \

-T - = J Tags-os- K. 'ss 75 N NY 'S 50 -Y. \\ Ne N \\ 1,3,5(10), 16 25 17o-Ethynyl-E, NS 17-Desoxy-E, YS Estratetraen-3-ol - - () O ) OOO O. O.O. C. O DO O. O. O. IO IO IOC Concentrations (nM) o ERO F.G. 6 Patent Application Publication Nov. 27, 2008 Sheet 7 of 7 US 2008/0293683 A1

Tamoxifen

s

Dibenzoylethane Hexes trol l SS CO ECO O ( IO IOC

O) 75 o ERO, 5) ERB 25 Die estrol

C Concentrations (nM) F.G. 7 US 2008/0293683 A1 Nov. 27, 2008

HORMONE REPLACEMENT THERAPY high-affinity , now commonly referred to as ERC, was cloned in 1986 from MCF-7 human cancer CROSS-REFERENCE TO RELATED cells, which abundantly expressed this ER subtype. For APPLICATIONS nearly a decade after its cloning, it was believed that the 0001. This application is based on and claims priority to signal through a single ER. However, a second ER U.S. Provisional Application Ser. No. 60/931,586, filed on (subtype B) was later identified in 1996 while studying the May 24, 2007 which is hereby incorporated herein by refer roles of estrogens in the prostate, gonads, and the immune system. The existence of two distinct ER subtypes indicated CCC. that the signaling pathways for endogenous estrogens are STATEMENT REGARDING FEDERALLY significantly more complex than previously thought. SPONSORED RESEARCH ORDEVELOPMENT 0008. The human ERC. is a 66 kDa hormone-inducible transcription factor that can act positively or negatively in 0002 The present invention was supported in part by the regulating the expression of genes involved in tissue growth National Institutes of Health Grant No. RO1-CA-92391 and and differentiation. The human ERB is a 53 kDa hormone RO1-CA-97109, and the government may have certain rights inducible transcription factor that shares high degrees of in the invention. sequence homology with the human ERC, especially in the DNA binding domain. Studies have shown that there are a BACKGROUND OF THE INVENTION number of functional similarities between human ERC. and 0003 Hormone replacement therapy has been known for ERB, and both receptor subtypes can bind 17 B- (E) Some time. One particular aspect of hormone replacement with similarly high affinities. See Kuiper et al., Comparison therapy, known generally as estrogen replacement therapy, of the binding specificity and transcript tissue distri has been used for over 30 years for women during or follow bution of estrogen receptors C. and 3, Endocrinology 138, ing . The reason for estrogen replacement, which 863-870 (1997); Katzenellenbogen et al., Hormone binding is usually accomplished through transdermal absorption or and transcription activation by estrogen receptors: analyses orally, is to make up for the decline in, or the low level of using mammalian and systems, J. . Biochem. endogenous estrogens produced by the body. Typically, estro Mol. Biol. 47,39-48 (1993). The activated ERC. and ERB (i.e., gen production decreases and then declines dramatically dur receptor bound with an such as E) can form ing and after menopause. It is during this time period that homodimers (ERC-ERC. or ERB-ERB) or heterodimers estrogen replacement is normally prescribed by a physician. (ERC-ERB), and these dimerized ERs can bind to various However, estrogen replacement can be prescribed in other estrogen response elements in highly similar fashions. circumstances where other causes account for a decline in 0009. However, there are also significant differences estrogen production or ifestrogen is produced at a lower than noted for human ERC. and ERB. For example, it has been desirable level. This could occur in women not yet in meno found that the tissue distribution pattern of these two ER pause. subtypes is quite different. See Katzenellenbogenetal. A new 0004. The reasons for estrogen replacement, which have actor in the estrogen receptor drama—Enter ER-3, Endocri been substantiated by scientific research over a number of nology 138,861-862 (1997); Spong et al., Maternal estrogen years, include the prevention and/or treatment of osteoporosis receptor-B expression during mouse gestation, Am. J. and cardiovascular disease, as well as preventing age-related Reprod. Immunol. 44, 249-252 (2000); Saunders et al., decline in mental function. Estrogen replacement has also Expression of Oestrogen receptor B (ERB) in multiple rat been used to decrease age-related changes in appearance. tissues visualised by immunohistochemistry, J. Endocrinol. 0005 For decades, the general scientific belief had been 154 R13-R16 (1997); Enmarket al., Human estrogen recep that “an estrogen is an estrogen, i.e., all estrogens would tor B-gene structure, chromosomal localization, and expres exert similar pharmacological actions in the body. As such, Sion pattern, J. Clin. Endocrinol. Metab. 82, 4258-4265 the most commonly prescribed estrogen forestrogen replace (1997); Shughrue et al., Comparative distribution of estrogen ment is actually concentrated from horse containing receptor-C. and-BmRNA in the rat central nervous system, J. many estrogenic compounds sold under the name Premarin R. Comp. Neurol. 388, 507–525 (1997); Denger et al., Tissue See generally, Hill et al., U.S. Pat. No. 6,855,703 entitled specific expression of human ERC. and ERB in the male, Mol. "Pharmaceutical compositions of and Endocrinol. 178, 155-160 (2001). In addition, an earlier methods of analyzing mixtures containing estrogenic com study has shown that 16? 3-hydroxyestradiol-17C. (16 B-OH pounds.” Many physicians and others have objected to equine E-17C., commonly known as 16, 17-epiestriol), an endog estrogen as being inappropriate for human use and even pos enous estrogen metabolite, has a preferential binding affinity sibly dangerous because of the fact that many individual for human ERB over ERC. Hence, the possibility exists that horse estrogens are not present in human bodies. There is also Some of the endogenously-formed estrogen metabolites/de Some evidence of the carcinogenic effect of . rivatives may have differential binding affinity for human 0006. In an attempt to duplicate or mimic the presence of ERC. or ERB, likely contributing to the differential activation natural estrogens in the human body by replacement therapy, of each signaling system in different target sites and/or under some physicians in the 1980s began to prescribe combina different physiological or pathophysiological conditions. tions of the three classical human estrogens, namely, a com 0010. In recent years, the present inventor and others have bination of (E), 17 B-estradiol (E), and (E). made considerable effort to systematically characterize the In addition, an estrogen formulation comprising 2-hydrox complete profiles of the metabolites of E and E that are yestrone, 17B-estradiol, and estriol was proposed. Seegener formed by human , non-hepatic tissues, as well as various ally Wright, U.S. Pat. No. 6,911,438 entitled “Hormone recombinant human cytochrome P450 isoforms . A replacement therapy. large number of endogenous estrogen metabolites have been 0007 More recently, the role of estrogens in the body has identified. In the present invention, the ability of these been further elucidated. In particular, it has been found that metabolites to stimulate ERC. and ERB was investigated. many of the well-known hormonal actions of estrogens are Those results were then used to develop and select certain mediated by specific estrogen receptors (“ERs). The first estrogenic compounds for use in estrogen replacement US 2008/0293683 A1 Nov. 27, 2008 therapy that are believed to mimic the naturally occurring RBA about 1.3), 2-hydroxyestriol (RBA/RBA about 2), estrogens in non-pregnant pre-menopausal women. 17)-estradiol 11-acetate (RBA/RBA of 1.2), 11 B-methoxy ethynyl estradiol (RBA/RBA of about 1.8), (RBA/ BRIEF SUMMARY OF THE INVENTION RBA of about 1.3), and 16B-hydroxyestradiol (RBA/RBA 0011. The present invention is directed to a novel hormone of about 1.3), 17c-estradiol (RBA/RBA about 7.3), 17a replacement therapy formulation and method of using the ethynylestradiol (RBA/RBA about 3.6). hormone replacement therapy formulation to treat diseases, 0019. The estrogenic compounds may also take the form disorders, and conditions in women having low endogenous of prodrugs. For example, 2-methoxyestrone is readily con estrogen levels, especially in peri-menopausal and post verted in the body to 2-methoxyestradiol (RBA/RBA about menopausal women. 2) by the 17 B-hydroxysteroid dehydrogenase. As 0012. In one aspect, the present invention is directed to a another example, estrone-3-sulfate, which has virtually no composition of matter comprising a mixture of estrogenic estrogen receptor binding affinity, can be readily hydrolyzed compounds endogenous to and normally circulating in the into estrone (RBA/RBA about 5). Still as another example, human female body. 17C.-estradiol-3-sulfate or 17C-estradiol-17-sulfate is rapidly 0013. In one aspect, the formulation consists essentially of one or more estrogenic compounds Such that the relative converted to 17C-estradiol (17C-E). binding affinity for ERC. (“RBA) of the estrogenic com 0020 Most preferred estrogenic compounds are selected pounds compared to 17B-estradiol (E) is less than about from the group consisting of comprises estrone (E), 17C.- 100%. For example, the estrogenic compounds may have an estradiol (17O.-E.), 2-hydroxyestrone (2-OH-E), 2-methox RBA, which is less than about 30%, 25%, 20%, 15%, 10%, yestrone (2-MeO-E), and/or 2-methoxyestradiol (2-MeO 5%, 4%, 2%, or 1% of 17 B-estradiol (E). In a more preferred E), as well as their sulfated or glucuronidated conjugates. In aspect, the formulation consists of estrogenic compounds another aspect, the formulation consists essentially of estrone such that the relative binding affinity for ERC. (“RBA) of (E), 17o-estradiol (17C-E), 2-hydroxyestrone (2-OH-E), the estrogenic compounds compared to 173-estradiol (E) is 2-methoxyestrone (2-MeO-E), and/or 2-methoxyestradiol less than about 100%. (2-MeO-E), as well as their sulfated or glucuronidated con 0014. In another aspect, the formulation consists essen jugates. tially of one or more estrogenic compounds such that the 0021. The estrogenic compounds in the mixture may be relative binding affinity for ERB (RBA) of the estrogenic present in a chemically pure form, or as prodrugs, especially compounds compared to 17 B-estradiol (E) is less than about Sulfated or glucuronidated conjugates, and their pharmaceu 100%. For example, the estrogenic compounds may have an tically acceptable salts. Thus, for example, the formulation RBA of less than about 10%, 5%, 4%, 3%, 2%, or 1% of may include pharmaceutically acceptable salts of conjugated 17 B-estradiol (E). In a preferred aspect, the formulation consists of estrogenic compounds such that the relative bind estrone (E), conjugated 17C-estradiol (17C-E), conjugated ing affinity for ERf (“RBA) of the estrogenic compounds 2-hydroxyestrone (2-OH-E), conjugated 2-methoxyestrone compared to 17? 3-estradiol (E) is less than about 100%. (2-MeO-E), and/or conjugated 2-methoxy-estradiol 0015. In one aspect, the formulation consists essentially of (2-MeO-E). In one aspect, the pharmaceutically acceptable one or more estrogenic compounds which preferentially is a salt. stimulate the ERC. over the ERB. In another aspect, the for 0022. According to embodiments of the present invention, mulation consists of estrogenic compounds which preferen the formulation may include the pharmaceutically acceptable tially stimulate the ERC. over the ERB. salts of , 17O.-, 2-hydrox 0016. In a further aspect, the estrogenic compounds have a yestrone Sulfate, 2-methoxyestrone sulfate, and/or 2-meth ratio of RBA/RBA which is greater than about 1,2,3,4,5, oxy-estradiol Sulfate. 6, 7, 8, 9, or 10. In another aspect, the ratio ranges between 0023. According to still other embodiments of the present about 1:1 to 20:1, more preferably 2:1 to 10:1, with exem invention, the formulation may include the Sodium salts of plary ranges being between about 1:1 to 3:1 and about 7:1 to estrone sulfate, 17C.-estradiol sulfate, 2-hydroxyestrone sul 20:1. fate, 2-methoxyestrone Sulfate, and/or 2-methoxy-estradiol 0017. In one aspect, the formulation comprises a mixture of at least one, two, or three estrogenic compounds that are sulfate. endogenous to the non-pregnant pre-menopausal human 0024. In another aspect, the invention provides a method female. of treating Subjects in need of treatments of various diseases 0018. In another aspect, the formulation comprises a mix and disorders associated with low levels of estrogenic com ture of at least four or five estrogenic compounds that are pounds. The method comprises administering an effective endogenous to the non-pregnant pre-menopausal human amount the estrogenic compounds of the present invention female. Preferred estrogens that may be used in the present (and formulations containing the same) to a subject in need invention include estrone (RBA/RBA about 5), 1-methyl thereof. Examples of treatments that are addressed by the estradiol (RBA/RBA about 1.8), 2-aminoestrone (RBA/ compositions of the invention include vasomotor symptoms, RBA about 7.5), 2-nitroestrone (RBA/RBA about 3), atrophic vaginitis, and osteoporosis. 2-hydroxyestrone (RBA/RBA about 10), 2-methoxyestra 0025. In another aspect, the estrogenic compounds of the diol (RBA/RBA about 2), 2-bromoestradiol (RBA/RBA about 10), 4-nitroestrone (RBA/RBA about 10): 4-hydrox present invention are co-administered with one or more pro yestrone (RBA/RBA about 2), A hydroxyestradio (RBA/ testing. Such as . RBA about 1.3), 4-methoxyestradiol (RBA/RBA about 0026. Additional aspects of the invention, together with 2), 6-ketoestrone (RBA/RBA about 2), 6.O-hydroxyestra the advantages and novel features appurtenant thereto, will be diol (RBA/RBA about 1.5), 6-ketoestradiol (RBA/RBA set forth in part in the description which follows, and in part about 1.3), 6-ketoestriol (RBA/RBA about 8.3), 6-ke will become apparent to those skilled in the art upon exami toestradiol-17C (RBA/RBA about 2), 7-dehydroestradiol nation of the following, or may be learned from the practice of (RBA/RBA about 1.3), 7-dehydroestradiol-17C. (RBA/ the invention. The objects and advantages of the invention US 2008/0293683 A1 Nov. 27, 2008

may be realized and attained by means of the instrumentali 0035. In general, the symptoms associated with treatment ties and combinations particularly pointed out in the methods of the present invention include, but are not limited appended claims. to osteoporosis, coronary heart disease, breast tenderness, oedema, fatigue, hot flashes, Sweating, headache, shortness BRIEF DESCRIPTION OF THE DRAWINGS of breath, depression, night Sweats, anxiety, sleep disorders, 0027 FIG. 1 shows the competition of HIE binding to vaginal dryness, vaginal shrinkage, dry skin and hair, hair recombinant human ERC. and ERB by E. E. E. (estriol or loss, mood Swings, urinary incontinence, nausea, heart pal 16C.-OH-E), or E-17C. The conditions for the in vitro ER pitations, short-term memory loss, frequent urinary tract binding assay were described in detail herein. For the data infections, yeast infections, painful intercourse, decreased shown in panels A-D, the concentration of the radioactive sexual activity, and inability to reach orgasm. ligand HE was 10 nM, and the concentrations of the com peting estrogens were 0, 0.24, 0.98, 3.9, 15.6, 62.5, 250, and 0036. In a preferred aspect, hormone replacement therapy 100 nM. For the data shown in panels E and F, the concen formulation consists essentially of estrogenic compounds trations of the radioactive ligand HE were 0.025, 0.1, 0.2, such that: (1) the relative binding affinity for ERC. (“RBA) 0.4, 0.8, 1.6, 3.2, 6.25, 12.5, and 25 nM. The non-specific of the estrogenic compounds compared to 173-estradiol (E) binding was determined in the presence of 400-fold excess of is less than about 100%; (2) the relative binding affinity for cold E. The K values for ERC. and ERB were calculated ERB (RBA) of the estrogenic compounds compared to according the S-shaped binding curves (curve regression 17 B-estradiol (E) is less than about 100%; and/or (3) the analysis). Abbreviations used: (“TB), total binding: estrogenic compounds preferentially stimulate the ERC. over (NSB), non-specific binding: (“SB), specific binding. the ERR such that the ratio of RBA/RBA is greater than Each data point in panels A-F was the mean of duplicate about 1. measurementS. I0028 FIG.2 shows competition of the binding of HIE, 0037. In one aspect, the formulation comprises a mixture to human ERC. and ERB by various catechol estrogens and of at least three estrogenic compounds and/or pharmaceuti methoxyestrogens. The conditions for the in vitro ER binding cally acceptable conjugates. In another aspect, the formula assay were described in detail herein. The concentration of tion comprises a mixture of at least five estrogenic com the radioactive ligand HE was 10 nM, and the concentra pounds and/or their pharmaceutically acceptable conjugates. tions of the competing estrogens were 0, 0.24, 0.98, 3.9, 15.6, Especially preferred estrogenic compounds are estrone (E), 62.5, 250, and 100 nM. Each data point was the mean of 17o-estradiol (17C-E), 2-hydroxyestrone (2-OH-E), duplicate measurements. 2-methoxyestrone (2-MeO-E), and 2-methoxyestradiol 0029 FIG. 3 shows the competition of the binding of (2-MeO-E), and pharmaceutically acceptable salts or pro 3HE to human ERC. and ERB by several other A-ring ana drugs thereof, including their Sulfated or glucuronidated con logs (most of them are synthetic analogs). The conditions for jugates. The structures of these preferred compounds are the receptor binding assay were the same as described in the below: legend to FIG. 2. 0030 FIG. 4 shows the competition of the binding of 3HE to human ERC. and ERB by several B-ring and C-ring (1) substitution metabolites or derivatives. The conditions for the receptor binding assay were the same as described in the legend to FIG. 2. 0031 FIG. 5 shows the competition of the binding of 3HE to human ERC. and ERB by several B-ring and C-ring dehydroestrogen metabolites or derivatives. The conditions for the receptor binding assay were the same as described in the legend to FIG. 2. HO 0032 FIG. 6 shows the competition of the binding of Estrone 3HE to human ERC. and ERB by several D-ring metabolites (2) or derivatives. The conditions for the receptor binding assay were the same as described in the legend to FIG. 2. Note that for 17-desoxy-E and 1,3,5(10), 16-estratetraen-3-ol, two more lower concentrations (0.015 and 0.06 nM) were also assayed. 0033 FIG. 7 shows the competition of the binding of 3HE to human ERC. and ERB by several , , and stilbene estrogens. The conditions for the receptor binding assay were the same as described in the 17 ot-estradiol legend to FIG. 2. (3) DETAILED DESCRIPTION OF PREFERRED EMBODIMENT 0034. The present invention is directed to a novel hormone CHO replacement therapy formulation, and method of using the hormone replacement therapy formulation, to treat diseases, disorders, and conditions associated with low estrogen levels HO in non-pregnant women, especially post-menopausal and 2-methoxyestrone peri-menopausal women. US 2008/0293683 A1 Nov. 27, 2008

ERB over the ERC. more than those in the normal pre-meno -continued pausal non-pregnant human female. In a preferred aspect, the (4) HO hormone replacement therapy compositions do not contain H any estrogenic compounds that preferentially stimulate the ERB over the ERC. 0043. The term “consists of or “consisting of indicates that only the recited estrogenic compounds or steps are CHO present, but does not foreclose the possibility that equivalents of the ingredients or steps can Substitute for those specifically recited. HO 0044) The term “menopause' is used throughout the speci 2-methoxyestradiol fication to describe the period in a woman's life between the (5) ages of approximately 45 and 50 (but not always) after which O O menstruation (menses) naturally ceases. The symptomology associated with menopause which is particularly relevant to the present invention includes bone loss associated with osteoporosis, for example. HO 0045. The terms peri-menopausal refers to that time in a women's life between pre-menopause (the reproductive years) and post-menopause. This time period is usually HO between the ages of 40-60, but more often several years on 2-hydroxyestrone either side of 45 to 50 years of age. This period is character ized by a rapid change in the hormonal balance in a woman. The hallmark of the ending of the peri-menopausal period and 0038. The estrogenic compounds are preferably in the the beginning of the post-menopausal period is the cessation form of conjugated estrogens, which function as prodrugs. of ovarian function or its inability to regulate the previously Other pharmaceutically acceptable prodrugs may also be normal cycle in the woman. This cessation of func used. The conjugates may be any Suitable conjugate known tion is clinically marked by the cessation of menses of a by those skilled in the art, including, but not limited to, period of one year or more. The time period over which this glucuronide and Sulfate. The estrogenic compounds may also cessation of ovarian function persists, i.e., the peri-meno be present as pharmaceutically acceptable salts of the conju pausal time, is usually not a Sudden or rapid event. The peri gated estrogens. The pharmaceutically acceptable salts may menopausal state can last from a few months to more typi be various salts understood by those skilled in the art, includ cally a year or more. ing, but not limited to, sodium salts, calcium salts, magne 0046. The term “patient is used throughout the specifica sium salts, lithium salts, and amine salts such as piperazine tion to describe an animal, preferably a human, to whom salts. The most preferred salts are sodium salts. treatment, including prophylactic treatment, with the estro 0039. The estrogenic compounds are administered in a genic compound formulation according to the present inven therapeutically effective amount to treat the specified condi tion is provided. For treatment of the symptomology, condi tion, for example in a daily dose preferably ranging from tions, or disease states which are specific for a specific animal about 1 to about 1000 mg per day, and more preferably about Such as a human patient, the term patient refers to that specific 5 to about 200 mg per day, given in a single dose or 2-4 animal. In most instances in the present invention, the patient divided doses. The plasma concentration for each of the estro is a human female exhibiting symptomology associated with genic compounds preferably ranges between about 10 and 50 menopause. While patients of the present invention are pref pg/ml. The exact dose, however, is determined by the attend erably post-menopausal woman, it will be appreciated that ing clinician and is dependent on Such factors as the potency estrogen replacement can be prescribed in other circum of the compound administered, the age, weight, condition, stances where other causes account for a decline in estrogen and response of the patient. production or ifestrogen is produced at a lower than desirable 0040. The term “co-administered” means the administra level. This could occur in women not yet in menopause. tion of the selected estrogenic compounds (or other agents, 0047. The term “pharmaceutically acceptable' is Such as progestins) to a Subject by combination in the same employed herein to refer to those compounds, materials, pharmaceutical composition or separate pharmaceutical compositions, and/or dosage forms which are, within the compositions. Thus, co-administration involves administra Scope of sound medical judgment, Suitable for use in contact tion at the same time of a single pharmaceutical composition with the tissues of human beings and animals without exces comprising the estrogenic compounds or administration of sive toxicity, irritation, allergic response, or other problem or two or more different compositions to the same subject at the complication, commensurate with a reasonable benefit/risk same or different times. ratio. 0041. The terms “comprising or “having indicate that 0048. The term “prodrug means a covalently-bonded any estrogenic compounds or steps can be present in addition derivative or carrier of the parent estrogenic compound which to those recited in the hormone replacement therapy formu undergoes at least Some biotransformation prior to exhibiting lations and methods. its pharmacological effect(s). In general. Such prodrugs have 0042. The term “consists essentially of or “consisting metabolically cleavable groups and are rapidly transformed essentially of indicates that unlisted ingredients or steps that to yield the parent compound, for example, by do not materially affect the basic and novel properties of the in blood. The prodrug is formulated with the invention can be employed in addition to the specifically objectives of improved chemical stability, improved patient recited estrogenic compounds. Typically, this means that the acceptance and compliance, improved bioavailability, pro hormone replacement therapy does not contain estrogenic longed duration of action, improved organ selectivity, compounds in an amount that preferentially stimulates the improved formulation (e.g., increased hydrosolubility), and/ US 2008/0293683 A1 Nov. 27, 2008 or decreased side effects (e.g., toxicity). In general, prodrugs magnesium carbonate, magnesium Stearate, talc, Sugar, lac themselves have weak or no biological activity and are stable tose, ethanol, glycerol, water, and the like. The amount of under ordinary conditions. Prodrugs can be readily prepared carrier in the formulation can range from about 5 to about 99 from the parent compounds using methods known in the art, weight percent of the total weight of the treatment composi such as those described in A Textbook of Drug Design and tion or therapeutic combination. Non-limiting examples of Development, Krogsgaard-Larsen and H. Bundgaard (eds.), Suitable pharmaceutically acceptable excipients and addi Gordon & Breach, 1991, particularly Chapter 5: “Design and tives include non-toxic compatible fillers, binders such as Applications of Prodrugs': Design of Prodrugs, H. Bund starch, polyvinyl pyrrolidone or cellulose ethers, disinte gaard (ed.), Elsevier, 1985, Prodrugs. Topical and Ocular grants such as Sodium starch glycolate, crosslinked polyvinyl Drug Delivery, K. B. Sloan (ed.), Marcel Dekker, 1998, pyrrolidone or croScarmellose sodium, buffers, preservatives, Methods in Enzymology, K. Widder et al., (eds.), Vol. 42, Academic Press, 1985, particularly pp. 309-396, Burger's anti-oxidants, lubricants, flavorings, thickeners, coloring Medicinal Chemistry and Drug Discovery, 5th Ed., M. Wolff agents, wetting agents such as Sodium lauryl Sulfate, emulsi (ed.), John Wiley & Sons, 1995, particularly Vol. 1 and pp. fiers, and the like. The amount of excipient or additive can 172-178 and pp. 949-982, Pro-Drugs as Novel Delivery Sys range from about 0.1 to about 95 weight percent of the total tems, T. Higuchi and V. Stella (eds.), Am. Chem. Soc., 1975; weight of the treatment composition or therapeutic combina and Bioreversible Carriers in Drug Design, E. B. Roche (ed.), tion. One skilled in the art would understand that the amount Elsevier, 1987, each of which is incorporated herein by ref of carrier(s), excipients, and additives (if present) can vary. erence in their entireties. Further examples of pharmaceutically acceptable carriers and 0049. The term “therapeutically effective amount” is methods of manufacture for various compositions can be understood to mean a sufficient amount of an estrogenic found in A. Gennaro (ed.), Remington. The Science and Prac compound(s) or composition that will positively modify the tice of Pharmacy, 20th Edition, (2000), Lippincott Williams symptoms and/or condition to be treated. The therapeutically & Wilkins, Baltimore, Md., which is periodically updated. effective amount can be readily determined by those of ordi 0053 Useful solid form preparations include powders, nary skill in the art, but of course will depend upon several tablets, dispersible granules, capsules, cachets, and Supposi factors. For example, one should consider the condition and tories. Useful liquid form preparations include Solutions, Sus severity of the condition being treated, the age, body weight, pensions, and emulsions. As an example may be mentioned general health, sex, diet, and physical condition of the patient water or water-propylene glycol solutions for parenteral being treated, the duration of the treatment, the nature of concurrent therapy, the particular active ingredient being injection or addition of Sweeteners and opacifiers for oral employed, the particular pharmaceutically-acceptable solutions, suspensions, and emulsions. Liquid form prepara excipients utilized, the time of administration, method of tions may also include Solutions for intranasal administration. administration, rate of , drug combination, and any 0054 Aerosol preparations suitable for inhalation may other relevant factors. include solutions and Solids in powderform, which may be in 0050. The estrogenic compounds are preferably adminis combination with a pharmaceutically acceptable carrier, Such tered to the patient in a continuous uninterrupted fashion. In as an inert compressed gas, e.g. nitrogen. one aspect, the frequency of administration is at least once 0055 Also useful are solid form preparations which are daily. The term “continuous” as applied in the specification intended to be converted, shortly before use, to liquid form means that the dosage is administered at least once daily. The preparations for either oral or parenteral administration. Such term “uninterrupted means that there is no break in the liquid forms include Solutions, Suspensions, and emulsions. treatment, and that the treatment is administered at least once 0056. The compounds of the invention may also be deliv daily in perpetuity until the entire treatment is ended. erable transdermally. The transdermal compositions can take 0051 Techniques for preparing the formulations compris the form of creams, lotions, aerosols, and/or emulsions can be ing the estrogenic compounds of the present invention are set included in a transdermal patch of the matrix or reservoir type forth, for example, in Huber et al., U.S. Pat. No. 5,908.638 as are conventional in the art for this purpose. Preferably, the entitled "Pharmaceutical compositions of conjugated estro compound is administered orally. gens and methods for their use': Potter et al., U.S. Pat. No. 0057. In one aspect, the delivery vehicle or formulation of 6,326,366 entitled “Hormone Replacement Therapy': Hoch the invention preferably provides for administration of estro berg, U.S. Pat. No. 6,476,012 entitled “Estradiol-16C.-Car genic composition by an oral. Subcutaneous, intravenous, boxylic Acid as Locally Active Estrogens: Luo et al., intramuscular, intraperitoneal, intrabuccal, vaginal, or trans U.S. Pat. No. 6,562,370 entitled “Transdermal Administra dermal route. Preferably, the carrier vehicle or device for each tion of Steroid Drugs. Using Hydroxide-Releasing Agents as component is selected from a wide variety of materials and Permeation Enhancers': Casper et al., U.S. Pat. No. 6,747, devices which are already known perse or may hereafter be 019 entitled “Low Dose Estrogen Interrupted Hormone developed which provide for controlled release of the com Replacement Therapy'. Lanquetin et al., U.S. Pat. No. 6,831, positions in the particular physiological environment. In par 073 entitled “Hormonal Composition Consisting of an Estro ticular, the carrier vehicle of the delivery system is selected gen Compound and of a Progestational Compound: Hill et such that near Zero-order release of the components of the al., U.S. Pat. No. 6,992,075 entitled “C(14) Estrogenic Com regimen is achieved. In the context of the present invention, pounds” which are incorporated by reference. The hormone the carrier vehicle should therefore also be construed to replacement therapy compositions may further comprise one embrace particular formulations of the compositions which or more pharmaceutically acceptable carriers, one or more are themselves suitable for providing near Zero-order release. excipients, and/or one or more additives. The hormone A targeted steady-state release can be obtained by suitable replacement therapy compositions may comprise about 1 to adjustment of the design or composition of the delivery sys about 99 weight percent of the estrogenic compounds. tem. Known devices Suitable for use as a delivery system in 0052. Useful pharmaceutically acceptable carriers can be accordance with the present invention include, for example, Solid, liquid, or gas. Non-limiting examples of pharmaceuti drug-delivery pump devices providing near Zero-order cally acceptable carriers include Solids and/or liquids such as release of the components of the regimen. US 2008/0293683 A1 Nov. 27, 2008

0058. The following examples are provided to illustrate 0060. In this example, endogenous E and E. metabolites, the present invention and are not intended to limit the scope along with Some of their synthetic analogs and phytoestro thereof. gens (structures shown in below in Table 1), were compared for their binding affinities for human ERC. and ERB. The Example 1 recombinant human ERS used in the present study were pro Determination of Estrogen Receptor Activation duced in a baculovirus expression system that yielded 0059. This example is set forth in Zhu et al., Ouantitative soluble, functionally-active recombinant ER proteins with Structure-Activity Relationship of Various Endogenous post-translational modification patterns (mainly phosphory Estrogen Metabolites for Human Estrogen Receptor C. and B lations and acetylations) similar to those found in mammalian Subtypes: Insights into the Structural Determinants Favoring cells. See Reid etal. Human estrogen receptor-C.: Regulation a Differential Subtype Binding, Endocrinology 147, 4132 by synthesis, modification and degradation, Cell. Mol. Life. 4150 (2006), which is incorporated by reference. Sci. 59,821-831 (2002).

TABLE 1.

Structure of E2 and Various Natural or Synthetic Estrogens

18 OH

HO Estradiol-17B (E)

O OH C)

HO O HO D-Equilenin 17 B-Dihydroequilenin City

HO -CIO 17-Desoxyestradiol 1,3,5(10), 16-Estratetraen-3-ol

OH OH

21

O HO

HO Dibenzoylmethane US 2008/0293683 A1 Nov. 27, 2008

TABLE 1-continued

Structure of E and Various Natural or Synthetic Estrogens

HO O O HO O

O O OH OH

OH

4.

ICI-182,780

OH OH

& w HO (CH3)6-OH HO (CH3)6-d

Raloxifene

Tamoxifen CSC OH CO OH HO HO US 2008/0293683 A1 Nov. 27, 2008

TABLE 1-continued Structure of E and Various Natural or Synthetic Estrogens OH HO O

OH

HO O OH OH O OH OH

OH O

0061 Chemicals and Reagents 0066. On the day of performing the ER binding assay, 0062) E, E, and most of their metabolites and derivatives 3HE solution was freshly diluted in the binding buffer, and listed in Table 2 were obtained from Steraloids (Newport, an aliquot (45 uL) of the HIE solution was added to a 1.5 R.I.). 7O-(6-Hydroxyhexanyl)-17? 3-estradiol E-7C-(CH2) mL microcentrifuge tube, giving a final HE concentration OH and 7.O-(6-benzyloxyhexanyl)-17 B-estradiol E-7C.- at 10 nM. Each of the competing ligands (in 50 u, volume) (CH2)OCHs were chemically Zed according to Jiang et al., was then added to the mixture for the intended final concen Synthesis of 7O-substituted derivatives of 17 B-estradiol, Ste trations at 0, 0.24, 0.98, 3.9, 15.6, 62.5, 250, and 1000 nM. roids 71 334-342 (2006). Dithiothreitol, glycerol, and Tris Note that all of the estrogens were initially dissolved in pure ethanol to a stock concentration of 1 mM, and then further HCl were obtained from the Sigma Chemical Co. (St. Louis, diluted to 100LLM with 20% aqueous ethanol. In this way, the Mo.). Hydroxylapatite and bovine serum albumin (BSA) final ethanol concentration in the incubation mixture was less were obtained from Calbiochem (through EMD Biosciences, than 0.2%. Immediately before the addition of the ERC. or Inc. San Diego, Calif.). (2,4,6,7,16,17-HIE (specific activ ERB protein, it was diluted in the binding buffer and mixed ity of 115 Ci/mmol) was obtained from NEN Life Sciences gently with repetitive pipettings. An aliquot (5 LL) of the (Boston, Mass.), and it was purified in our laboratory using a diluted ERC. or ERB solution was precisely added to the high-pressure liquid chromatography (HPLC)-based method mixture containing 45 uL of the HIE, and 50 uL of the prior to its use in the in vitro receptor binding assays. See Lee competing ligand, giving a final receptor concentration of 1-2 et al. Characterization of the oxidative metabolites of 17 B fmol/mL. The incubation mixture was then mixed gently and estradiol and estrone formed by 15 selectively expressed thoroughly with repetitive pipettings. Nonspecific binding human cytochrome p450 isoforms, Endocrinology 144,3382 (NSB) by the HE was determined in separate tubes by 3398 (2003). inclusion of a 400-fold concentration of the nonradioactive E. 0063. The recombinant human ERC. and ERB proteins and (at a final concentration of4 uM). Based on UV spectrometric bovine serum albumin (BSA) were obtained from PanVera monitoring of E in water, this estrogen at 4LM concentration Corporation (Madison, Wis.). According to the supplier, the (the highest steroid concentration used) appeared to be recombinant human ERC. and ERB were produced in a bacu readily soluble in the aqueous solution. The binding mixture lovirus-mediated expression system, and they were soluble was incubated at room temperature for two hours. At the end and functionally active, with post-translational modifications of the incubation, 100 uL of the hydroxylapatite slurry was similar to those found in mammalian cells. See Reid et al., added to each tube and the tubes were incubated on ice for 15 Human estrogen receptor-C.: Regulation by synthesis, modi minutes with 3 times of brief vortexing. An aliquot (1 mL) of fication and degradation, Cell. Mol. Life. Sci. 59,821-831 the appropriate washing buffer was added, mixed, and cen (2002). trifuged at 10,000 g for five minutes, and the supernatants 0064 ERC. and ERB Binding Assays were discarded. This wash step was repeated twice. Hydroxy 0065. The following buffer solutions were used in the ER lapatite pellets were then resuspended in 200 uL ethanol binding assays, and they were prepared beforehand and (followed by another rinse with 200 uLethanol), and then the stored at 4°C. The binding buffer consisted of 10% glycerol, content was transferred to Scintillation vials (containing 4 mL 2 mM dithiothreitol, 1 mg/mL BSA and 10 mM Tris-HCl at of the scintillation fluid) for measurement of H-radioactivity pH 7.5. The ERC. washing buffer contained 40 mM Tris-HCl with a liquid scintillation counter (Packard Tri-CARB 2900 and 100 mM KCl (pH 7.4), but the ERG washing buffer TR: Downers Grove, Ill.). contained only 40 mM Tris-HCl (adjusted to pH 7.4). The 0067. To calculate the specific binding (pmol/mL) of the 50% hydroxylapatite slurry was prepared first by vigorously human ERC. or ERB protein at each concentration point, the mixing 10 g hydroxylapatite with 60 mL of the Tris-HCl following equation was used: solution (50 mM, pH 7.4). Hydroxylapatite was then allowed to settle for 20 minutes at room temperature, and the Super natant was decanted. The above procedures were repeated 10 times, and afterwards hydroxylapatite was kept in the 50 mM ERa or ERf8 = Tris-HCl solution overnight at 4°C. Hydroxylapatite slurry (d. p.m. for total binding- d. p.m. for NSB)x dilution factor was then adjusted to an approximate final concentration of final volume of the mixturex (d. p.m. f.pnol 3 HE) 50% (v/v) using the same Tris-HCl solution and stored at 4 C., and the slurry was stable for up to several months. US 2008/0293683 A1 Nov. 27, 2008

0068. The ICs value for each competing estrogen was sponding ICso value will also increase. Because the radioli calculated according to the sigmoidal inhibition curve, and gand HE concentration used in this study was 10 nM the relative binding affinity (RBA) was calculated against E. (which was 10-100 times higher than the previously-reported using the following equation: K, values for human ERC), the absolute ICs values would also be higher if they were compared with values reported in some of the earlier studies when lower concentrations of the Cso for E RBA = - Y - - radioligand were used. The reason that a higher concentration Cso for the test compound of HIE was used was simply because it would yield more reproducible readings of the radioactivity counts. Since the 0069. It should be noted that the absolute ICs values are RBA value is a parameter that is independent of the radioli affected by the concentrations of the radioligand (HE) gand concentration used, we thus have placed more emphasis used. When a lower radioligand concentration is used, the on the RBA values instead of the absolute ICs values in corresponding ICso value will also be relatively lower, but interpreting the physiological meanings of the data from in when the radioligand concentration increases, the corre vitro receptor competition assays.

TABLE 2 The ICso and RBA values of various hydroxylated, keto, and dehydrogenated metabolites of E2 and E as well as some other natural or synthetic derivatives for the recombinant human ER, and ER. ERC ERB Figure ICso ICso RBA, RBA/ Chemical names Abbreviations No. (nM) RBA,% (nM) (%) RBAp Estradiol-17B (or Estradiol) E. FIG. 1A 11.2 100 8.9 100 1 Estrone E1 FIG. 1B 112.2 10 446.7 12 5 A-RING METABOLITES

1- 1-Methyl-E2 FIG. 3C 79.4 14 112.2 8 1.8 2-Aminoestrone 2-NH2-E FIG. 3 398.1 3 2S11.9 0.4 7.5 2-Nitroestrone 2-NO.-E. FIG.3H 1584.9 1 ND ND ND 2-Hydroxyestrone 2-OH-E FIG. 2A 316.2 4 1995.3 0.4 10 2-Hydroxyestradiol 2-OH-E- FIG. 2B SO.1 22 25.1 35 O.6 2-Hydroxyestriol 2-OH-E. FIG. 2C SO1.2 2 794.3 2 2-Methoxyestrone 2-MeO-E. FIG. 2F ND ND ND ND ND 2-Methoxyestradiol 2-MeO-E, FIG. 2 SO1.2 2 631.1 2 2-Ethoxyestradiol 2-Ethoxy-E, FIG.3E ND ND ND ND ND Estrone 2,3-dimethyl ether 2,3-diMeO-E. FIG.3F ND ND ND ND ND 17B-Estradiol 2,3-dimethyl ether 2,3-diMeO-E, FIG. 3G ND ND ND ND ND 2-Hydroxyestrone 3-methyl ether 2-OH-3-MeO-E. FIG. 2G ND ND ND ND ND 2-Hydroxyestradiol 3-methyl ether 2-OH-3-MeO-E, FIG. 2 ND ND ND ND ND 2-Bromoestradiol 2-Br-E2 FIG. 3A 281.1 4 2S11.1 0.4 10 4-Aminoestrone 4-NH.-E. FIG. 3K 3S4.8 3 ND ND ND 4-Nitroestrone 4-NO-E, FIG. 3 446.7 3 ND ND ND 4-Hydroxyestrone 4-OH-E FIG. 2D 708.1 2 708.1 2 4-Hydroxyestradiol 4-OH-E- FIG. 2E 15.9 70 15.9 56 1.3 4-Methylestradiol 4-Methyl-E, FIG. 3D 125.9 9 25.1 35 O.3 4-Methoxyestrone 4-MeO-E. FIG. 2H ND ND ND ND ND 4-Methoxyestradiol 4-MeO-E, FIG. 2K 708.1 2 891.3 2 4-Methoxyestriol 4-MeO-E. FIG. 2L 1258.1 1 631.1 1 4-Bromoestradiol 4-Br-E, FIG. 3B 158.5 7 25.1 35 O.2 Estrone 3-sulfate E-3-sulfate FIG. 3L ND ND ND ND ND Estradiol 3-sulfate E-3-sulfate FIG. 3M ND ND ND ND ND B-RING METABOLITES (C-6) 6-Ketoestrone 6-Keto-E FIG. 4C 489.8 2 891.3 2 6C-Hydroxyestradiol 6C-OH-E FIG. 4A 199.5 6 251.2 4 1.5 6B-Hydroxyestradiol 6?-OH-E- FIG. 4B 794.3 1 S62.3 2 O.S 6-Ketoestradiol 6-Keto-E2 FIG. 4D 15.9 71 15.9 56 1.3 6-Ketoestriol 6-Keto-Es FIG. 4E 44.7 25 3.16.2 3 8.3 6-Ketoestradiol-17C. 6-Keto-E-17C. FIG. 4F S62.3 2 1122.1 1 2 6-Dehydroestrone 6-Dehydro-E FIG. SA 1OOO.1 1 SO1.2 2 O.S 6-Dehydroestradiol 6-Dehydro-E2 FIG.SB 22.4 50 10.2 89 O.1 7-Dehydroestrone () 7-Dehydro-E FIG. 6C 251.2 4 70.8 13 O.3 7-Dehydroestradiol (17B 7-Dehydro-E, FIG. 6D 7.9 142 7.9 113 1.3 Dihydroequilin) 7-Dehydroestradiol-17C (17C 7-Dehydro-E-17C. FIG. 6 63.1 18 63.1 14 1.3 Dihydroequilin) 9(11)-Dehydroestrone 9(11)-Dehydro-E FIG. 6E 223.9 5 1413 6 O.8 9(11)-Dehydroestradiol 9(11)-Dehydro-E FIG. 6F 12.9 64 7.5 119 O.S D-Equilenin D-Equilenin FIG. 6G S62.3 2 125.9 7 O.3 17B-Dihydroequilenin 17B- FIG. 6H 31.6 35 8.9 100 0.4 Dihydroequilenin US 2008/0293683 A1 Nov. 27, 2008 10

TABLE 2-continued The ICso and RBA values of various hydroxylated, keto, and dehydrogenated metabolites of E2 and E as well as some other natural or synthetic derivatives for the recombinant human ER, and ER. ERC ERB Figure ICso ICso RBA, RBA, Chemical names Abbreviations No. (nM) RBA,% (nM) (%) RBA C-RING METABOLITES (C-11) 1C-Hydroxyestrone 1C-OH-E FIG. 4G ND ND ND ND ND 1B-Hydroxyestrone 1B-OH-E FIG. 4H ND ND ND ND ND 1-Ketoestrone 1-Keto-E FIG. 4 ND ND ND ND ND 1C-Hydroxyestradio 1C-OH-E FIG. 4 ND ND ND ND ND 1B-Hydroxyestradio 1?-OH-E- FIG. 4K ND ND ND ND ND 7B-Estradiol 11-acetate 1-Acetate-E- FIG. 4L 2O1 56 19.9 45 1.2 1B-Methoxyethynylestradiol 1B-MeO-EE. FIG. 4M 31.6 35 44.7 2O 1.8 D-RING METABOLITES

5C-Hydroxyestriol (Estetrol) 5C-OH-E. FIG. 6F 281.8 4 3S4.8 3 1.3 6C-Hydroxyestrone 6C-OH-E FIG. 6A 56.2 2O 25.1 35 O.1 6-Ketoestrone 6-Keto-E FIG. 6B 631.1 2 89.1 10 O.2 6C-Hydroxyestradiol (Estriol) 6C-OH-E (E) FIG. 1C 1OO 11 25.1 35 O.3 FIG. 6C 6B-Hydroxyestradiol (16- 6B-OH-E2 FIG. 6D 17.8 63 17.8 50 1.3 Epiestriol) 6-Ketoestradio 6-Keto-E, FIG. 6E 1122 1 SO.1 18 O.1 6C-Hydroxyestradiol-17C (16, 6C-OH-E-17C. FIG. 6H 15.8 71 11.2 79 O.9 7-Epiestriol) 6B-Hydroxyestradiol-17C. 6B-OH-E-17C. FIG. 6 1258.9 1 70.8 13 O.1 (16,17-Epiestriol) Estradiol-17C. E-17C. FIG. 1D SO-1 22 281.8 3 7.3 FIG. 6G 7C-Ethynylestradiol 7C-EE FIG. 6 S.6 2OO 15.9 56 3.6 7-Desoxyestradio 7-Desoxy-E, FIG. 6K 70.8 16 19.9 45 0.4 3,5(10), 16-Estratetraen-3-ol 6-Estratetraen FIG. 6L 31.6 35 14.1 8O 0.4 SOME OTHER ANALOGS

CI-182,780 CI-182,780 FIG. 7A 25.1 45 25.1 35 1.3 7C-(6-Hydroxyhexanyl)-17B- E-7C-(CH2)6OH FIG. 7B 4.1.8 27 22.3 40 0.7 estradiol 7C-(6-Benzyloxyhexanyl)-17B- E-7C-(CH2)OCHs FIG.7C 39.8 28 63.1 14 2.O estradiol Tamoxifen Tamoxifen FIG. 7D 3S4.8 3 251.2 4 O.8 Railoxifene FIG.7E 22.4 50 63.6 14 3.6 Genistein Genistein FIG.7F 1995 6 11.2 79 O.1 Coumestrol Columestrol FIG. 7G SO2 22 25.1 35 O.6 Myricetin Myricetin FIG. 7H ND ND ND ND ND Daidzein Daidzein FIG.7 1OOO O.1 631 2 O.OS Dibenzoylmethane DBM FIG.7 ND ND ND ND ND Diethylstilbestrol DES FIG. 7K 11.2 1OO 5.4 166 O.6 Dienestrol Dienestrol FIG. 7L 30.2 37 1995 56 0.7 Hexestrol Hexestrol FIG. 7M 36.3 31 18.62 60 O.S

0070 The “ND” indicates that the corresponding ICs HE as ligand, the apparent K values was also determined values could not be determined because the maximal inhibi for the recombinant human ERC. and ERB (FIG. 1E, 2F). tion of the receptor binding at the highest concentration tested Based on curve regression analysis of the receptor binding (namely, 1000 nM) did not reach 50%. However, it will be data, the K, of E for human ERC. was 0.7nM, and its K, for appreciated that the RBA/RBA for some compounds may ERB was 0.75 nM. The values are slightly higher than some of be determined by extrapolating the curves set forth in FIGS. the earlier measurements (average about 0.3 nM) using the crude ER protein preparations from various human tissues or 3-7. For example, it was determined that the RBA/RBA for cell lines. See Katzenellenbogen et al. In vivo and in vitro 2-methoxyestrone, 2-nitorestrone, and 4-nitroestrone, was steroid receptor assays in the design of estrogen pharmaceu about 1, 3, and 10, respectively. ticals, In: Eckelman W C (editor), Receptor-Binding 0071 Binding Affinities of E, E, and E for Human ERC. Radiotracers 1, CRC, Boca Raton, Fla., pp. 93-126 (1982); and ERB Fishman, Biological action of catecholestrogens, J. Endocr. 0072 E (estrone), E, (estradiol-17B), and Es (estriol or 85, 59P-65P (1981). This difference likely was due to the 16C.-OH-E) are three well-known human estrogens. Among relatively higher concentration of the recombinant ERC. and all estrogens analyzed in this study, E was found to have ERB proteins used in our in vitro receptor binding assays, and nearly the highest binding affinity for both ERC. and ERB, and perhaps also due to the absence of other cellular proteins or its binding affinities for these two ER subtypes were very components that usually partner the steroid receptors in Sub similar (FIG. 1A, Table 2). Using different concentrations of cellular crude extracts or in vivo. US 2008/0293683 A1 Nov. 27, 2008

0073 E had 10% of the binding affinity of E for human over ERB. Taking together the ER-binding data for E and ERC, and had 2% of the affinity ofE for ERB (FIG. 1B, Table 2-OH-E, it is interesting to see that these two quantitatively 2). E also had markedly diminished binding affinity for ERC. predominant estrogens normally present in non-pregnant compared to E (RBA 10% of E), but it had rather high woman would consistently produce a preferential activation binding affinity for ERB (RBA35% ofE) (FIG.1C, Table 2). of ERO. over ERB. For comparison, the binding affinity of E-17C. (a C-17 iso (0079. Different from 2-OH-E, 4-OH-E is known to mericanalog of E) was determined for human ERC. and ERB. retain strong estrogenic activity and high ER binding affinity, While E-17C. retained considerable binding affinity for and the data from this example also showed that this catechol human ERC. (RBA 22% of E), its binding affinity for ERB E. metabolite retained high and almost identical binding was much lower (RBA only 3% of E) (FIG. 1D, Table 2). affinity for ERC. and ERB, with RBAS 70% and 56% of E. Notably, the relative binding affinities and binding preference respectively (FIG. 2E, Table 2). In comparison, 2-OH-E and of E-17C. for human ERC. and ERB mirror those of E. 4-OH-E (a quantitatively-minor metabolite) each had mark 0074. Notably, E and E are perhaps the two best known edly weaker binding affinity for ERC. and ERB. While 4-OH metabolites of E inhumans. Although these two endogenous E had almost identical binding affinity for ERC. and ERB E. derivatives had markedly lower binding affinities for (FIG. 2D), 2-OH-E (the quantitatively-predominant endog human ERC. and ERB than E. (FIG.1), it is of interest to point enous oxidative metabolite of E) had a substantially higher out that the facile metabolic conversion of E to E or of E to affinity for ERC. than for ERB (FIG. 2A). 2-OH-E had weak E. in a woman may confer differential activation of the ERC. and similar binding affinity for ERC. and ERB (FIG. 2C). or ERB signaling system under different physiological con ditions. For instance, E had 4-fold higher relative binding 2- or 4-Methoxyestrogens affinity for human ERC. than for ERB, and this estrogen metabolite is present in larger quantities than E in circulation 0080 All of the monomethylated catechol-E metabolites as well as in most tissues of a non-pregnant woman, largely tested in this study (2-MeO-E, 2-OH-3-MeO-E, and due to the actions of high levels of the oxidative 17 B-hydrox 4-MeO-E) did not have appreciable binding affinity for ysteroid dehydrogenase(s). Hence, the facile metabolic con human ERC. and ERB at concentrations up to 1000 nM (FIGS. version of E to E would effectively produce a preferential 3F, 3G, 3H). However, the two major monomethylated cat activation of the ERC. signaling system over the ERB system echol-E metabolites (2-MeO-E, and 4-MeO-E.) each in most target tissues of a non-pregnant woman. In contrast, retained weak but similar binding affinities for both ERC. and ERB (FIGS. 2I, 3K and Table 2), with RBAS 1-2% of E.The E, has a more than five-fold preference for the activation of estimated binding affinities are considerably higher than ear human ERB over ERC, and it is a quantitatively-predominant lier measurements using prepared from human estrogen metabolite produced during . It is of inter . The weak ER-binding activity of 2-MeO-E, is est to suggest that the very high levels of E. present during believed to be mainly responsible for its moderate growth pregnancy may produce a differential activation of the ERB stimulatory effect in ER-positive human breast cancer cells signaling system in the pregnant woman and fetus for fulfill when exogenous estrogens were not present. See Liu et al., ing various unique physiological functions. Concentration-dependent mitogenic and antiproliferative 0075 A-Ring Metabolites. actions of 2-methoxyestradiol in estrogen receptor-positive 0076 Catechol Estrogens. human breast cancer cells, J. Steroid. Biochem. Mol. Biol. 0077 2-OH-E is the most abundant hydroxy-E metabo 88,265-275 (2004). In comparison, 2-OH-3-MeO-E. (a close lite formed in human liver. Largely because of its rather low structural analog of 2-MeO-E) had a substantially weaker estrogenic activity as measured earlier in laboratory animals binding affinity for ERC. and ERB than 2-MeO-E. (FIG. 2J). (ovariectomized or immature rats or mice) and also in cul 4-MeO-E also retained weak but similar binding affinity for tured human breast cancer cells, this catechol-E metabolite ERC. and ERB (FIG. 2L), and its affinity was comparable to was generally considered to have a very weak estrogenic those of 2-MeO-E, and 4-MeO-E. activity in human. It has been widely accepted the notion that I0081 2-Ethoxy-E is an analog of 2-MeO-E, with strong increased metabolic formation of 2-OH-E in viva as opposed anticancer activity (29, 30). See Wang et al., Synthesis of to the formation of other oxidative metabolites such as 4-OH B-ring homologated estradiol analogues that modulate tubu E. 16C.-OH-E, or 16-OH-E (E), would significantly lin polymerization and microtubule stability, J. Med. Chem. reduce estrogen's hormonal activity in human and thus would 43, 2419-2429 (2000); Cushman et al., Synthesis, antitubulin be beneficial for the reduction of breast cancer risk. In the and antimitotic activity, and cytotoxicity of analogs of present experiments, 2-OH-E had comparable binding affin 2-methoxyestradiol, an endogenous mammalian metabolite ity for ERC. and ERB, and its RBAs for ERC. and ERB were of estradiol that inhibits tubulin polymerization by binding to 22% and 35%, respectively, of E. (FIG. 2B, Table 2). The the colchicine binding site, J. Med. Chem. 38, 2041-2049 assays were repeated twice, and highly consistent results (1995). The compound retained a weak binding affinity for were obtained. ERC. and ERB (FIG. 3E), and its affinity is slightly weaker 0078. Despite its relatively high ER binding affinity, than 2-MeO-E, probably due to the bulkier size of the ethoxy 2-OH-E, may still be a highly beneficial metabolite of E in group at the C-2 position compared to a methoxy group. human owing to its rapid metabolic O-methylation in vivo I0082 Some Other A-Ring Analogs. which deactivates its hormonal activity and also concomi I0083. The binding affinities of several semi-synthetic tantly forms the anticarcinogenic 2-MeO-E. See Zhu et al., A-ring derivatives of E (data shown in FIG. 3) were also Functional role of estrogen metabolism in target cells. compared. Notably, some earlier studies have Suggested that Review and perspectives, Carcinogenesis 19, 1-27 (1998); Substitution of Small functional groups at the C-2 and C-4 Zhu et al., Is 2-methoxyestradiol an endogenous estrogen positions are reasonably well tolerated, whereas largergroups metabolite that inhibits mammary carcinogenesis, Cancer may readily reduce ER binding affinity because they may Res. 58,2269-2277 (1998). It is of note that although 2-OH involve the formation of an intra-molecular hydrogen bond E has relatively low binding affinity for human ERC. and ERB with the C-3 hydroxyl group. See Anstead etal. The estradiol (significantly lower than that of 2-OH-E), this oxidative E pharmacophore. ligand structure-estrogen receptor binding metabolite has a significant preference for binding to ERC. affinity relationships and a model for the receptor binding US 2008/0293683 A1 Nov. 27, 2008

site, 62,268-303 (1997). However, it was observed I0088 B-Ring and C-Ring Metabolites. that, in Some cases, Substitution of even a very Small group I0089) C-6 Substituted Estrogens. such as bromine at the C-2 position of E (2-Br-E) drastically 0090 The data shows that addition of a hydroxyl group to reduced its binding affinity for ERB (RBA only <0.5% ofE), the C-6C, or C-6B position of E markedly reduced its binding while this substitution reduced its binding affinity for ERC. to affinity for both ERC. and ERB, but addition of a keto group to a relatively lesser degree (RBA 4% of E)) (FIG. 3A). This the C-6 positions of E, or E did not significantly affect the observation is rather interesting since C-2 bromine substitu original binding affinity of these estrogens for ERC. or ERB. tion had a far stronger negative effect on ER binding (particu 0091 Among six B-ring hydroxylated or keto metabolites larly for ERB) than the C-2 hydroxyl substitution. Notably, of E or E tested in this study, all of them retained certain bromine substitution at the C-4 position of E (4-Br-E) degrees of binding affinity for both ERC. and ERB (FIG. affected its binding affinity for ERC. and ERB in an opposite 4A-4F and Table 2). 6C.-OH-E- or 6 B-OH-E had markedly manner as what was observed for 2-Br-E. The 4-Br-E com reduced binding affinities for ERC. and ERB compared to E pound had a decreased binding affinity for ERC. about 5 times (FIG. 4A, 4B). However, addition of a keto group to the C-6 position of E did not markedly affect its original binding more than for ERB. Similarly, addition of a methyl group to affinity for ERC. and ERB(FIG. 4C). In comparison, addition the C-4 position of E (4-Methyl-E) also decreased its bind of a C-6 keto group to E differentially altered its binding ing affinity for ERC. (RBA only 7% of E) more than for ERA affinity for ERC. and ERB (RBAS 23% and 50% of E, respec (RBA35% of E), See FIGS. 3B and 3D. tively) (FIG. 4D, Table 2). Similarly, addition of a C-6 keto to 0084. Addition of a methyl group to the C-1 position of E. E-17C (6-keto-E-17C) also markedly reduced its binding (1-Methyl-E) decreased its binding affinity to a similar affinity for ERC. (RBA9% of E-17C), but its binding affinity degree (by approximately 90%) for ERC. and ERB (FIG.3C). for ERB was decreased to a relatively lesser extent (RBA 25% Several earlier studies have also shown that the C-1 substitu of E-17C) (FIG. 4F, Table 2). However, addition of a C-6 tion of E (regardless of polarity of the substituents) all had a keto group to E slightly increased its binding affinity for ERC. negative effect on the binding affinity for crude ER proteins (RBA 224% of Es), but it drastically reduced its binding from rabbit or human. See Anstead et al., The estradiol phar affinity for ERB (RBA 8% of E) (FIG. 4E, Table 2). macophore: ligand structure-estrogen receptor binding affin 0092 C-11 Substituted Estrogens. ity relationships and a model for the receptor binding site, (0093. The data with the C-11 position derivatives were Steroids 62,268-303 (1997). This influence was thought to be rather interesting and revealing. Addition of a hydrophilic due to a direct interaction of the C-1 substituting group with group (such as a hydroxyl or keto group) to the C-11 position the ER protein rather than a structural perturbation of the of E or E almost completely abolished their binding affini ligand conformations. ties for both ERC. and ERB. This was true regardless of whether the substitution was 11C. or 11B (FIG. 4G-4J). How I0085. The 2,3-dimethylated catechol E and E, derivatives ever, substitution of a lipophilic group with even a bulkier size did not have any appreciable binding affinity for ERC. and (such as the acetate or methoxy group) did not significantly ERB (FIG.3F, 4G). This is in accord with earlier studies using affect the binding affinity for either ERC. or ERB (FIG. 4L, the rat uterine ER protein preparations. See Ball et al., 4M, Table 2). These data indicated that the drastic decrease in Calecholoestrogens (2- and 4-hydroxyOestrogens): Chemis the binding affinities of 11C.-OH-E, 11-OH-E, or 11-keto try, biogenesis, metabolism, occurrence and physiological E. for human ERC. and ERB is not due to steric hindrance significance, Acta. Endocrinol. 232, 1-127 (1980). caused by the C-11 position substitutions, but it is primarily I0086. As expected, several synthetic C-2 or C-4 substitu due to alterations of the lipophilicity near the C-11 position. It tion analogs containing an amino (—NH) or nitro (—NO) is of note that earlier studies have also shown that the C-113 group resulted in diminished its binding affinity for ERC. and position of E was tolerant of even very large Substituents, if ERB (FIG. 3H-K). In particular, E (2-NH-E, 2-NO-E, the polar functional groups were placed at a distance from the 4-NH-E, and 4-NO.-E.) derivatives only retained very weak steroid core structure (reviewed in ref. Anstead et al., The binding affinities for human ERC. and ERB. It is of note that estradiol pharmacophore: ligand structure-estrogen recep the NO, and—NH substitutions ofE produced inhibition tor binding affinity relationships and a model for the receptor curves with rather shallow slopes, which likely suggests that binding site, Steroids 62, 268-303 (1997); and Gao et al., these were not pure competitive inhibition. Comparative OSAR analysis of estrogen receptor ligands, Chem. Rev. 99,723-744 (1999)). These observations agree I0087. The C-3 sulfated estrogens (E-3-sulfate and E-3- well with recent homology modeling data for human ERC. sulfate) were found to be basically devoid of appreciable and ERB showing that there is considerable space near the binding affinity for ERC. and ERB (FIG.3L, 3M), which was C-7C. binding site of E which can readily accommodate consistent with earlier findings. Like the C-3 sulfated estro various estrogen analogs with a rather bulky/lengthy Substi gens, earlier studies have shown that E3-ethyl ether (43,44) tution (data not shown). or 2-desoxy-E (see Fanchenko et al. The specificity of 0094 Dehydroestrogens. human estrogen receptor, Acta. Endocrinol. 7, 232-240 0095. In addition to the B-ring and C-ring substitution (1979); and Brooks et al., Estrogen structure-receptor func metabolites described above, this example also investigated tion relationships, Moudgil V K (ed.), Recent Advances in several common B- or C-ring dehydrogenated E or E Action, Walter de Gruyter, Berlin, pp. 443 metabolites. It was found that found that most of the dehy 466 (1987)), each had very low binding affinity for ER com droestrogen metabolites retained rather high binding affini pared to E. It was suggested that the C-3 hydroxyl group of ties for both ERC. and ERB compared to their respective E functions primarily as an H-bond donor in its interactions non-dehydrogenated precursors, and some of them Such as with ERC. and ERB. According to more recent X-ray crystal 6-dehydro-E, and 9(11)-dehydro-E retained high binding lography study of the human ERC. and ERB bound with E, it affinities for human ERs. The data summarized in FIG.5 and appears that the very low binding affinities of various C-3 Table 2. More specifically, 6-dehydro-E and 9(11)-dehydro modified E. derivatives are due to a combination of distur E had similar or somewhat higher binding affinity for human bance of H-bond formation and steric hindrance. ERB compared to E (RBAS 89% and 119%, respectively, of US 2008/0293683 A1 Nov. 27, 2008

E), but their binding affinities for ERC. were slightly reduced, Taking together all the information we have gathered, it with RBAS 50% and 64% of E, respectively (FIG. 5B, 5F, appears that while the presence of the C-17? hydroxyl group Table 2). (but not a C-17C. hydroxyl or C-17 keto group) increases the 0096. Notably, several of the dehydrogenated estrogens binding affinity of an aromatic steroid for human ERC. and tested in this study are the major components (in their conju ERB, its relative influence likely is not as strong as that of the gated forms) present in Premarin R, the commonly-used hor C-3 hydroxyl group. mone replacement therapy in peri-menopausal and post 0101. The binding affinity of 16C.-OH-E for human ERC. menopausal women. As discussed more fully below, the main was twice as high as that of E, but its affinity for ERB was estrogenic ingredients include Sodium El Sulfate, sodium 18-fold higher than E (FIG. 6A). Further, its binding affinity equaling Sulfate, and the Sodium sulfate conjugates of was still lower than that of E (with RBAs 56% and 25%, E-17C, 17C.-dihydroequilenin, and 17 B-dihydroequilin. respectively, of E). This is one of the most notable eases that Equilin (7-Dehyro-E) and 9(11)-dehydro-E, each had hydroxylation of an endogenous estrogen markedly enhanced slightly decreased binding affinity for ERC. compared to E its binding affinity for human ERC. and/or ERB than the (RBAs 45% and 50% of E, respectively), but they had a respective parent hormone. In addition, an earlier study drastically increased binding affinity for ERB (RBAs 63.1% reported that this E metabolite may be able to bind covalently and 316% of E, respectively). to the ER protein through the formation of a Schiff's base, 0097. The binding affinities of 17 B-dihydroequilin (i.e., likely resulting in Sustained ER-mediated growth stimulation 7-dehydro-E) for human ERC. and ERB were actually of the target cells. These biochemical properties of 16C.-OH slightly higher than E (its RBAS 142% and 113%, respec E have been the basis for the well-known hypothesis that tively, of E) (FIG. 5D). Similarly, while the binding affinity increased metabolic formation of 16C.-OH-E in a woman of 17C.-dihydroequilin (i.e., 7-dehydro-E-17C) for ERC. may increase the risk for development of estrogen-inducible remained the about same as that of E-17C, this equine estro cancers. Notably, despite its much higher binding affinities gen had a more than fourfold higher binding affinity for ERB than those of E for human ERC. and ERB, they were still than E-17C (RBA 447% of E-17C) (FIG. 5I, Table 2). slightly lower than E (with RBAs of 56% and 25%, respec Compared to E, 6-dehydro-E had nearly the same binding tively, of E). affinity for ERB, but its binding affinity for ERC. was signifi I0102 Interestingly, while 16-keto-E only had 18% of the cantly decreased, with its RBA only 10% of E (FIG. 5A, binding affinity of E for ERO, its binding affinity for ERB Table 2). was five-fold higher than that of Et (RBA501% of E) (FIG. 0098 D-Equilenin had a much weaker binding affinity 6B). Thus, the relative preference of 16-keto-E for human than E for human ERC. (RBA 20% of E), but its binding ERB over ERC. is approximately 25 times higher than E. affinity for ERB was more than 3 times higher than that of E. Addition of C-16 keto group to E increased its binding affin Very similarly, while 17 B-dihydroequilenin had a low bind ity for ERB but decreased its binding affinity for ERC. ing affinity for ERC. (35% ofE), it had a high binding affinity 0103) Addition of a C-16 keto or a C-15C. hydroxyl to E for ERB (RBA 100% of E) (FIG. 5H). Taken together, it is each significantly decreased the binding affinity for ERC. and evident that many of the equine estrogens contained in Pre ERB compared to E. 16-Keto-E and 15C.-OH-E (estetrol) marin have a differential binding affinity for human ERB over each had a reduced binding affinity for both ERC. and ERB ERO. compared to E and E, respectively (FIG. 6E, 6F) 0099 D-Ring Metabolites. 0104. As Already Mentioned Earlier, E. (Estriol 16C.-OH 0100. A total of twelve D-ring metabolites/derivatives of E), a Major D-Ring Metabolite in humans (particularly dur E, and E, were studied (data summarized in FIG. 6A-6L, ing pregnancy), had a markedly decreased binding affinity for Table 2). The data showed that E only had 5-10% of the ERC. compared to E (RBA 11% ofE), but it retained a rather binding affinity of E for human ERC. and ERB, and it had a high binding affinity for ERB (RBA 35% of E) (FIG. 6C, significant preference for binding to ERC. The markedly Table 2). By contrast, substitution of a C-16? hydroxyl group reduced binding affinity of E for ERs has previously been to E (namely, 16.f3, 17 B-OH-E, also called 16-epiestriol) did Suggested to reflect the unique importance of the C-17B not noticeably affect its binding affinity for either ERC. or hydroxyl in enhancing its interactions with the ER molecules. ERB (FIG. 6D). This suggestion was also Supported by other studies showing 0105. As already mentioned earlier, E-17C. retained con that when the C-17B hydroxyl ofE was converted to a methyl siderable binding affinity for ERC. (RBA 22% of E), but it ether or an acetate, their ER binding affinities were greatly had substantially lower binding affinity for ERB (3% of E) diminished. See Katzenellenbogen et al., Photoafinity labels (FIG. 1D or 7G). Interestingly, addition of a hydroxyl group for estrogen binding proteins of rat , Biochemistry 12, to the C-16C. or C-16? position of E-17C affected its binding 4085-4092 (1973); Kaspar et al., Shielding effets at 17o affinity for ERC. and ERB rather differently (FIG. 6H-6I). substituted estrogens. A tentative explanation for the low 16C.-OH-E-17C. (17-epiestriol) had very high, almost iden biological activity of 17O-ethyl-estradiol based on IR and tical binding affinities for both ERC. and ERB (RBAS 71% and NMR spectroscopic studies, J. Steroid. Biochem. 23, 611-616 79%, respectively, of E), which were 3 and 16 times higher, (1985). In this example, the data also showed that when the respectively, than its precursor E-17C. However, 16.f3-OH entire C-17f8 hydroxyl group was absent, the derivatives i.e., E-17C. (16,17-epiestriol) had low binding affinity for ERC. 17-desoxy-E and 1.3.5(10), 16-estratetraen-3-ol actually preferential binding affinity for ERB over ERC, and the dif had quite high binding affinity for ERC. and ERB, which was ference in the binding affinities is 18-fold. much higher than that of E, but lower than E. (FIG. 6, Table I0106 17o-Ethynylestradiol (17C-EE), a semi-synthetic 2). The data are also consistent with a few earlier reports on steroidal estrogen commonly used as an estrogenic compo the binding affinity of 17-desoxy-E, for human and ratestro nent in various oral contraceptives, had very high binding gen receptors. See Fanchenko et al., The specificity of human affinity for both ERC. and ERB compared to E. The binding estrogen receptor, Acta. Endocrinol. 7, 232-240 (1979); affinity of 17O.-EE for ERC. was twice as high as that of E. Brooks et al., Estrogen structure-receptor fitnction relation but its affinity for ERB was only about half of that of E (Table ships, Moudgil V K (ed.), Recent Advances in Steroid Hor 2 and FIG. 6J). The same receptor binding assay with this mone Action, Walter de Gruyter, Berlin, pp. 443-466 (1987). estrogen was repeated twice, and consistent results were US 2008/0293683 A1 Nov. 27, 2008

obtained. Accordingly, the relative ratio of preference for high and similar binding affinities for ERC. and ERB as the ICI binding to ERC. and ERB by 17 O.-EE is approximately 4 compound (FIG.7C, Table 2). This data is in agreement with times of that for E. Interestingly, the removal of the C-17 the earlier Suggestion that the human ER is tolerant of large? hydroxyl group from E (17-desoxy-E) did not drastically lengthy substitution at the C-7C. position of E, if the polar reduce its binding affinity for human ERC. and ERB (FIG. group is placed away from the steroid core. Ongoing homol 6K). Similarly, 1,3,5(10), 16-estratetraen-3-ol, which is also ogy modeling studies of the binding of various bioactive without a C-17 substitution, had a very similar binding affin estrogen derivatives with human ERC. and ERB also showed ity as that of 17-desoxy-E for ERC. and ERB (FIG. 6L). that there is considerable space near E.'s C-7C.-binding 0107. It is worth noting that the 16C.-hydroxylated estro pocket which can accommodate ligands with a bulky Substi gens (16C-OH-E and E), epiestriols (16C.-OH-E-17C, tution. Since the C-7C.-binding position is mainly composed 16.f3-OH-E, and 16 B-OH-E-17C), and other C-16 metabo of lipophilic amino acid residues, this also explains that polar lites (e.g., 16-keto-E) are usually quantitatively-minorestro groups need to be placed away from the C-7C. position in gen metabolites in non-pregnant woman, but some of them order to retain a high binding affinity with the receptor. are formed in unusually large quantities during pregnancy, 0114 Tamoxifen and raloxifene are two well-known non particularly at late stages of pregnancy. The data from this steroidal ER antagonists (partial ). Tamoxifen had example revealed that many of these estrogen metabolites almost identical binding affinities for human ERC. and ERB (e.g., E 16 B-OH-E-17C) had high preferential binding (FIG. 7D), although its binding affinities for these two recep affinities for human ERB over ERC. It is possible that they tors were only 3-4% of those of E and 7-10% of ICI-182,780. may jointly serve as important endogenous ligands for the 0.115. In comparison, while raloxifene had a similar bind preferential activation of the ERB signaling pathway during ing affinity for ERB as tamoxifen, the former had 16-fold human pregnancy. Such a preferential activation of ERB may higher binding affinity for ERC. than the latter and was com play an indispensable role in mediating the various actions of parable to ICI-182,780 (FIG. 7E, Table 2). Therefore, ralox the endogenous estrogens required for the development of the ifene actually had a strong preferential binding affinity for fetus as well as for fulfilling other physiological functions of human ERC. than for ERB. Since raloxifene and tamoxifen are pregnancy. This Suggestion is in line with some of the obser very different from each other in that the former had a strong vations showing that the ERB has a wide distribution inmater preferential binding affinity for ERC, this may be one of the nal rat reproductive organs as well as the fetus. important underlying factors that determine their different 0108 Based on all of the endogenous estrogen metabo pharmacological profiles in various targettissues. In addition, lites/derivatives analyzed in this example, it is apparent that it is possible that differences in their metabolic conversion to the D-ring (particularly at the C-16 and C-17 positions) of E. derivatives with differing ER-binding affinities may also con is the most sensitive target where modifications of its struc tribute to some of the known pharmacological differences of ture may differentially modify its binding affinity for the these two antiestrogens in vivo. human ERC. or ERB. This property will have important physi 0116 Phytoestrogens. ological as well as pharmacological implications. From a 0117 Genistein, a well-known abundantly physiological point of view, it is known that E is perhaps the present in soy products, had an extremely high binding affin most potent endogenous estrogen which has similar binding ity for ERB (almost identical to that of the endogenous hor affinity for ERC. and ERB, but it is not the predominant estro mone E), but its binding affinity for ERC. was only 6% of its gen(s) present in the body. In non-pregnant woman, the pre binding affinity for ERB. This data is consistent with earlier dominantform of estrogens invarious tissues is E (which has reports. If one assumes that a significant portion of the a higher ERC. activity over ERB), whereas in a pregnant ingested genistein is Subsequently uptaken into target cells woman, E along with several other D-ring metabolites without degradation, then the practice of using dietary phy become the quantitatively-predominant forms of estrogens toestrogens (e.g., genistein) as the sole or main source of (which have strong preference for ERB). From a pharmaco estrogens for female hormone replacement therapy may logical point of view, selective modifications of the D-ring of unwittingly confer a long-term predominant ERB stimulation a steroidal estrogen may represent an efficient strategy for the in postmenopausal women. Before the health benefits or rational design of selective/preferential agonists or antago potential side effects associated with a long-term ERB stimu nists for human ERC. and particularly for ERB. lation in peri-menopausal or postmenopausal women are 0109. In summary, most of the D-ring metabolites retained known, it may be risky to use dietary phytoestrogens as the rather high binding affinity for human ERC. and ERB, but sole or main source of estrogens for female hormone replace several of them (16(3-OH-E-17C, 16C.-OH-E-17C, 16-keto ment therapy. Likewise, more studies are urgently needed to E. 16C.-OH-E, and 16C.-OH-E) had markedly increased determine if there are any potential side effects in newborns or binding affinity for human ERB over ERC. compared to their infants who feed entirely on soymilk (rich in genistein) respective precursors (namely, E, E, and E-17C). instead of human or cow milk. 0110 Antiestrogens, Phytoestrogens, and Stilbene Estro 0118 Coumestrol, another well-known phytoestrogen, genS. had very high binding affinity for human ERC. and ERB, and 0111 For the purpose of comparison, the binding affinities its relative binding affinity for ERB was slightly higher than of a number of steroidal and antiestrogens, phy its affinity for ERC. (FIG. 7G). Myricetin basically had no toestrogens, stilbene estrogens and nonaromatic steroids for appreciable binding affinity for human ERC. and ERB (FIG. human ERC. and ERB were also determined. Their data were 7H). Daidzein had very weak binding affinities for both ERC. summarized in FIG. 7A-M. and ERB, but its relative affinity for ERB was significantly 0112 Steroidal and Nonsteroidal Antiestrogens. higher than its affinity for ERC. (FIG. 7I). Dibenzoylmethane 0113. The binding affinities of ICI-182,780 for both ERC. (DBM) had a weak overall binding affinity for ERC. and ERB and ERB were very high and nearly the same (RBAs 45% and (FIG.7J). 35%, respectively, of E) (FIG. 7A and Table 2). Similarly, 0119) Stilbene Estrogens. another two synthetic C-7C. Substituted analogs, E-7.C.- I0120 Many earlier animal studies as well as invitro recep (CH)OH and E-7C-(CH)OCHs, which have shorter tor binding assays have shown that diethylstilbesterol (DES), side chains at the C-7C. position than ICI-182,780, retained dienestrol, and hexestrol are very potent synthetic estrogens US 2008/0293683 A1 Nov. 27, 2008 with similarestrogenic potency and efficacy as E. The results Example 2 from this example also showed that each of these stilbene estrogens had very high binding affinity (similar to that of E) Comparison of Endogenous Estrogens in Pregnant for both human ERC. and ERB. The three well-known non and Non-Pregnant Women steroidal stilbene estrogens (diethylstilbesterol DES). dienestrol, and hexestrol) had very high binding affinities I0123. A large number of endogenous estrogen derivatives (similar to that of E) for human ERC. and ERB (FIG. 7K-M, Table 2). We noted that DES and hexestrol had a slightly are known to be present in humans. In this example, the higher binding affinity for ERB than for ERC, although the human urinary excretion of various estrogens (mostly as con difference was only very small. jugates) as a global indicator of the and metabo 0121. In this example, the activity of a large number of lism of endogenous estrogens in vivo was investigated. It is endogenous estrogen metabolites, including those contained estimated that the total daily amount of various urinary estro in Premarin R, for human ERC. and ERB was investigated. It gens excreted from a late pregnant woman is about 300 times was found that while E (perhaps the best-known endogenous higher than the amount excreted by a non-pregnant woman of estrogen) has nearly the highest and almost identical binding the same age group. In addition, the composition of the uri affinities for human ERC. and ERB, many of its metabolites nary estrogens in women are also widely different. Represen have widely different preference for the activation of human tative profiles of various endogenous estrogens found in the ERC. and ERB. It is of particular interest to note that the urine of pregnant and non-pregnant young women are sum predominant estrogens that are present in a pregnant woman marized in Table 3.

TABLE 3 The levels of endogenous estrogen metabolites present in the urine samples from pregnant and non-pregnant women. Pregnant woman (ng/mL) Non-pregnant woman (1g24 h a month before Day 6-10 Day 16 Day 21-25 delivery Average S.D. (peak) Average SD Average SD E1 (estrone) 340 O.81 11.13 3.12 O.27 39.9 6.1 E2 (17B-estradiol) 144 O.54 4.17 1.64 O.16 20.4 3.3 2-OH-E 6.06 1.7O 42.74 11.13 3.27 13.4 2.5 4-OH-E 2.20 1.26 S.40 1.75 O.69 19.6 1.9 16C-OH-E 3.15 1.61. 21.18 3.91 1.SS 1358.6 210.3 2-MeO-E. ND ND 7.38 ND ND 12.O S.1 2-OH-E- O.71 O.22 3.59 1.09 O.23 3.0 0.7 4-OH-E- O.69 O.09 1.42 O.95 0.26 ND ND 2-MeO-E, 0.97 O.38 140 0.77 0.29 3.4 1.2 E (estriol or 16C.-OH-E) 4.28 1.29 22:32 3.61 O.44 8177.5 763.1 16-EpiE. ND ND ND ND ND 45.9 7.6 17-EpiE. 2.96 O.48 4.69 3.43 1.21 17.3 6.4 16,17-EpiE. ND ND ND ND ND 55.5 9.5 2-OH-E. ND ND ND ND ND 7.9 1.9 15C-OH-Es (estetrol) ND ND ND ND ND 30.7 6.5 *Twenty-four hour urine sample from pregnant women was not available, and the data is orga nized in the concentration of ng estrogen metabolite/mL urine. *It is possible that the estrogen concentrations in the urine were increased to higher levels before delivery, but the concentrations of estrogens in urine returned to normal levels very quickly after delivery. are very different from those present in a non-pregnant 0.124. In the urine samples obtained from non-pregnant woman. Furthermore, these estrogens have widely different young women, the conjugated forms of 2-hydroxy-estrone, preference for activation of human ERC. and ERB. followed by 16O-hydroxy-estradiol (E), 16C.-hydrox 0122 Many of the endogenous estrogen metabolites yestrone, and estrone (E), are the predominant estrogens. retained varying degrees of similar binding affinity for ERC. The amount of E and its major metabolites 2-hydroxy-estra and ERB, but some of them retained differential binding affinity for the two subtypes. For instance, several of the diol and 2-methoxy-estradiol was much less than that of D-ring metabolites, such as 16C.-OH-E 16 B-OH-E-17C, estrone and its corresponding metabolites. The relative com and 16-keto-E, had distinct, preferential binding affinity for position of various estrogens in circulation is believed to be human ERB over ERC. (difference up to 18-fold). Notably, largely comparable to what is seen in the urine. The presence while E has nearly the highest and equal binding affinity for of higher levels of estrone (E) over estradiol (E) in a non ERC. and ERB, E and 2-OH-E (two quantitatively-predomi pregnant woman is largely attributable to the high levels of the nant endogenous estrogens in non-pregnant woman) have oxidative 17f8-hydroxysteroid dehydrogenase (“17 B-HSD), preferential binding affinity for ERC. over ERB, whereas 16C.- which catalyzes the facile conversion of estradiol (E) to OH-E (estriol) and other D-ring metabolites (quantitatively estrone (E). The conversion of estrone (E) to 2-hydroxy predominant endogenous estrogens formed during preg estrone or estradiol (E) to 2-hydroxy-estradiol is catalyzed nancy) have preferential binding affinity for ERB over ERC. by various cytochrome P450 , and the subsequent US 2008/0293683 A1 Nov. 27, 2008

O-methylation to form 2-methoxy-estrone or 2-methoxy-es some of the observations showing that the ERB has a wide tradiol is catalyzed by catechol-O-methyltransferase distribution in maternal reproductive organs in rats as well as (“COMT). their fetus. 0125 There is a drastic change in the endogenous estrogen composition during pregnancy. Estriol (E) becomes the pre Comparative Example 3 dominant estrogen and it is produced in unusually large quan tities. The daily amount of this estrogen (in its conjugated Analysis of PremarinR) forms) released into the urine of a late pregnant woman is I0131 Premarin R, the commonly-used hormone replace 200-1000 times higher than any of the quantitatively-major ment therapy, contains a mixture of conjugated estrogens estrogens produced in a non-pregnant woman. Notably, sev obtained from pregnant mare's urine. As shown in the follow eral other D-ring estrogen derivatives, such as 17-epi-Es, ing table, the majorestrogens produced in a pregnant mare are 16-epi-Es, 16,17-epi-E and estetrol (15C-hydroxy-estriol), quite different from those produced in a pregnant woman. are also produced in readily detectable quantities at late stages of pregnancy. These D-ring derivatives are usually only present at low or undetectable levels in non-pregnant TABLE 4 young Women. Composition of Premarin B. 0126 From Example 1, it was found that E and 2-OH-E, two of the quantitatively-major estrogen derivatives present Sodium estrogen Sulfate MgTablet ERC. RBA ERB RBA Ratio in a non-pregnant woman, have a modest but significant pref Estrone (E) O.370 10 2 5 erence for binding to human ERC. over ERB. More specifi 7-dehydroestrone (Equilin) O.168 4 13 O.3 cally, E had five-fold higher relative binding affinity for 17C-Dihydroequilin O.1O2 18 14 1.3 17C-Estradiol O.O27 22 3 7.3 human ERC. than for ERB. Similarly, 2-OH-E (the 2-hy 17B-Dihydroequilin O.O11 142 113 1.3 droxylated metabolite of E) also has a about ten-fold pref 17C-Dihydroequilenin O.O21 erence for activation of ERC. over ERB. Equilenin O.O15 2 7 O.3 0127. Notably, E and 2-OH-E have markedly lower 17B-Estradiol O.OOS 1OO 100 1 binding affinity for human ERC. and ERB compared to E. In A8.9-Dehydroestrone O.O26 the present invention, it is theorized that the relatively lower binding affinity ofE and 2-OH-E is an advantage rather than 0.132. The exact total amount of various estrogenic com a disadvantage, because Such compounds would pose a lower ponents contained in each Premarin R tablet is not known. It risk for causing over-stimulation of the ERC. and ERB signal has been traditionally assumed to each Premarin R tablet con ing systems in vivo. tained a mixture of estrogen Sulfates that are biologically 0128. In contrast, E, the quantitatively-predominant equivalent to 0.625 mg of estrone 3-sulfate, according to an estrogen produced during pregnancy, has a significant pref earlier uterotropic assay using ovariectomized female rats. erence for binding to ERB over ERC. E. had a rather low Further, a synthetic Premarin R formulation is set forth in binding affinity for human ERC. compared to E (RBA 11% of Hill, U.S. Pat. No. 6,855,703, which is incorporated by ref E), but it retained a relatively high binding affinity for ERB CCC. (RBA 35% of E). Similarly, 16C.-OH-E, another well 0.133 Table 4 shows that the major estrogens present in known hydroxy-E metabolite that is also formed in increased pregnant mare's urine do not include E. Rather, they include quantity during pregnancy, has a higher binding affinity than a number of other equine estrogens. Some of these equine E for both ERC. and ERB. estrogens are basically not produced in humans. Several of 012.9 16,17-Epiestriol, another estrogen in pregnant the equine estrogens contained in PremarinR are functionally women, had a very low binding affinity for human ERC, but similar to human pregnancy estrogens with respect to their it had a preferential affinity for ERB. The difference of its preferential affinity for human ERB over ERC. binding affinity for ERB over ERC. is 18-fold. The relative 0.134 Similarly, D-equilenin had a weaker binding affinity quantity of this estrogen in pregnant woman's urine is rather for human ERC. than E (RBA 20% of E), but its binding small (Table 3). affinity for ERB was much higher than that of E (RBA355% 0130. In sum, although 17 B-estradiol (E) is perhaps the of E). Also, 17(3-dihydroequilenin only had a weak binding best-known endogenous estrogen in humans, it is not the affinity for ERC. (RBA 10% of E), it retained very high predominant estrogen produced in the body of a pregnant binding affinity for ERB (RBA 100% of E). woman or of a non-pregnant woman. Based on the informa 0.135 For example. Example 1 showed that equilin (i.e., tion discussed above, it is evident that the major endogenous 7-dehyro-E) had slightly decreased binding affinity for ERC. estrogens that are produced in a non-pregnant woman are compared to E (its RBA 40% of E), but it had drastically vastly different in composition and quantity from those pro increased binding affinity for ERB (its RBA 63.1% of E). duced in a pregnant woman, Further, it is evident that there is Similarly, D-equilenin had a much weaker binding affinity a marked difference in the ratio and also intensity of ERC. and than E for human ERC. (RBA 20% of E), but its binding ERB activation in a non-pregnant young woman compared to affinity for ERB was more than 3 times higher than that of E. a pregnant woman. The major estrogens produced in a non Also, while 17 B-dihydroequilenin had a low binding affinity pregnant woman would modestly favor the activation of the for ERC. (35% of E), it had a high binding affinity for ERB ERC. system over the ERB system. However, during preg (RBA 100% of E). The binding affinities of 17 B-dihy nancy, there is a preponderance of activation of ERB over droequilin (i.e., 7-dehydro-E) for human ERC. and ERB were ERC, which is exerted by various pregnancy estrogens, actually slightly higher than E (its RBAS 142% and 113%, mainly estriol, which is produced in unusually large quanti respectively, of E). ties. Such a preferential activation of ERB is believed to play 0.136 Further, while 6-dehydro-E and 9(11)-dehydro-E an indispensable role in mediating the various actions of had slightly reduced binding affinity for ERC. (RBAS 50% and endogenous estrogens that are required for the development 64% of E, respectively), their binding affinity for human of the fetus as well as for fulfilling other physiological func ERB was compared to E (RBAS 89% and 119%, respectively, tions related to pregnancy. This Suggestion is in line with of E). Compared to E, 6-dehydro-E had nearly the same US 2008/0293683 A1 Nov. 27, 2008 binding affinity for ERB, but its binding affinity for ERC. was gens are inactive themselves (with little or no binding affinity significantly decreased, with its RBA only 10% of E. 7-De for human ERC. and ERB), but they can be enzymatically hyro-E and 9(11)-dehydro-E each had slightly decreased hydrolyzed to release bioactive estrogens in a variety of tis binding affinity for ERC. compared to E (RBAs 45% and Sues in the body. As such, oral administration of estrogen 50% of E, respectively), but they had a drastically increased sulfates would have the natural cushion effect which would binding affinity for ERB (RBAs 631% and 316% of E, avoid causing unwanted over-stimulation of the ER system respectively). Similarly, D-equilenin had a weaker binding throughout the body. Instead, they usually would only acti affinity for human ERC. than E (RBA 20% of E), but its binding affinity for ERB was much higher than that of E vate those target tissues or cells that are most in need of (RBA 355% of E). Also, 17(3-dihydroequilenin only had a estrogenic stimulation. Here it is also of note that several weak binding affinity for ERC. (RBA35% of E), it retained recent studies have already shown that the estrogen target very high binding affinity for ERB (RBA 100% of E). cells can actively transport E-3-sulfate into the cells. More 0.137 Taken together, it is evident that many of the equine over, these cells may selectively adjust their ability to actively estrogens contained in Premarin Rhave a preferential binding transport E-3-sulfate into the cells to release bioactive estro affinity for human ERB over ERC. gens, depending on the level of their hormonal needs. See Pizzagalli et al., Identification of Transport Example 5 Process in the Human Mammary Gland, J. Clin. Endocrinol. Metab. 88, 3902-3912 (2003). Theoretically, such a mecha Hormone Replacement Formulations nism would offer certain degrees of target organ selectivity of estrogenic stimulation. Compared to estrogen glucuronides, 0.138. In the present invention, a primary criterion that estrogen sulfates are probably better because they usually determines whether a given estrogen or combination of estro gens is ideal for postmenopausal hormone replacement have longer half-lives (t) in the body, thereby making them therapy is that the estrogen(s) should be able to restore the pharmacologically more useful. hormonal environment to those in a normal non-pregnant 0.143 Based on the discussion given above, it is Suggested young woman, but not that in a pregnant woman. Because that modest levels of stimulation of both ERC. and ERB sys very different types of estrogens are produced in pregnant tems with a slight preference for the ERC. system would be compared to non-pregnant women and they serve very differ betterfor postmenopausal hormone replacement therapy than ent physiological functions, it is theorized the use of endog estrogens that confer a predominant activation of the ERB enous estrogens found in a non-pregnant young woman system. It is apparent that Premarin R, the most widely pre would be more ideal for hormone replacement therapy than scribed hormone replacement therapy, is less ideal for achiev those predominantly produced during pregnancy. ing this clinical purpose. While there is considerable amount 0.139. In a preferred aspect, hormone replacement therapy of E-3-sulfate contained in Premarin R, which presumably is formulation consisting essentially of estrogenic compounds good for its intended purpose as a hormone replacement such that: (1) the relative binding affinity for ERC. (“RBA) therapy, the fact is that it also contains many other very potent of the estrogenic compounds compared to 173-estradiol (E) pregnancy equine estrogens which would jointly produce a is less than about 100%; (2) the relative binding affinity for strong over-stimulation of the ERB system. Similarly, ERB (RBA) of the estrogenic compounds compared to genistein, a potent and preferential partial agonist of human 17 B-estradiol (E) is less than about 100%; and/or (3) the ERB, would be even less suitable than Premarin R) for use in estrogenic compounds preferentially stimulate the ERC. over postmenopausal hormone replacement therapy because it the ERB such that the ratio of RBA/RBA is greater than would essentially provide a near selective ERB stimulation. about 1. Most preferred estrogenic compounds are estrone This Suggestion is in agreement with recent clinical observa (E), 17C-estradiol (17C-E), 2-hydroxyestrone (2-OH-E), tions showing that the singular use of genistein is ineffective 2-methoxyestrone (2-MeO-E), and/or 2-methoxyestradiol as a hormone replacement therapy in postmenopausal (2-MeO-E), as well as their sulfated or glucuronidated con WOa. jugates. 0144. The following references to the extent that they pro 0140 For example, one preferred hormone replacement vide exemplary procedural or other details Supplementary to formulation comprises about 0.1-0.3 mg estrone (E) sulfate, those set forth herein, are specifically incorporated herein by and/or about 0.1-0.3 mg 17C.-estradiol (17C-E) sulfate, and/ reference. or about 0.1 to 0.5 mg 2-hydroxyestrone (2-OH-E) sulfate, 0145 Anstead et al., 2-Arylindenes and 2-arylindenones: and/or about 0.1 to 1 mg 2-methoxyestrone (2-MeO-E), Molecular structures and considerations in the binding and/or about 0.1 to 1 mg 2-methoxyestradiol (2-MeO-E). Orientation of unsymmetrical nonsteroidal ligands to the 0141. It should also be noted that some endogenous estro estrogen receptor, J. Med. Chem. 32, 2163-2172 (1989). gens (such as the conjugates of 2-methoxyestradiol) are ben eficial antitumorigenic estrogen metabolites. Given that 014.6 Anstead et al., The estradiol pharmacophore: many of the endogenous estrogens may have a rather rapid ligand structure-estrogen receptor binding affinity rela metabolic disposition in the body, some other naturally-oc tionships and a model for the receptor binding site, Ste curring or synthetic estrogens that have longer half-lives and roids 62,268-303 (1997). can also provide a similar preferential activation of the ERC. 0147 Ball et al., Catecholoestrogens (2- and 4-hydroxy system as E may also be useful as alternatives. For instance, Oestrogens). Chemistry, biogenesis, metabolism, occur since 17 O.-E. has similar ER-binding preference as E but it rence and physiological significance, Acta. Endocrinol. cannot be readily converted to E by 17 B-hydroxysterpoid 232, 1-127 (1980). dehydrogenase, its sulfate conjugates may serve as alterna 0148 Bradlow et al., Oestradiol 16C.-hydroxylase. A risk tives to E sulfate to achieve similar biological functions. marker for breast cancer, Cancer Surv. 5,573-583 (1986). 0142. In the present invention, using conjugated estro 0149 Brooks et al., Estrogen structure-receptor function gens. Such as Sulfated estrogens for human hormone replace relationships, Moudgil V K (editor), Recent Advances in ment therapy is also preferable to using the corresponding Steroid Hormone Action. Walter de Gruyter, Berlin 443 parent estrogens. The main reasons are: (i) The Sulfated estro 466 (1987). US 2008/0293683 A1 Nov. 27, 2008

0150 Brzozowski et al., Molecular basis of agonism and 0172 Katzenellenbogen et al., Photoafinity labels for antagonism in the Oestrogen receptor, Nature 389, 753-758 estrogen binding proteins of rat uterus, Biochemistry 12, (1997). 4085-4092 (1973). 0151 Cramer et al., Cross validation, bootstrapping, and 0173 Katzenellenbogen et al., In vivo and in vitro steroid partial least squares compared with multiple regression in receptor assays in the design of estrogen pharmaceuticals, conventional OSAR studies, Quant. Struct. Act. Relat. 7, In: Eckelman W C (editor), Receptor-Binding Radiotrac 18-25 (1988). ers. Vol. 1. CRC, Boca Raton, Fla. 93-126 (1982). 0152 Cushman et al., Synthesis, antitubulin and antimi 0.174 Katzenellenbogen et al., Hormone binding and totic activity, and cytotoxicity of analogs of 2-methox transcription activation by estrogen receptors: analyses vestradiol, an endogenous mammalian metabolite of estra using mammalian and yeast systems, J. Steroid. 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0193 Shughrue et al., Comparative distribution of estro 2. The estrogen formulation of claim 1 consisting essen gen receptor-C. and -f mRNA in the rat central nervous tially of at least two estrogenic compounds which preferen system, J. Comp. Neurol. 388, 507–525 (1997). tially stimulate the (ERC) compared 0194 Spong et al., Maternal estrogen receptor-f expres to the (“ERB’) and a pharmaceutically Sion during mouse gestation, Am. J. Reprod. Immunol. 44. acceptable carrier. 249-252 (2000). 3. The estrogen formulation of claim 1 consisting essen 0.195 Swanecket al., Covalent binding of the endogenous tially of at least three estrogenic compounds which preferen estrogen 16C.-hydroxyestrone to estradiol receptor in tially stimulate the estrogen receptor alpha (ERC) compared human breast cancer cells. Characterization and intra to the estrogen receptor beta (“ERB’) and a pharmaceutically nuclear localization, Proc. Natl. Acad. Sci. USA 85,7831 acceptable carrier. 7835 (1988). 0196. Tsvetan, Ouantitative structure-activity relation 4. The estrogen formulation of claim 3 wherein said estro ships/comparative molecular field analysis (OSAR/ genic compounds have a relative binding affinity for ERC. CoMFA) for receptor-binding properties of halogenated (“RBA) compared to 17 B-estradiol (E) which is less than estradiol derivatives, J. Med. Chem. 37, 4164-4176 about 100%. (1994). 5. The estrogen formulation of claim 3 wherein said estro 0.197 Wakeling et al., ICI 182,780, a new antioestrogen genic compounds have an RBA, compared to 17 B-estradiol of with clinical potential, J. Steroid. Biochem. Mol. Biol. 43. less than about 30%. 173-177 (1992). 6. The estrogen formulation of claim 5 wherein at least two 0198 Wang et al., Synthesis of B-ring homologated estra of said at least three estrogenic compounds wherein said diol analogues that modulate tubulin polymerization and estrogenic compounds have an RBA, compared to 173-es microtubule stability, J. Med. Chem. 43, 2419-2429 tradiol of about 10% or less. (2000). 0199 Woldet al., Multivariate data analysis in chemistry, 7. The estrogen formulation of claim 3 wherein said estro In Chemometrics: Mathematics and Statistics in Chemis genic compounds have a relative binding affinity for ERB try: Kowalski B, Ed, Reidel: Dordrecht, The Netherlands (“RBA) compared to 1713-estradiol (E.) which is less than (1984). about 100%. 0200 Wolohan et al., CoMFA and docking study of novel 8. The estrogen formulation of claim 3 wherein said estro estrogen receptor subtype selective ligands, J. Comput. genic compounds have an RBA compared to 1713-estradiol Aided Mol. Des. 17, 313-328 (2003). of less than about 10%. 0201 Zhu et al., Functional role of estrogen metabolism in 9. The estrogen formulation of claim 8 wherein at least two target cells. Review and perspectives, Carcinogenesis 19, of said at least three estrogenic compounds wherein said 1-27 (1998). estrogenic compounds have an RBA compared to 17|3-es 0202 Zhu et al., Is 2-methoxyestradiol an endogenous tradiol of about 5% or less. estrogen metabolite that inhibits mammary carcinogen 10. The estrogen formulation of claim3 wherein said estro esis, Cancer Res. 58, 2269-2277 (1998). genic compounds have an RBA compared to 1713-estradiol 0203 Zhu, et al., NADPH-dependent metabolism of 17 B of less than about 5%. estradiol and estrone to polar and nonpolar metabolites by human tissues and cytochrome P450 isoforms, Steroids 70, 11. The estrogen formulation of claim3 wherein said estro 225-244 (2005). genic compounds have a ratio of RBA/RBA which is 0204 From the foregoing, it will be seen that this inven greater than about 2. tion is one well adapted to attain all ends and objectives herein 12. The estrogen formulation of claim 3 wherein at least above set forth, together with the other advantages which are one of said estrogenic compounds has a ratio of RBA/RBA obvious and which are inherent to the invention. Since many which is greater than about 5. possible embodiments may be made of the invention without 13. The estrogen formulation of claim 3 wherein at least departing from the scope thereof, it is to be understood that all one of said estrogenic compounds has a ratio of RBA/RBA matters herein set forth herein are to be interpreted as illus which is greater than about 10. trative, and not in a limiting sense. While specific embodi ments have been shown and discussed, various modifications 14. The estrogen formulation of claim 1 wherein said estro may of course be made, and the invention is not limited to the genic compounds are selected from the group consisting of specific forms or arrangement of parts and steps described estrone (RBA/RBA about 5), 1-methylestradiol (RBA/ herein, except insofar as Such limitations are included in the RBA about 1.8), 2-aminoestrone (RBA/RBA about 7.5), following claims. Further, it will be understood that certain 2-nitroestrone (RBA/RBA about 3), 2-hydroxyestrone features and Subcombinations are of utility and may be (RBA/RBA about 10), 2-methoxyestradiol (RBA/RBA employed without reference to other features and subcombi about 2), 2-bromoestradiol (RBA/RBA about 10), 4-nitro nations. This is contemplated by and is within the scope of the estrone (RBA/RBA about 10): 4-hydroxyestrone (RBA/ claims. RBA about 2), 4-hydroxyestradiol (RBA/RBA about 1.3), 4-methoxyestradiol (RBA/RBA about 2), 6-ketoestrone What is claimed and desired to be secured by Letters Patent ((RBA/RBA about 2), 6O-hydroxyestradiol (RBA/RBA is as follows: about 1.5), 6-ketoestradiol (RBA/RBA about 1.3), 6-ke 1. An estrogen formulation for use in hormone replacement toestriol (RBA/RBA about 8.3), 6-ketoestradiol-17c. therapy consisting essentially of (RBA/RBA about 2), 7-dehydroestradiol (RBA/RBA a therapeutically effective amount of at least one or more about 1.3), 7-dehydroestradiol-17c. (RBA/RBA about 1.3), estrogenic compounds which preferentially stimulate 2-hydroxyestriol (RBA/RBA about 2), 1713-estradiol the estrogen receptor alpha (ERC) compared to the 11-acetate (RBA/RBA of 1.2), 11-?3-methoxyethynyl estrogen receptor beta (“ERB) and a pharmaceutically estradiol (RBA/RBA of about 1.8), estetrol (RBA/RBA acceptable carrier. of about 1.3), and 1613-hydroxyestradiol (RBA/RBA of US 2008/0293683 A1 Nov. 27, 2008 20 about 1.3), 17c-estradiol (RBA/RBA about 7.3), 17o-ethy stimulate the estrogen receptor alpha (ERC) compared to the nylestradiol (RBA/RBA about 3.6). estrogen receptor beta (“ERB) and a pharmaceutically 15. The estrogen formulation of claim 1 wherein said estro acceptable carrier. genic compounds are selected from the group consisting of 21. The estrogen formulation of claim 20 wherein said estrone (E), 17C-estradiol (17C-E), 2-hydroxyestrone three to five estrogenic compounds are selected from the (2-OH-E), 2-methoxyestrone (2-MeO-E), and 2-methox group consisting of estrone (E), 17C.-estradiol (17C-E), yestradiol (2-MeO-E) and their corresponding conjugates. 2-hydroxyestrone (2-OH-E), 2-methoxyestrone (2-MeO E), and 2-methoxyestradiol (2-MeO-E) and their corre and pharmaceutically acceptable salts thereof. sponding conjugates, and pharmaceutically acceptable salts 16. The estrogen formulation of claim3 wherein said estro thereof. genic compounds are selected from the group consisting of 22. A method for the treatment of peri-menopausal or estrone (E), 17C-estradiol (17C-E), 2-hydroxyestrone post-menopausal symptoms in a human female which com (2-OH-E), 2-methoxyestrone (2-MeO-E), and 2-methox prises administering the estrogen formulation of claim 1. yestradiol (2-MeO-E) and their corresponding conjugates, 23. The method of claim 22 wherein said formulation com and pharmaceutically acceptable salts thereof. prises at least three estrogenic compounds, said estrogenic 17. The estrogen formulation of claim 15 wherein said compounds selected from the group consisting of estrone conjugates are sulfated or glucuronidated conjugates. (E), 17o-estradiol (17C-E), 2-hydroxyestrone (2-OH-E), 18. The estrogen formulation of claim 1 wherein said estro 2-methoxyestrone (2-MeO-E), and 2-methoxyestradiol genic compounds are endogenous to non-pregnant pre-meno (2-MeO-E) and their corresponding conjugates, and phar pausal human females. maceutically acceptable salts thereof. 19. The estrogen formulation of claim 1 wherein said for 24. The method of claim 23 wherein said at least three mulation is in tablet form. estrogenic compounds are co-administered at the same time 20. The estrogen formulation of claim 1 wherein said for in a single formulation. mulation consists of a therapeutically effective amount of three to five estrogenic compounds which preferentially c c c c c