WO2008101030A1

WO2008101030A1 - Methods for amplifying steroid hormone effects

Info

Publication number: WO2008101030A1
Application number: PCT/US2008/053881
Authority: WO
Inventors: Bill L. Lasley; Bruce Hammock; Jiangang Chen; Ki Chang Ahn; Nancy A. Gee; Shirley J. Gee
Original assignee: The Regents Of The University Of California
Priority date: 2007-02-13
Filing date: 2008-02-13
Publication date: 2008-08-21

Abstract

The present invention provides methods for enhancing signal transduction through steroid hormone superfamily receptors and methods for identifying compounds that enhance signal transduction through these receptors.

Description

METHODS FORAMPLIFYING STEROID HORMONE EFFECTS

CROSS-REFERENCES TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. US60/889,720, filed February 13, 2007, and U.S. Provisional Application No. US60/991.154, filed November 29, 2007, the disclosures of which are incorporated by reference in their entireties for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with Government support under Grant Nos. P42ES04699 and P30ES005707 awarded by the National Institute of Environmental Health Sciences (NIEHS). The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] Lipid-soluble hormones such as steroid hormones, retinoids, and thyroid hormones, among others, are hormones that bind to and regulate specific members of a large superfamily of related transcription factors, termed the steroid hormone receptor superfamily. As lipid-soluble molecules, members of this class of hormones are able to diffuse through plasma and nuclear membranes to interact directly with intracellularly located members of the steroid hormone receptor superfamily. Binding of a hormone to its cognate receptor results in transcriptional activation of target genes containing DNA binding sites, or response elements, for the hormone receptor complex, although the precise mechanism by which this occurs varies depending on the particular hormone/receptor complex involved. [0004] For example, in the absence of hormone, the glucocorticoid receptor is anchored in the cytoplasm in a large protein aggregate complexed with an inhibitor protein. In this state, the receptor cannot interact with and activate target genes. Binding of hormone releases the glucocorticoid receptor from its cytoplasmic anchor, allowing it to enter the nucleus and bind to response elements associated with target genes. Other members of the steroid receptor superfamily interact with inhibitors in the nucleus from which they are released when they bind their specific hormone. [0005] In contrast, the thyroid hormone receptor binds to its DNA response elements in the absence of hormone, and the bound protein represses transcription rather than activating it. Thus, when thyroid hormone binds to the thyroid hormone receptor, the protein is converted from a repressor to an activator. [0006] Examples of lipid-soluble hormone receptors include those for androgens, estrogens, progesterone, glucocorticoids, thyroid hormone, vitamin D, and retinoids, among others. The diversity of lipid-soluble hormones implicates them in a variety of physiological processes, and accordingly, a variety of disease states arise when these hormonal signaling systems function aberrantly. For example, the androgen, testosterone, is involved in the maturation and normal function of accessory male sex organs and the development of male sex characteristics. Thus, underproduction of this hormone can result in medical conditions such as infertility and delayed puberty, among others.

[0007] Estradiol is an estrogen involved in the differentiation of the uterus and other female sex organs, the maintenance of secondary female sex characteristics, and the development of the duct system in mammary glands, among other functions. Progesterone is involved in the differentiation of the uterus in preparation for implantation of the early embryo, the maintenance of early pregnancy, and the development of the alveolar system in mammary glands, among other functions. Accordingly, these hormones play prominent roles in the regulation of the menstrual cycle, child birth, birth control, and hormonal replacement therapies, among others.

[0008] In contrast, thyroid hormone is involved in heat production, regulation of metabolism, and has a broad effect on gene and protein expression. Deficiencies in thyroid hormone production, or hypothyroidism, such as in Hashimoto's thyroiditis, results in a number of clinical manifestations, including changes in skin, loss of hair and teeth, increased bulk of body, excess subcutaneous fat, as well as, problems with speech, movement, sensation, consciousness, and intellect.

[0009] Given the prominent roles that lipid-soluble hormones, such as steroid hormones, retinoids, and thyroid hormones, play in health and disease, it would be desirable to find new methods to modulate the function of these hormones, while avoiding the side effects associated with many existing treatments. The present invention satisfies these and other needs.

BRIEF SUMMARY OF THE INVENTION

[0010] Many xenobiotics have been associated with endocrine effects in a wide range of biological systems. These associations are usually between small non-steroid molecules and steroid receptor signaling systems. As described herein, triclocarban (TCC; 3,4,4'- trichlorocarbanilide), a common ingredient in personal care products that is employed as an antimicrobial agent, was evaluated and found to represent a new category of endocrine- disrupting substance (EDS). A cell-based androgen receptor-mediated bioassay was used to demonstrate that TCC and other urea compounds with a similar structure, which have little or no endocrine activity when tested alone, act to enhance testosterone (T) induced androgen receptor-mediated transcriptional activity in vitro. This amplification effect of TCC was also apparent in vivo when 0.25% TCC was added to the diet of castrated male rats that were supported by exogenous testosterone treatment for ten days. All male sex accessory organs increased significantly in size following the T+TCC treatment compared to T or TCC treatments alone. The data presented herein suggest that the bioactivity of endogenous and exogenous hormones may be amplified by exposure to commercial personal care products containing sufficient levels of TCC and related compounds. [0011] Accordingly, the present invention provides compositions and methods for enhancing steroid receptor-mediated signal transduction.

[0012] One embodiment of the invention provides methods of enhancing steroid hormone signal transduction in a mammal, the method comprising contacting a cell with a composition comprising: (a) a therapeutically effective amount of a steroid; and (b) a therapeutically effective amount of a compound having the Formula I:

R² R³ (I) wherein

R¹ is a member selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^a, CO₂H, NHC(O)R², OC(O)NHR³ and NHC(O)OR³; wherein R^a is Ci_-6 alkyl and each

R¹ group is optionally substituted with 1 to 2 R^m substituents, and wherein optionally any two R¹ group located on adjacent atoms can be combined to form a 5- to 6-membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; n is an integer from 0-4; R² and R³ are each members independently selected from the group consisting of hydrogen, Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy and Ci_-8 alkoxy;

R⁴ is a member selected from the group consisting OfCi_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively, R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, C_]-8 dialkylamino, NO₂, CN, CO₂R^b, CO₂H, NHC0R^b,

0C0NHR^b and NHCOOR^b; wherein R^b is Ci_-6 alkyl and each R⁰ group is optionally substituted with 1 to 2 R^p substituents, and wherein optionally any two R⁰ group located on adjacent atoms can be combined to form a 5- to 6- membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; and wherein R^m, Rⁿ and R^p are each independently selected from the group consisting of halogen, Ci_-8 alkyl, aryl, hydroxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino and Ci_-8 alkoxy. [0013] Another embodiment of the invention provides a method of providing androgen replacement therapy, comprising administering to a subject in need thereof, (a) a therapeutically effective amount of an androgen replacement agent; and (b) a therapeutically effective amount of a compound having the Formula I:

wherein

R¹ is a member selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy,

NH₂, Ci_-8 alkylamino, C,_-8 dialkylamino, NO₂, CN, CO₂R³, CO₂H, NHC(O)R³, OC(O)NHR^a and NHC(O)OR³; wherein R^a is Ci_-6 alkyl and each R¹ group is optionally substituted with 1 to 2 R^m substituents, and wherein optionally any two R¹ group located on adjacent atoms can be combined to form a 5- to 6-membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; n is an integer from 0-4;

R² and R³ are each members independently selected from the group consisting of hydrogen, C_1-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy and Ci_-8 alkoxy; R⁴ is a member selected from the group consisting of Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively, R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy,

NH₂, C_1-8 alkylamino, C_-8 dialkylamino, NO₂, CN, CO₂R^b, CO₂H, NHCOR^b, OCONHR^b and NHCOOR^b; wherein R^b is Ci_-6 alkyl and each R⁰ group is optionally substituted with 1 to 2 R^p substituents, and wherein optionally any two R⁰ group located on adjacent atoms can be combined to form a 5- to 6- membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; and wherein R^m, Rⁿ and R^p are each independently selected from the group consisting of halogen, Ci_-8 alkyl, aryl, hydroxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino and Ci_-8 alkoxy. [0014] Another embodiment of the invention provides a method of preparing the cervix for parturition or child birth comprising administering to a subject in need thereof, (a) a therapeutically effective amount of an estrogen; and (b) a therapeutically effective amount of a compound having the Formula I:

wherein R¹ is a member selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^a, CO₂H, NHC(O)R³, OC(O)NHR³ and NHC(O)OR³; wherein R^a is Ci_-6 alkyl and each R¹ group is optionally substituted with 1 to 2 R^m substituents, and wherein optionally any two R¹ group located on adjacent atoms can be combined to form a 5- to 6-membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; n is an integer from 0-4;

R² and R³ are each members independently selected from the group consisting of hydrogen, Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy and

Ci_-8 alkoxy;

R⁴ is a member selected from the group consisting of Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively, R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, KH₂, C_1-8 alkylamino, C_1-8 dialkylamino, NO₂, CN, CO₂R^b, CO₂H, NHCOR^b, OCONHR^b and NHCOOR^b; wherein R^b is Ci_-6 alkyl and each R⁰ group is optionally substituted with 1 to 2 R^p substituents, and wherein optionally any two R⁰ group located on adjacent atoms can be combined to form a 5- to 6- membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; and wherein R^m, Rⁿ and R^p are each independently selected from the group consisting of halogen, Ci_-8 alkyl, aryl, hydroxy, NH₂, Ci_-8 alkylamino, Cj_-8 dialkylamino and Ci_-8 alkoxy.

[0015] Another embodiment of the invention provides a method of preparing the cervix for parturition or child birth comprising administering to a subject in need thereof, (a) a therapeutically effective amount of an estrogen; and (b) a therapeutically effective amount of a compound having the Formula I for internal delivery:

R² R³ (I) wherein

R¹ is a member selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^a, CO₂H, NHC(0)R^a, OC(O)NHR³ and NHC(O)OR³; wherein R^a is Ci_-6 alkyl and each

R¹ group is optionally substituted with 1 to 2 R^m substituents, and wherein optionally any two R¹ group located on adjacent atoms can be combined to form a 5- to 6-membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; n is an integer from 0-4; R² and R³ are each members independently selected from the group consisting of hydrogen, Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy and Ci_-8 alkoxy; R⁴ is a member selected from the group consisting of Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively, R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, C_-8 dialkylamino, NO₂, CN, CO₂R^b, CO₂H, NHCOR^b, OCONHR^b and NHCOOR^b; wherein R^b is Ci_-6 alkyl and each R⁰ group is optionally substituted with 1 to 2 R^p substituents, and wherein optionally any two R⁰ group located on adjacent atoms can be combined to form a 5- to 6- membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; and wherein R^m, R" and R^p are each independently selected from the group consisting of halogen, Ci_-8 alkyl, aryl, hydroxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino and Ci_-8 alkoxy. [0016] Another embodiment of the invention provides a method of enhancing the efficacy of topical corticoid administration, the method comprising the step of administering to a subject in need thereof: (a) a therapeutically effective amount of a topically administered corticoid, and (b) a therapeutically effective amount of a compound having the Formula I:

wherein

R¹ is a member selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R³, CO₂H,

NHC(O)R³, OC(O)NHR³ and NHC(O)OR³; wherein R³ is Ci_-6 alkyl and each R¹ group is optionally substituted with 1 to 2 R^m substituents, and wherein optionally any two R¹ group located on adjacent atoms can be combined to form a 5- to 6-membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; n is an integer from 0-4;

R² and R³ are each members independently selected from the group consisting of hydrogen, Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy and Ci_-8 alkoxy;

R⁴ is a member selected from the group consisting of Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively, R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^b, CO₂H, NHCOR^b,

OCONHR^b and NHCOOR^b; wherein R^b is Ci_-6 alkyl and each R⁰ group is optionally substituted with 1 to 2 R^p substituents, and wherein optionally any two R⁰ group located on adjacent atoms can be combined to form a 5- to 6- membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; and wherein R^m, Rⁿ and R^p are each independently selected from the group consisting of halogen, Ci_-8 alkyl, aryl, hydroxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino and Ci_-8 alkoxy. hi some aspects, the topical corticoid comprises Cortisol or hydrocortisone. [0017] Another embodiment of the invention provides a method of treating delayed puberty, the method comprising the step of administering to a subject in need thereof, (a) a therapeutically effective amount of a steroid hormone; and (b) a therapeutically effective amount of a compound having the Formula I:

wherein R¹ is a member selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R³, CO₂H, NHC(O)R³, OC(O)NHR³ and NHC(O)OR³; wherein R^a is C_1-6 alkyl and each R¹ group is optionally substituted with 1 to 2 R^m substituents, and wherein optionally any two R¹ group located on adjacent atoms can be combined to form a 5- to 6-membered saturated or unsaturated ring having from O to 2 heteroatoms selected from N, O and S; n is an integer from 0-4; R² and R³ are each members independently selected from the group consisting of hydrogen, Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy and Ci_-8 alkoxy;

R⁴ is a member selected from the group consisting of Ci_-8 alkyl, C_1-8 haloalkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively, R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, C_1-8 dialkylamino, NO₂, CN, CO₂R^b, CO₂H, NHC0R^b, 0C0NHR^b and NHC00R^b; wherein R^b is Ci_-6 alkyl and each R⁰ group is optionally substituted with 1 to 2 R^p substituents, and wherein optionally any two R⁰ group located on adjacent atoms can be combined to form a 5- to 6- membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; and wherein R^m, Rⁿ and R^p are each independently selected from the group consisting of halogen, Ci_-8 alkyl, aryl, hydroxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino and Cj_-8 alkoxy. [0018] Another embodiment of the invention provides a method of treating hypothyroidism, the method comprising the step of administering to a subject in need thereof, (a) a therapeutically effective amount of a thyroid hormone; and (b) (b) a therapeutically effective amount of a compound having the Formula I:

wherein R¹ is a member selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^a, CO₂H, NHC(O)R³, OC(O)NHR³ and NHC(O)OR³; wherein R³ is C_]-6 alkyl and each R¹ group is optionally substituted with 1 to 2 R^m substituents, and wherein optionally any two R¹ group located on adjacent atoms can be combined to form a 5- to 6-membered saturated or unsaturated ring having from O to 2 heteroatoms selected from N, O and S; n is an integer from 0-4; R² and R³ are each members independently selected from the group consisting of hydrogen, Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy and Ci_-8 alkoxy; R⁴ is a member selected from the group consisting of Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively, R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^b, CO₂H, NHCOR^b, OCONHR^b and NHC00R^b; wherein R^b is Ci_-6 alkyl and each R⁰ group is optionally substituted with 1 to 2 R^p substituents, and wherein optionally any two R⁰ group located on adjacent atoms can be combined to form a 5- to 6- membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; and wherein R^m, Rⁿ and R^p are each independently selected from the group consisting of halogen, Ci_-8 alkyl, aryl, hydroxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino and Ci_-8 alkoxy. [0019] Another embodiment of the invention provides a method of enhancing the efficacy of weak estrogen in hormone replacement therapy, the method comprising the step of administering to a subject in need thereof, (a) a therapeutically effective amount of a weak estrogen; and (b) a therapeutically effective amount of a compound having the Formula I:

(I) wherein

R¹ is a member selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^a, CO₂H, NHC(O)R^a, OC(O)NHR^a and NHC(O)OR^a; wherein R^a is Ci_-6 alkyl and each

R¹ group is optionally substituted with 1 to 2 R^m substituents, and wherein optionally any two R¹ group located on adjacent atoms can be combined to form a 5- to 6-membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; n is an integer from 0-4;

R⁴ is a member selected from the group consisting of C_L8 alkyl, C]_-8 haloalkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively, R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, C_-8 dialkylamino, NO₂, CN, CO₂R^b, CO₂H, NHC0R^b,

OCONHR^b and NHCOOR^b; wherein R^b is Ci_-6 alkyl and each R⁰ group is optionally substituted with 1 to 2 R^p substituents, and wherein optionally any two R⁰ group located on adjacent atoms can be combined to form a 5- to 6- membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; and wherein R^m, Rⁿ and R^p are each independently selected from the group consisting of halogen, Ci_-8 alkyl, aryl, hydroxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino and Ci_-8 alkoxy. [0020] Another embodiment of the invention provides a pharmaceutical composition for use as a systemic anti-inflammatory agent comprising (a) a therapeutically effective amount of a corticoid; and (b) a therapeutically effective amount of a compound having the Formula I:

wherein

R¹ is a member selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^a, CO₂H,

NHC(O)R³, OC(O)NHR^a and NHC(O)OR^a; wherein R^a is Ci_-6 alkyl and each R¹ group is optionally substituted with 1 to 2 R^m substituents, and wherein optionally any two R¹ group located on adjacent atoms can be combined to form a 5- to 6-membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; n is an integer from 0-4;

R² and R³ are each members independently selected from the group consisting of hydrogen, Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy and Ci_-8 alkoxy; R⁴ is a member selected from the group consisting Of Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively, R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy,

NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^b, CO₂H, NHCOR^b, OCONHR^b and NHCOOR^b; wherein R^b is Ci_-6 alkyl and each R⁰ group is optionally substituted with 1 to 2 R^p substituents, and wherein optionally any two R⁰ group located on adjacent atoms can be combined to form a 5- to 6- membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; and wherein R^m, Rⁿ and R^p are each independently selected from the group consisting of halogen, Ci_-8 alkyl, aryl, hydroxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino and Ci_-8 alkoxy. [0021] Another embodiment of the invention provides a pharmaceutical composition comprising (a) a therapeutically effective amount of a steroid; and (b) a therapeutically effective amount of a compound having the Formula I:

wherein

NHC(O)R³, OC(O)NHR³ and NHC(O)OR³; wherein R^a is Ci_-6 alkyl and each R¹ group is optionally substituted with 1 to 2 R^m substituents, and wherein optionally any two R¹ group located on adjacent atoms can be combined to form a 5- to 6-membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; n is an integer from 0-4;

R² and R³ are each members independently selected from the group consisting of hydrogen, Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy and Ci_-8 alkoxy; R⁴ is a member selected from the group consisting Of Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively, R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Cj_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy,

NH₂, C_1-8 alkylamino, C_1-8 dialkylamino, NO₂, CN, CO₂R^b, CO₂H, NHC0R^b, 0C0NHR^b and NHC00R^b; wherein R^b is Ci_-6 alkyl and each R⁰ group is optionally substituted with 1 to 2 R^p substituents, and wherein optionally any two R⁰ group located on adjacent atoms can be combined to form a 5- to 6- membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; and wherein R^m, R" and R^p are each independently selected from the group consisting of halogen, Ci_-8 alkyl, aryl, hydroxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino and Ci_-8 alkoxy. [0022] Another embodiment of the invention provides methods of enhancing steroid hormone signal transduction in a mammal, the method comprising administering to said mammal a composition for internal delivery comprising: a therapeutically effective amount of a compound having the Formula I:

wherein R¹ is a member selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R³, CO₂H, NHC(O)R³, OC(O)NHR³ and NHC(O)OR³; wherein R^a is Ci_-6 alkyl and each R¹ group is optionally substituted with 1 to 2 R^m substituents, and wherein optionally any two R¹ group located on adjacent atoms can be combined to form a 5- to 6-membered saturated or unsaturated ring having from O to 2 heteroatoms selected from N, O and S; n is an integer from 0-4;

Ci_-8 alkoxy;

R⁴ is a member selected from the group consisting of Ci_-8 alkyl, Cj_-8 haloalkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively, R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^b, CO₂H, NHC0R^b, 0C0NHR^b and NHC00R^b; wherein R^b is Ci_-6 alkyl and each R⁰ group is optionally substituted with 1 to 2 R^p substituents, and wherein optionally any two R⁰ group located on adjacent atoms can be combined to form a 5- to 6- membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; and wherein R^m, Rⁿ and R^p are each independently selected from the group consisting of halogen, Ci_-8 alkyl, aryl, hydroxy, NH₂, Ci_-8 alkylamino, C_1-8 dialkylamino and Ci_-8 alkoxy. [0023] Another embodiment of the invention provides a pharmaceutical composition for internal delivery comprising a therapeutically effective amount of a compound having the Formula I:

wherein

R¹ is a member selected from the group consisting of halogen, C_1-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, C_-8 alkylamino, C_-8 dialkylamino, NO₂, CN, CO₂R^a, CO₂H, NHC(O)R^a, OC(O)NHR³ and NHC(O)OR³; wherein R^a is C_-6 alkyl and each R¹ group is optionally substituted with 1 to 2 R^m substituents, and wherein optionally any two R¹ group located on adjacent atoms can be combined to form a 5- to 6-membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; n is an integer from 0-4; R² and R³ are each members independently selected from the group consisting of hydrogen, C_-8 alkyl, C_-8 haloalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy and C_-8 alkoxy; R⁴ is a member selected from the group consisting of Ci_-8 alkyl, C_-8 haloalkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively, R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, C_-8 alkyl, C_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, C_-8 alkoxy, NH₂, C_1-8 alkylamino, C_-8 dialkylamino, NO₂, CN, CO₂R^b, CO₂H, NHCOR^b, OCONHR^b and NHCOOR^b; wherein R^b is C_1-6 alkyl and each R⁰ group is optionally substituted with 1 to 2 R^p substituents, and wherein optionally any two R⁰ group located on adjacent atoms can be combined to form a 5- to 6- membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; and wherein R^m, Rⁿ and R^p are each independently selected from the group consisting of halogen, Ci_-8 alkyl, aryl, hydroxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino and C_-8 alkoxy. [0024] In some aspects of the above embodiments, in Formula I, R¹ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN, wherein each R¹ group is optionally substituted with 1 to 2 R^m substituents; n is an integer from 1 to 3; R² and R³ are each independently selected from the group consisting of hydrogen and Ci_-8 alkyl; R⁴ is Ci_-8 alkyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN; and each R⁰ group is optionally substituted with 1 to 2 R^p substituents.

[0025] In other aspects of the above embodiments, in Formula I, R⁴ is selected from the group consisting Of Ci_-8 alkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 R" substituents. In some embodiments, in Formula I, R³ and R⁴ are each independently selected from the group consisting of methyl, ethyl, propyl and butyl. In some embodiments, in Formula I, in Formula I, R⁴ is selected from the group consisting of aryl and heteroaryl; and is optionally substituted with 1 to 4 R⁰ substituents.

[0026] In other aspects of the above embodiments, the compound of Formula I has the Subformula Ia:

'ιm

(Ia) wherein m is an integer from 0 to 4.

[0027] In other aspects of the above embodiments, in Subformula Ia, R¹ and R⁰ are each independently selected from the group consisting Of Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN; m and n are each an integer from 1 to 3; and R² and R³ are each independently hydrogen or Ci_-8 alkyl. In some embodiments, in Subformula Ia, R¹ and R⁰ are independently selected from the group consisting of fluoride, chloride, bromide and iodide; and m and n are each independently an integer from 1 to 3.

[0028] In other aspects of the above embodiments, the compound of Formula I is selected from the group consisting of:

[0029] In other aspects of the above embodiments, the steroid is testosterone. In other aspects, the steroid is an androgen, such as testosterone, an estrogen, such as estradiol, estriol, or estrone, a glucocorticoid, a corticoid, such as beclometasone, prednisone, or dexamethasone, Cortisol, or a thyroid hormone, such as levothyroxine. Li some aspects, the cell is in a mammal, hi other aspects, the mammal is a human, hi some aspects, the human has a disease or disorder associated with low levels of endogenous steroids (e.g., menopause, peri-menopause, sexual dysfunction, delayed puberty, infertility, hypoandrogenism, and combinations thereof). In other aspects, the disease, disorder, or condition is symptoms resulting from post-menopause, child birth or parturition, and prostate cancer. The symptoms of post-menopause can include: osteoporosis, hot flashes, vaginal dryness, urinary stress incontinence, chilly sensations, dizziness, fatigue, irritability, and sweating, hi other aspects, the disease, disorder, or condition can be allergic, inflammatory, and autoimmune disorders, acute transplant rejection, and graft-versus-host disease, hi other aspects, the internal delivery is by means of oral delivery, injectable delivery, nasal delivery, or transmucosal delivery.

[0030] Another embodiment of the invention provides in vitro methods of identifying modulators of steroid hormone signal transduction, said method comprising: (a) contacting a cell expressing an androgen receptor with an androgen and a test compound suspected of having the ability to modulate steroid hormone signal transduction; and (b) determining whether the test compound modulates signal transduction through the androgen receptor, wherein a compound that increases signal transduction through the androgen receptor relative to a control is identified as a compound that enhances steroid hormone signal transduction and a compound that reduces signal transduction through the androgen receptor relative to a control is identified as a compound that represses steroid hormone signal transduction, hi some embodiments, the androgen is testosterone, hi some embodiments, the cell is transfected with the androgen receptor. In some embodiments step (b) comprises measuring the signal from a reporter gene following step (a). In some embodiments, the reporter gene is luciferase.

[0031] A further embodiment of the invention provides pharmaceutically acceptable compositions comprising a therapeutically effective amount of a steroid and a therapeutically effective amount of a compound having Formula I or Subformula Ia as set forth herein. In some embodiments, the steroid is testosterone.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 illustrates the structure of triclocarban (3,4,4'-Trichlorocarbanilide) (TCC). [0033] Figure 2 illustrates data demonstrating that TCC alone has little, if any androgenic activity, but when combined with testosterone a 45% of increase in T induced signal was observed.

[0034] Figure 3 illustrates data from competitive binding experiments demonstrating that the amplification of transcriptional activity by TCC is hAR dependent. [0035] Figure 4 illustrates data demonstrating that amplification of the T-induced signal by

TCC is upstream through the AR rather than the result of a post-translational modification of androgen receptor signaling through the cAMP/PKA pathway.

[0036] Figure 5 illustrates the chemical structure of testosterone and dihydrotestosterone and the conventional numbering of carbons in the steroid nucleus.

[0037] Figure 6 illustrates the effect of TCC on cell proliferation. 2933 Y cells were treated for 16 hours with TCC (1.0 μM) alone or in combination with testosterone (0.125 nJVI). The cytotoxicity or cell proliferation was evaluated by the 3-(4,5-dimethylthiazolyl-2)-2, 5- diphenyltetrazolium bromide (MTT) assay. The absorbance was measured at 570 ran with a reference wavelength of 650 nm using an EMax Spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). Absorbance (optical density) at 570 nm is expressed as mean ± SD (n=6). No significant difference in cell proliferation was observed in cells with TCC alone or in combination with T when compared to the vehicle control.

[0038] Figure 7 illustrates the effect of TCC on AR-mediated transcriptional activity induced by testosterone. 2933 Y cells were treated for 16 hours with and without TCC (1.0 μM) and in combination with testosterone (0.125 nM) and/or flutamide (10 μM). Cell lysates were assessed for luciferase activity which is expressed as mean + SD (n=4) of relative light units (RLU). a: significantly different from vehicle control; b: significantly different from vehicle control and from T treatment. [0039] Figure 8 illustrates, in Panel a., the time course effect of TCC on AR-mediated transcriptional activity induced by testosterone. 2933 Y cells were treated with T (0.125 nM) alone (•) or with a combination of TCC (1.0 μM) (o) for different time periods; Panel b. The dose response effect of TCC on AR-mediated transcriptional activity induced by testosterone. 2933 Y cells were treated with T (0 - 1.0 nM) alone (•) or with a combination of TCC (1.0 μM) (o) for 16 hours; Panel c. The dose response effect of TCC on AR-mediated transcriptional activity induced by testosterone. 2933Y cells were treated with T (0.125 nM) alone or with a combination of various concentrations of TCC for 16 hours. In each experiment, cell lysates were measured for luciferase activity which is expressed as mean + SD (n=3) of relative light units (RLU). *: Significantly different from T treatment.

[0040] Figure 9 illustrates the augmentation of TCC analogs on AR-mediated transcriptional activity. Closed bar: T at 0.125 nM alone; open bar: vehicle control; checkered bar: TCC analogs at 1.0 μM alone; hatched bar: TCC analogs at 1.0 μM in the presence of 0.125 nM of T. 1 : Carbanilide; 2: 4,4'-Dichlorocarbanilide; 3: TCC (3,4,4'- Trichlorocarbanilide); 4: 3,3',4,4'-Tetrachlorocarbanilide and 5: 4'-Methoxy-3,4-dichloro- carbanilide.

[0041] Figure 10 illustrates competitive binding of TCC in AR fluorescence polarization (FP) assay. Rat AR ligand binding domain (ARLBD)/Fluormone complex was incubated with TCC at various concentrations. A: maximum fluorescence polarization (FP) in the absence of any competitor. B and C: FP values in the presence of DHT, a strong AR competitor (B: 10 nM and C: 100 nM); D-I: FP values in the presence of increasing concentrations of TCC (D: 2 nM; E: 2OnM; F: 200 nM; G: 2 μM; H: 20 μM; I: 200μM). Data presented as mean + SD of triplicates.

[0042] Figure 11 illustrates the effect of TCC on the amount of immunoreactive AR protein. MDA-kb2 or 2933Y cells were treated with vehicle, T (1.0 nM), TCC (1.0 μM) or a combination of T+TCC for 48 hours. Whole-cell lysates were probed by western blot analysis with antibody against amino acids 299-315 of human AR. Each lane contained either 60 μg (for MDA-kb2) or 15 μg (for 2933Y) of protein. Veh: vehicle control.

[0043] Figure 12 illustrates the effect of TCC on cAMP/PKA-mediated transcriptional activity induced by human chorionic gonadotrophin (hCG). JK293 cells were treated for 16 hours with and without TCC (1.0 μM) and in combination with hCG (3.2 ng/mL), T (0.125 nM) and/or flutamide (10 μM). Cell lysates were measured for luciferase activity which is expressed as mean ± SD (n=4) of relative light units (RLU). Neither T nor flutamide induced any effect on cAMP/PKA-mediated transcriptional activity. No significant differences in luciferase activities were observed in TCC and hCG combinations when compared to hCG treatment alone.

[0044] Figure 13, in Panel A, illustrates enhancement of estradiol (E2) induced transcriptional activity by TCC in an ER alpha-mediated bioassay (Rogers J et al, In Vitro MoI Toxicol. 2000 13(l):67-82.) Treated with E2 alone (open circles); treated with E2 and 1.0 μM of TCC (closed circles); Panel B: Enhancement of the cross-reactivity of Cortisol by TCC in the AR-mediated transcriptional activity in 2933Y cells. 2933Y cells also have low endogenous glucocorticoid receptor expression (Chen J et al, 2006 JCEM 2006

Nov;91(l l):4387-94). Treated with various concentrations of T alone (open circles); treated with various concentrations of T and 1.0 μM of TCC (closed circles); treated with various concentrations of Cortisol alone (open triangles); treated with various concentrations of Cortisol and 1.0 μM of TCC (closed triangles).

[0045] Figure 14 illustrates the effect of enhancement of estrogenic activity by TTC. Each panel depicts the dose response of each of the three primary estrogens with (closed circles) and without (open circle) the addition of TCC. A constant amount of TCC ( 1 μM) was added to each standard concentration of estrone, estradiol, and estriol, respectively.

[0046] Figure 15 illustrates the effect of TCC on Cortisol induced transcriptional activity. A constant amount of TCC (1 uM) was added to various medium containing various amount of Cortisol. Open circle: Cortisol only; closed circle: TCC and Cortisol co-treatment.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

[0047] The growth of high-production- volume compounds in our daily life has continued to raise public concern on their potential ecological and human health impact. Numerous reports have revealed associations between environmental exposures and reduced fecundity, abnormal fetal development, delayed onset of puberty, disruption of ovarian function, abnormal lactation, early onset of reproductive senescence, and cancer. It is speculated that the etiologies of these conditions are environmental in origin as a result of persistent contaminations and many of these adverse effects are mediated through the interference of xenobiotics with sex steroid hormone action. These environmental xenobiotics are generally referred to as endocrine-disrupting substances (EDSs).

[0048] Efforts to identify and characterize these environmental compounds has led to the classification of a relatively large number of EDSs that have estrogenic activity. In contrast, comparatively few compounds have been associated with androgenic activity despite increasing public concern regarding environmental influences on male reproductive health. Thus, the recent reports of a large number of non-steroidal compounds that have the ability to activate the human androgen receptor (hAR) are of particular concern because human exposures to many of these compounds are ubiquitous and constant. [0049] Triclocarban, an antimicrobial compound (TCC; 3,4,4'-trichlorocarbanilide), is a high-production-volume chemical, commonly added to a wide range of household and personal care products including bar soaps, detergents, body washes, cleansing lotions, and wipes for its sanitizing properties. TCC containing products have been marketed broadly for more than 45 years, and thus, have a long history of use in Europe and the U.S. [0050] As a result of studies designed to characterize the endocrine-disrupting activity of TCC, we have discovered that TCC amplifies the action of a number of lipid-soluble hormones that bind to members of the steroid hormone receptor superfamily, including steroid hormones that bind to androgen, estrogen, and glucocorticoid receptors. [0051] Thus, the present invention provides compositions and methods for enhancing androgen receptor-mediated signal transduction. In particular, the invention provides methods of using compounds of Formula (I) to enhance steroid receptor-mediated signal transduction. The invention is based on the surprising discovery that compounds of Formula (I) enhance steroid receptor-mediated signal transduction. Thus compounds of Formula (I) can be administered in combination with steroids to enhance steroid receptor-mediated signal transduction, i.e., to enhance the effect of the steroid so that lower doses of steroid can be administered to a subject. The invention also provides methods for identifying additional compounds that enhance steroid receptor-mediated signal transduction.

II. Definitions

[0052] A "steroid" or "steroid hormone" as used herein refers generally to a class of lipid- soluble compounds capable of binding to a member of the steroid hormone receptor superfamily. Examples of steroids can include androgens, estrogens, glucocorticoids, thyroid hormones, retinoids, among others, as described herein. This term encompasses synthetic forms, mimics, and analogs, as well as, naturally occurring forms of steroids. [0053] "Androgen" as used herein refers to any natural or synthetic compound, usually a steroid hormone, that stimulates or controls the development and maintenance of masculine characteristics (i.e., development of male sex organs and male secondary sex characteristics) in vertebrates by binding to androgen receptors. Androgens include, for example, testosterone, adrenal androgens such as dehydroepiandrosterone (DHEA), androstenedione, androstenediol, androsterone, and dihydrotestosterone (DHT). [0054] "Estrogen" as used herein refers to any natural or synthetic compound, usually a steroid hormone, that stimulates or controls the estrous cycle and functions as the primary female sex hormone. Estrogens include, for example, the naturally occurring compounds, estradiol, estriol, and estrone. While estrogens are present in both men and women, they are usually present at significantly higher levels in women of reproductive age. They promote the development of female secondary sex characteristics, such as breasts, and are also involved in the thickening of the endometrium and other aspects of regulating the menstrual cycle. In males estrogen regulates certain functions of the reproductive system important to the maturation of sperm and may be necessary for a healthy libido.

[0055] A "weak estrogen" as used herein refers to natural or synthetic estrogens as defined above (e.g., C-18 steroids and non-steroidal estrogenic compounds) that have less bioactivity than estradiol. Examples of weak estrogens include: Estrone, Estriol, 16-hydroxyestrone, 2- hydroxyestrone, 2,4- hydroxyestrone, and 4- hydroxyestrone, as well as, non-steroidal estrogenic compounds such as phytoestrogens and their congeners.

[0056] "Corticosteroid" or "corticoid" as used herein refers to a class of natural or synthetic steroid hormones that are produced in the adrenal cortex. Corticosteroids are involved in a wide range of physiologic processes, such as stress responses, immune responses and in the regulation of inflammation, carbohydrate metabolism, protein catabolism, blood electrolyte levels, and behavior. One class of corticosteroids or corticoids includes glucocorticoids, such as Cortisol, which controls carbohydrate, fat and protein metabolism, and which has anti- inflammatory activity by functioning to prevent phospholipid release, decreasing eosinophil action, and through a number of other mechanisms. Examples of corticosteroids or corticoids that are used as anti-inflammatory agents include, but are not limited to, beclometasone dipropionate, prednisone, and dexamethasone.

[0057] "Thyroid hormone" as used herein refers to tyrosine-based hormones produced by the thyroid gland, including thyroxine (T4) and triiodothyronine (T3). Both natural and synthetic forms of thyroid hormone are encompassed by this term. For example, levothyroxine is a synthetic form of thyroxine which is a stereoisomer of physiological thyroxine. Also included, for example, are natural desiccated thyroid hormones, which are used as a "natural" hypothyroid treatment and contain 20% T3 and traces of T2 (diiodothyronine), Tl (monoiodothyronine), and calcitonin.

[0058] "Steroid hormone receptor superfamily" as used herein refers to a class of nuclear receptors that binds to lipid-soluble hormones, such as steroid hormones, retinoids, and thyroid hormones, among others, as described elsewhere herein and known in the art. As known in the art, members of the steroid hormone receptor superfamily share a common domain structure which includes a hormone binding domain and a DNA binding domain, and generally, these receptors function by regulating transcriptional activity of target genes upon hormone binding. See generally, e.g., Lodish et al, Molecular Cell Biology, 3th edition, Scientific American Books (1995), for a review. [0059] "Contacting" when used in reference to contacting a cell with a steroid or a compound of Formula I refers to actual physical contact between the cell and the antagonist or to bringing antagonist into proximity with the cell.

[0060] "Enhancing steroid hormone signal transduction" as used herein refers to modulating, activating, or repressing signal transduction by e.g., modulating, activating, or repressing gene expression of steroid hormone responsive genes. [0061] "Reduced attendant liver toxicity" as used herein refers to a lowered risk of developing liver toxicity as compared to patients who have an increased risk for developing liver toxicity as a result of androgen replacement therapy.

[0062] "Enhancing the efficacy", as used herein in the context of administration of a therapeutic agent refers to increasing the therapeutic effectiveness of a particular therapeutic agent, e.g., topical corticoids, by the additional administration of a second therapeutic agent, e.g., TCC.

[0063] "For internal delivery" as used herein refers to a non-transdermal delivery method (i.e., through the external skin) such as oral delivery, injectable delivery, rectal delivery, transmucosal delivery, nasal delivery, and the like. [0064] A 'therapeutically effective amount" or an "effective amount" of a steroid or a compound of Formula I is an amount sufficient to provide a therapeutic effect, i.e., an amount of effective for reducing, ameliorating, or inhibiting the symptoms of diseases or disorders associated with inappropriate levels of steroid receptor-mediated signal transduction {e.g., menopause and delayed onset of puberty) by at least 10%, preferably by at least 25%, more preferably by at least 50%, even more preferably by at least 60%, yet more preferably by at least 75%. Typically an effective amount of a compound of Formula I is about 1 μM to about 100 μM, more typically about 5 μM to about 75 μM, even more typically about 7.5 μM to about 50 μM, most typically, about 1 μM to about 10 μM. Typically an effective amount of a steroid is about 0.1 nM to about 100 nM, more typically about 5 nM to about 75 nM, even more typically about 7.5 nM to about 50 nM, most typically, about 0.1 nM to about 5OnM. [0065] "Mammal" as used herein refers to any warm blooded vertebrate born with a placenta, such as, for example, a rodent {e.g., a mouse, a rat, a hamster, a guinea pig, or a rabbit), a feline (e.g., a cat, a tiger, a lion, a lynx, or a panther), a canine (e.g., a dog, a wolf, a coyote), or a primate (e.g. , a monkey, a chimpanzee, a gorilla, or a human).

[0066] The term "composition" as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. By "pharmaceutically acceptable" it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0067] The term "alkyl", alone or in combination, refers to a straight-chain or branched- chain alkyl group having the indicated number of carbon atoms. For example, C_1-10 alkyl refers to an alkyl group having from one to ten carbon atoms with the remaining valences filled occupied by hydrogen atoms. Preferred alkyl groups are those with 1 to 8 carbon atoms, more preferably a straight or branched-chain alkyl group with 1 to 6 carbon atoms and particularly preferred are straight or branched-chain alkyl groups with 1 to 4 carbon atoms. Examples of straight-chain and branched Cj_-1O alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert. -butyl, the isomeric pentyls, the isomeric hexyls, the isomeric heptyls and the like.

[0068] The term "alkenyl", alone or in combination refers to a straight-chain or branched hydrocarbon residue comprising an olefinic bond and the indicated number of carbon atoms. Preferred alkenyl groups have up to 8, preferably up to 6, particularly preferred up to 4 carbon atoms. Examples of alkenyl groups are ethenyl, 1-propenyl, 2-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl and isobutenyl.

[0069] The term "alkynyl", alone or in combination refers to a straight-chain or branched hydrocarbon residue having a carbon carbon triple bond and the indicated number of carbon atoms. Preferred alkynyl groups have up to 8, preferably up to 6, particularly preferred up to 4 carbon atoms. Examples of alkynyl groups are ethynyl, 1-propynyl, 1-butynyl and 2- butynyl.

[0070] The terms "alkoxy," and "alkylamino" are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom or an amino group, respectively. Examples of alkoxy group include methoxy, ethoxy, n- propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy and tert-butoxy. Preferred alkoxy groups are methoxy and ethoxy. Examples of alkylamino groups include, methylamino, diethylamino, and the like. Additionally, for dialkylamino groups, the alkyl portions can be the same or different and can also be combined to form a 3-7 membered ring with the nitrogen atom to which each is attached.

[0071] The term "aryl" means, unless otherwise stated, a polyunsaturated, typically aromatic, hydrocarbon group which can be a single ring or multiple rings (up to three rings) which are fused together or linked covalently. Non-limiting examples of aryl groups include phenyl, naphthyl and biphenyl. The aryl groups are optionally substituted with, for example, groups such as alkyl, haloalkyl, heteroalkyl, alkenyl, alkynyl, hydroxy, alkoxy, amino, alkylamino, nitro, cyano, carboxy, carboxyester and carbamates, among others. [0072] The term "heteroaryl", alone or in combination, signifies aromatic 5- to 10- membered heterocycle which contains one or more, preferably one or two hetero atoms selected from nitrogen, oxygen and sulfur, wherein nitrogen or oxygen are preferred. Examples of heteroaryl groups include pyridinyl, pyrrolyl, imidazolyl, thienyl and the like. The heteroaryl groups are optionally substituted with substituents, such as, alkyl, haloalkyl, heteroalkyl, alkenyl, alkynyl, hydroxy, alkoxy, amino, alkylamino, nitro, cyano, carboxy, carboxyester and carbamates, among others. [0073] The term "heteroalkyl," by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or combinations thereof, consisting of the stated number of carbon atoms and from one to three heteroatoms selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. [0074] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl," are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "C^₄ haloalkyl" is mean to include trifluoromethyl, 2,2,2-trifluoroethyl, 4- chlorobutyl, 3-bromopropyl, and the like. III. Compositions Comprising Enhancers of Steroid Receptor-Mediated Signal Transduction

[0075] In one aspect, the present invention provides for a pharmaceutically acceptable composition that enhances steroid hormone mediated signal transduction in a mammal. The composition comprises: (a) a therapeutically effective amount of a steroid; and (b) a therapeutically effective amount of a compound having the Formula I:

[0076] In a compound having Formula I, R¹ is a member selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, C₁. g alkoxy, NH₂, C_1-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^a, CO₂H, NHC(O)R^a, OC(O)NHR³ and NHC(O)OR³; wherein R³ is C_1-6 alkyl and each R¹ group is optionally substituted with 1 to 2 R^m substituents, and optionally any two R¹ group located on adjacent atoms can be combined to form a 5- to 6-membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S. The subscript n is an integer from 0-4. R² and R³ are each members independently selected from the group consisting of hydrogen, Ci_-8 alkyl, C]_-8 haloalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy and Ci_-8 alkoxy. R⁴ is a member selected from the group consisting of Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents. Alternatively, R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^b, CO₂H, NHC0R^b, OCONHR^b and NHCOOR^b; wherein R^b is Ci_-6 alkyl. Each R⁰ group is optionally substituted with 1 to 2 R^p substituents, and optionally any two R⁰ group located on adjacent atoms can be combined to form a 5- to 6-membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S. R^m, Rⁿ and R^p are each independently selected from the group consisting of halogen, Ci_-8 alkyl, aryl, hydroxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino and C]_-8 alkoxy. In one embodiment, R⁴ is selected from the group consisting of Ci_-8 alkyl, C_2-8 alkenyl and C_2-8 alkynyl and is optionally substituted with from 1 to 2 Rⁿ substituents. In one embodiment, R³ and R⁴ are each independently selected from the group consisting of methyl, ethyl, propyl and butyl. In another embodiment, R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with 1 to 4 R⁰ substituents, wherein in certain aspects of this embodiment, R⁰ is halogen.

[0077] In certain embodiments, in Formula I, R¹ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN, wherein each R¹ group is optionally substituted with 1 to 2 R^m substituents; n is an integer from 1 to 3; R² and R³ are each independently selected from the group consisting of hydrogen and Ci_-8 alkyl; R⁴ is Ci_-8 alkyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN; and each R⁰ group is optionally substituted with 1 to 2 R^p substituents.

[0078] In other embodiments, the compound of Formula I has Sub formula Ia:

wherein the subscript m is an integer from 0 to 4. hi Subformula Ia, R¹ and R⁰ are each independently selected from the group consisting of Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN. The subscripts m and n are each an integer from 1 to 3. R² and R³ are each independently hydrogen or Ci_-8 alkyl. hi certain aspects of this embodiment, R¹ and R⁰ are independently selected from the group consisting of fluoride, chloride, bromide and iodide, and the subscripts m and n are each independently an integer from 1 to 3. hi certain other aspects of this embodiment, R¹ and R⁰ are independently selected from the group consisting of fluoride, chloride and bromide, the subscripts m and n are each independently an integer from 1 to 2; and R² and R³ are each hydrogen. [0079] In another embodiment, the compounds having Formula I are selected from the group consisting of

[0080] Compounds suitable for use in the present invention include natural or synthetic lipid-soluble hormones that bind to members of the steroid hormone receptor superfamily. Examples of steroid compounds suitable for use in the composition are androgens, including but not limited to, testosterone, adrenal androgens such as dehydroepiandrosterone, androstenedione, androstenediol, androserone, and dihydrotestosterone; and derivatives thereof. Derivatives of testosterone derivatives include 17β-esters, 7α-methyl, 17α-alkyl or methyl, 19-normethyl and D-homo-androgens, and the like (see, Handelsman, "Testosterone and Other Androgens: Physiology, Pharmacology, and Therapeutic Use," in Endocrinology — Volume 3, Ed's DeGroot et al. (1995), 2351-2361). Other testosterone derivatives include testosterone substituted at the Cl position with methyl (e.g., methenolone and mesterolone); compounds with substitutions in and additions to, the A ring, e.g., oxandrolone and stanozolol, and the like (see, Catlin, D.H., "Anabolic Steroids," in Endocrinology —Volume 3, ed's DeGroot et al. pp. 2362-2376 (1995).); and glycosidic derivatives of testosterone (e.g., as described in U.S. Pat. No. 6,916,791); medroxyprogesterone and its acetate salt; norethindrone, and its acetate salt; and progestins.. In certain preferred embodiments, the steroid is testosterone or dihydrotestosterone (DHT) (see, Figure 4).

[0081] Other suitable compounds include estrogens, such as estradiol, estriol, and estrone. Also suitable for use in the invention are thyroid hormones, such as T4, T3, or lexothyroxine. Corticoids, such as naturally occurring or synthetic glucocorticoids, can also be used in the practice of the invention. Examples of such compounds include, but are not limited to, Cortisol, hydrocortisone, beclometasone, prednisone and dexamethasone. These compounds may be used in their various active salt and addition forms and in the form of other chemical derivatives and active stereoisomers.

Preparation of Compounds of Formula I

[0082] Compounds of Formula I that can enhance a steroid hormone mediated signal transduction may be available from commercial sources, although in certain case, de novo synthesis may be desirable. Generally, compounds of Formula I or Sub formula Ia can prepared as shown in Scheme 1 below.

Scheme 1

Pd Catalyst Carbon monoxide

[0083] For example, compounds of Formulae I and/or Ia can be prepared by combining a suitable isocyanate compound i with an amine ii in the presence of a base to form the urea compound iii of Formula I and Ia (See, Scheme 1, A). A skilled artisan would recognized that other electrophilic carbonyl derivatives, besides isocyanate i, would also be suitable for use in this reaction to produce compounds of iii. Other examples of carbonyl derivatives suitable for the reaction shown in Scheme 1 (A) include a N-arylcarbamoyl chloride, a N- arylcarbamate, and the like, Also, compounds of Formulae I and Ia can be prepared by the transition metal catalyzed (e.g., Pd, etc) oxidative carbonylation of aniline iv in the presence of carbon monoxide to form compound v (See, Scheme 1, B). The substituents in R, R' and R" are non-interfering substituents such as, for example, hydrogen, alkyl, aryl alkoxy, halogen, and the like.

[0084] A skilled artisan would recognize other modifications of the above described routes as well as additional synthetic routes that would also produce compounds of Formula I. For example, it is apparent that one of the reagents (e.g., i, iv) could be tethered to a solid phase linker and the synthetic reactions could be carried out in combinatorial manner.

IV. Methods of Enhancing Steroid Receptor-Mediated Signal Transduction

[0085] One embodiment of the invention provides methods for enhancing steroid receptor- mediated signal transduction by administering a urea compound of Formula I. In some embodiments, pharmaceutical compositions comprising the urea compounds of Formula I and the steroids disclosed herein (e.g., testosterone) may be delivered to an individual in need of such treatment, e.g., a menopausal individual or an individual diagnosed with delayed onset of puberty. The composition is administered to enhance androgen receptor-mediated signal transduction in that individual. The individual is typically a mammal such as, for example, a human.

[0086] Formulation of pharmaceutically-acceptable excipients and carrier solutions is well- known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, and intramuscular administration and formulation. [0087] The dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial therapeutic response {e.g., a reduction the symptoms of disorders associated with low levels of androgen receptor-mediated signal transduction) in the patient over time. Such a therapeutically effective dose will be determined by the efficacy of the particular enhancer of androgen receptor-mediated signal transduction employed and the condition of the patient, as well as the body weight or surface area of the patient to be treated. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of an enhancer of androgen receptor-mediated signal transduction in a particular patient. The appropriate doses of androgens {e.g., testosterone) are those doses that create sustained normal circulating levels that are appropriate for age, life-stage and gender. The appropriate dose of TCC will be doses that exceed the natural ligand by approximately two to three orders of magnitude. [0088] In determining the effective amount {i.e., therapeutically effective amount) of the enhancer of androgen receptor-mediated signal transduction to be administered, the physician evaluates circulating plasma levels of the enhancer of androgen receptor-mediated signal transduction, toxicities of the enhancer of androgen receptor-mediated signal transduction, and progression of the disease.

[0089] For administration, enhancers of androgen receptor-mediated signal transduction of the present invention can be administered at a rate determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a candidate compound; the LD50 of the enhancer of androgen receptor-mediated signal transduction; and the side-effects of the enhancer of androgen receptor-mediated signal transduction at various concentrations, as applied to the mass and overall health of the patient. In general, the dose will range from 0.1-50 mg per kg, preferably 1-25 mg per kg, most preferably 1-20 mg per kg body weight. Administration can be accomplished via single or divided doses. A. Compositions for Oral Delivery

[0090] In certain applications, the pharmaceutical compositions disclosed herein may be delivered via oral administration to the individual. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft-shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated into the diet.

[0091] The active compounds may even be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al, 1997; Hwang et al, 1998; U.S. Patent 5,641,515; U.S. Patent 5,580,579 and U.S. Patent 5,792,451). The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. A syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations. [0092] Typically, these formulations may contain at least about 0.1% of the active compound or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 60% or 70% or more of the weight or volume of the total formulation. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable. [0093] For oral administration the compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally-administered formulation. For example, a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically- effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants. Alternatively the compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.

B. Compositions for Injectable Delivery

[0094] In certain circumstances it will be desirable to deliver the pharmaceutical compositions disclosed herein parenterally, intravenously, intramuscularly, or even intraperitoneally as described in U.S. Patent 5,543,158; U.S. Patent 5,641,515 and U.S. Patent 5,399,363. Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0095] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U. S. Patent 5,466,468). In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0066] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion {see, e.g., Remington 's Pharmaceutical Sciences, 15th Edition, pp. 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biologies standards.

[0096] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above, hi the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.

[0097] The compositions disclosed herein may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.

[0098] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. [0099] The phrase " pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified.

C. Compositions for Nasal Delivery

[0100] In certain embodiments, the pharmaceutical compositions may be delivered by intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering genes, nucleic acids, and peptide compositions directly to the lungs via nasal aerosol sprays has been described e.g., in U. S. Patent 5,756,353 and U. S. Patent 5,804,212. Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U. S. Patent 5,725,871) are also well-known in the pharmaceutical arts. Likewise, transmucosal drug delivery in the form of a polytetrafluoroetheylene support matrix is described in U. S. Patent 5,780,045.

D. Compositions for Transdermal or Transmucosal Delivery

[0101] In other embodiments, the pharmaceutical compositions may be delivered by transdermal or transmucosal means. For transdermal or transmucosal administration, penetrants appropriate to the barrier to be permeated can be used in the formulation. For topical administration, the compositions are formulated into ointments, creams, salves, powders and gels. Transdermal delivery systems can include, e.g., patches. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. Exemplified transdermal delivery formulations that can find use in the present invention include those described in U.S. Patent Nos. 6,589,549; 6,544,548; 6,517,864; 6,512,010; 6,465,006; 6,379,696; 6,312,717; and 6,310,177, each of which are hereby incorporated herein by reference. Examples of transdermal patches that may be used in the practice of the invention include those described in U.S. Patent Nos. 5,302,395; 5,262,165; 5,248,501; 5,232,702; 5,230,896; 5,227,169; 5,212,199; 5,202,125; 5,173,302; 5,154,922; 5,139,786; 5,122,383; 5,023,252; and 4,978,532, each of which are hereby incorporated herein by reference. V. Identification of Additional Enhancers of Steroid Receptor-Mediated Signal Transduction

[0102] One embodiment of the invention provides methods for identification of additional compounds that enhance or potentiate steroid receptor-mediated signal transduction. A. Assays to Identify Enhancers of Steroid Receptor-Mediated Signal Transduction

[0103] Assays to detect androgen receptor-mediated signal transduction are well known in the art (see, e.g., Chen et al., J. Clin. Endocrin. Metab.). For example, cells transfected with androgen receptors and/or reporter genes (e.g., luciferase or /3-galactosidase) can be used to identify compounds that enhance androgen receptor signal transduction. Compounds that increase signals from the reporter gene are identified as enhancers of androgen receptor signal transduction. B. Confirmation that Identified Compounds Act Via a Steroid Receptor

[0104] Competitive binding assays using compounds that competitively inhibit steroid binding to a steroid receptor (e.g., flutamide and the like in the case of the androgen receptor) can be used to confirm that compounds initially identified as enhancers of steroid receptor- mediated signal transduction act via a steroid receptor-dependent mechanism. In some cases, the compounds can be labeled.

[0105] The particular label or detectable group used in the assay is not a critical aspect of the invention, as long as it does not significantly interfere with the specific binding of the compound to the androgen receptor. The detectable group can be any material having a detectable physical or chemical property. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, electrical, optical or chemical means. A wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions. Useful labels in the present invention include magnetic beads (e.g., DYNABEADSTM)₅ fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g., ^H, 125^ 35$_^ 14Q _or 32p^ _and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.). [0106] The molecules can be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore. Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol. For a review of various labeling or signal producing systems that may be used, see U.S. Patent No. 4,391,904. [0107] Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Finally simple colorimetric labels may be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.

[0108] Throughout the assays, incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, optionally from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, antigen, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 1O⁰C to 4O⁰C.

[0109] One of skill in the art will appreciate that it is often desirable to minimize nonspecific binding in immunoassays. Particularly, where the assay involves an antigen or antibody immobilized on a solid substrate it is desirable to minimize the amount of nonspecific binding to the substrate. Means of reducing such non-specific binding are well known to those of skill in the art. Typically, this technique involves coating the substrate with a proteinaceous composition, hi particular, protein compositions such as bovine serum albumin (BSA), nonfat powdered milk, and gelatin are widely used with powdered milk being most preferred.

VI. High Throughput Screening to Identify Compounds that Enhance Steroid Receptor-Mediated Signal Transduction

[0110] In certain embodiments, combinatorial libraries of compounds (e.g. those having Formula I) will be screened for an ability to enhance androgen receptor-mediated signal transduction. Conventionally, new chemical entities with useful properties are generated by identifying a chemical compound (called a "lead compound") with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. Often, high throughput screening (HTS) methods are employed for such an analysis.

[0111] In one preferred embodiment, high throughput screening methods involve providing a library containing a large number of potential therapeutic compounds (candidate compounds). Such "combinatorial chemical libraries" are then screened in one or more assays to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity, in for example, a signal transduction assay. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics. [0112] A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building blocks" such as reagents. Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks (Gallop et al., J. Med. Chem. 37(9): 1233-1251 (1994)). [0113] Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al, Proc. Nat. Acad. ScL USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with a Beta-D-Glucose scaffolding (Hirschmann et al. , J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al, J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho, et al, Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al, J. Org. Chem. 59:658 (1994)). See, generally, Gordon et al, J. Med. Chem. 37:1385 (1994), carbohydrate libraries (see, e.g., Liang et al, Science 274:1520-1522 (1996), and U.S. Patent No. 5,593,853), and small organic molecule libraries (see, e.g., benzodiazepines, Baum, C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent No. 5,569,588; thiazolidinones and metathiazanones, U.S. Patent No. 5,549,974; pyrrolidines, U.S. Patent Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Patent No. 5,506,337; benzodiazepines, U.S. Patent No. 5,288,514, compounds that regulate adenyl cyclase and cyclic AMP, such as, for example, forskolin and its derivatives, U.S. Patent Nos. 5,789,439; 5,350,864, and 4,954,642. [0114] Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433 A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA).

[0115] A number of well known robotic systems have also been developed for solution phase chemistries. These systems include automated workstations like the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif), which mimic the manual synthetic operations performed by a chemist. The above devices, with appropriate modification, are suitable for use with the present invention, hi addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, NJ., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, MO, ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).

[0116] The assays to identify compounds that enhance androgen receptor-mediated signal transduction are amenable to high throughput screening. High throughput assays for evaluating the presence, absence, quantification, or other properties of particular nucleic acids or protein products are well known to those of skill in the art. Similarly, binding assays and reporter gene assays are similarly well known. Thus, e.g., U.S. Patent No. 5,559,410 discloses high throughput screening methods for proteins, U.S. Patent No. 5,585,639 discloses high throughput screening methods for nucleic acid binding (i.e., in arrays), while U.S. Patent Nos. 5,576,220 and 5,541,061 disclose high throughput methods of screening for ligand/antibody binding.

[0117] In addition, high throughput screening systems are commercially available (see, e.g., Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA, etc.). These systems typically automate procedures, including sample and reagent pipeting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for various high throughput systems. Thus, e.g., Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like.

EXAMPLES

[0118] The following examples are provided by way of illustration only and not by way of limitation. Those of skill will readily recognize a variety of noncritical parameters which could be changed or modified to yield essentially similar results. EXAMPLE 1 : Materials and Methods

[0119] Chemicals and cell culture reagents: TCC (reported purity of 99.3%), carbanilide (reported purity of 99.9%), flutamide (non-steroid antiandrogen) and testosterone propionate (TP) were purchased from Sigma- Aldrich (St. Louis, MO, USA). Other TCC analogs (purity >99%) were synthesized in the laboratory of Dr. Bruce D. Hammock by the condensation of the appropriate isocyanate and amine (Morisseau C et al., Proc Natl Acad Sd USA, 96:8849-8854 (1999); Newman JW et al., Environmental health perspectives, 109:61-66 (2001)). 17β-Hydroxy-4-androsten-3-one (testosterone, T) and 5α-Androstan-17β-ol-3-one (dihydrotestosterone, DHT) were purchased from Steraloids (Newport, RI, USA). MDA- kb2, a cell line expressing endogenous androgen receptor (AR) and 3-(4,5-Dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The testosterone and DHT were dissolved in absolute ethyl alcohol while all other compounds were dissolved in dimethylsulfoxide (DMSO). Phenol-red free Dulbecco's Modified Eagle Medium (DMEM), L- 15 (Leibovitz) medium, fetal bovine serum (FBS), L-glutamine, penicillin/streptomycin sulfate, blasticidin and geneticin sulfate (G418) were obtained from Invitrogen (Carlsbad, CA, USA). Dextran- coated charcoal-treated (DCC) FBS was purchased from Hyclone (Logan, UT, USA). Cell lysis buffer was purchased from Promega (Madison, WI, USA). AR (441) mouse monoclonal IgG raised against human AR amino acids 299-315 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Human chorionic gonadotropin (CG) standard (CR 121) was provided by Dr. John O'Connor (Columbia University, NY) and diluted in DMEM. [0120] Cell-based human AR-mediated bioassay: A full detailed description of the development and application of the cell-based human AR-mediated bioassay has been published by Chen et al. (Chen J et al., The Journal of clinical endocrinology and metabolism, 91 :4387-4394 (2006)). Briefly, the bioassay system employs human embryonic kidney (HEK) 293 cells that lack critical steroid metabolizing enzymes. The cells are stably transfected with PCDNA6-hAR and an MMTV-Luc.neo plasmid containing a luciferase reporting gene (Chen J et al., The Journal of clinical endocrinology and metabolism, 91 :4387-4394 (2006)). The cells (designated as 2933Y) are highly responsive to endogenous steroids as well as synthetic compounds. The signal induction is stable for more than 60 passages under double antibiotic selection conditions (Chen J et al., The Journal of clinical endocrinology and metabolism, 91:4387-4394 (2006)).

[0121] The details of the in vitro procedures to evaluate the androgenic/antiandrogenic activity of the EDS as well as the concentration selection for testosterone in the AR-mediated cell system have been previously described (Chen J et al., Toxicology and applied pharmacology, 221 :278-284 (2007)). The lower limit of detection of this assay is 15 pM T in cell culture medium (blank + 3 SD) with intra- and inter-assay coefficients of variation of 7.4% and 7.5% at 0.25 nM T and 4.9% and 6.4% at 0.03 nM T, respectively (Chen J et al., The Journal of clinical endocrinology and metabolism, 91 :4387-4394 (2006)). [0122] Androgen Receptor Competitor Assay: The competition of TCC with endogenous hormone for AR binding was evaluated using the PolarScreen AR Fluorescence Polarization (FP) Assay with a Beacon 2000 Fluorescence Polarization System according to the manufacturer's instructions (Invitrogen Catalog P3018, Carlsbad, CA, USA). [0123] Western blot analysis: The expression of AR protein in MDA-kb2 and 2933Y cells was analyzed by western blot. Briefly, after treatment with either T (1.0 nM), TCC (1.0 μM) or T and TCC in combination for 48 hours, the cells were lysed and whole cell lysates were prepared and subjected to 7.5% SDS-PAGE and transfered to a polyvinylidene difluoride membrane. The membrane was then blocked in 20 mM Tris-HCl, 137 mM NaCl, and 0.1% (v/v) Tween 20 (pH 7.4) containing 5% non-fat milk. The membrane was immunoblotted with AR (441) mouse anti-human monoclonal antibody overnight, followed by secondary antibody (donkey anti -mouse antibody) coupled to horseradish peroxidase from Amersham Biosciences (Piscataway, NJ, USA) for 1 hour. The membrane was exposed on X-ray film (Eastman Kodak Co.) using ECL Western blot detection reagents (Amersham Biosciences, Piscataway, NJ, USA). To reprobe with beta actin, the membrane was stripped in stripping buffer at 53 ⁰C for 30 minutes. [0124] cAMP/PKA-mediated luciferase transcriptional activity: Luciferase transcriptional activity mediated by cAMP/PKA pathway was measured by the in vitro bioassay described by Jia et al. (Jia XC et al., Molecular endocrinology (Baltimore, Md), 5:759-768 (1991)) and modified as described below. This assay utilizes HEK 293 cells stably transfected with the human luteinizing/chorionic gonadotropin receptor gene and the luciferase reporter gene (pCRE-luc) (JK293) (Chen J et al., Reproductive toxicology (Elmsford, NY) 17:87-93 (2003)). JK293 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin sulfate, and 100 μg/mL geneticin sulfate. After the JK293 cells were cultured to 80-100% confluence in 100x20 mm cell culture dishes, cells were counted and incubated in 96-well plates. Each well contained 10⁵ cells in 100 μL DMEM. Then, 50 μL human CG standard CR 121 at a concentration of 3.2 ng/mL and/or test compounds at designated concentrations were added to each well containing 150 μL DCC-FBS containing phenol-red free DMEM. Cells were further incubated for 16 hours. The media was then removed and 100 μL of cell lysis buffer was added to each well and allowed to incubate for 30 minutes. Cell lysates (60 μL) were transferred to 96-well Microfluor II plates (Fisher Scientific, Santa Clara, CA, USA). Luciferin substrate was then injected into each well and the luciferase activity induced by test compounds was measured by a Veritas Luminometer (Turner Biosystems, Sunnyvale, CA, USA) (Chen J et al., Toxicology and applied pharmacology, 221 :278-284 (2007)).

[0125] To compensate for any organic solvent effects, the final content of ethyl alcohol in both the AR-mediated and cAMP/PKA-mediated assay systems was 0.1% (v/v) for all studies and the total DMSO concentration in the final culture media was no more than 0.2% (v/v). The total concentration of organic solvent (v/v) was maintained at the same level for both controls and test compounds.

[0126] Androgen receptor mediated signal transduction assay: 2933 Y cells were cultured in DMEM with 10% FBS. When cells reached 80% confluence, cells were trypsinized and equal numbers of cells (density of 25,000 cells/50 μL) were placed in 96-well tissue culture plates containing 150 μl/well of phenol-red free DMEM supplemented with 10% DCC-FBS. On the following day, media were removed and replaced. On day 3, media were again removed and replaced with 200 μl phenol red-free cell culture media containing 20 μl solutions of the test compounds or testosterone at the designated concentrations. TCC was dissolved in DMSO. Testosterone was dissolved in ethyl alcohol. To compensate for any organic solvent effects, the final content of ethyl alcohol in the assay system was 0.1% (v/v) for all studies and the total DMSO concentration in the final culture media was no more than 0.2% (v/v). The total concentration of organic solvent (v/v) was maintained at the same level for both controls and test compounds. Cells were further cultured for 18 h and luciferase activity was evaluated by a Veritas luminometer (Turner Biosystems, Sunnyvale, CA, USA). [0127] MTT assay: The MTT assay for cell proliferation or cytotoxicity testing under varying concentrations of test compounds was performed according to the manufacturer's instructions (ATCC, Catalog Number 30-101 OK, USA) and has been described previously (Chen J et al., Toxicology and applied pharmacology, 221:278-284 (2007)). Briefly, the 2933Y cells were plated at the same density and cultured by the same procedure as described above for each 96-well plate. After 18 hours of treatment, 20 μl of MTT were added to each well and the plate was incubated at 37 °C for 4 h. The yellow tetrazolium MTT was reduced by metabolically active cells, in part by the action of dehydrogenase enzymes. The resulting intracellular purple formazan was solubilized by adding 100 μL of detergent reagent and the plate was incubated at room temperature in the dark for 2 hours. At the end of this period, the absorbance was measured at 570 nm with a reference wavelength of 650 nm using an EMax Spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). [0128] In vitro bioassay for CAMP/PKA signal transduction: Human fetal kidney cells (cell line 293, ATCC) were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco BRL, Grand Island, NY) containing 5% calf serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin sulfate, and the cells were cotransfected with pCRE-luc, pCDNA3hLHR containing the gene for neomycin resistance. After transfection, the cells were cultured in medium containing 600 μg/ml geneticin (Gibco BRL, Gaithersburg, MD). Several single-cell colonies from transfected cells were isolated, and the luciferase activity and LH receptor content were measured. One cell colony (JK 293) had both receptor binding and luciferase activity and was used for development of the CAMP/PKA mediated signal transduction assay.

[0129] After the JK 293 cells were cultured to 80-100% confluence in 100^χ20-mm cell culture dishes (Falcon, BD Labware, USA), cells were counted and incubated in Micro-well cell plates (Nunclon TM Surface; Applied Scientific). Each well contained 10⁵ cells in 100 μl DMEM. For bioassay, 50 μl hCG CR 127 standards at concentration of 3.2 mg/mL(provided by R. Canfield, Columbia University, NY), 50 uL of internal controls, and 150 uL of TCC at concentration of 1.7 uM TCC were added. The final concentration of TCC is 1.0 uM. The plates were incubated at 37 °C for 10 h. At the end of incubation, the medium was aspirated out and 100 μl Ix lysis buffer (5^χ Cell Culture Lysis Reagent, Promega Co., USA) were added. The plates were put on a rotator at 150 rpm at room temperature for 30 min. To determine the luciferase activity, 60 μl of the cell lysate was transferred to Micro Fluor plates (Dynatech Laboratories, Inc., Chantilly, VA), and then mixed with 100 μl assay buffer (0.5 mM luciferin, 20 mM tricine (Sigma), 1.07 mM [(MgCOs)₄Mg(OH)₂] -5H₂O, 2.67 mM MgSO₄, 0.1 mM EDTA, 33.3 mM DTT, 0.27 mM Coenzyme A, and 0.5 mM ATP). Light production was measured for 2 s by a microtiter plate luminometer (Turner Biosystems). [0130] Flutamide competitive binding assay: 2933Y cells were cultured in DMEM with 10% FBS. When cells reached 80% confluence, cells were trypsinized and equal numbers of cells (density of 25,000 cells/50 μL) were placed in 96-well tissue culture plates containing 150 μl/well of phenol-red free DMEM supplemented with 10% DCC-FBS. On the following day, media were removed and replaced. On day 3, media were again removed and replaced with 200 μl phenol red-free cell culture media containing 20 μl solutions of the TCC, testosterone or combination of testosterone and TCC the designated concentrations. To compensate for any organic solvent effects, the final content of ethyl alcohol in the assay system was 0.1% (v/v) for all studies and the total DMSO concentration in the final culture media was 0.2% (v/v). The total concentration of organic solvent (v/v) was maintained at the same level for both controls and test compounds. Cells were further cultured for 18 h and luciferase activity was evaluated by a Veritas luminometer (Turner Biosystems, Sunnyvale, CA, USA). [0131] In vivo effect of TCC on accessory sex organ weight: Forty-eight male Sprague Dawley rats 48-52 days old (castrated at 42-46 days old) were randomly assigned to four treatment groups with 12 rats in each group. All animals were maintained on their respective treatment regimen for ten days. Animals in Group One served as controls and received sham treatments of sesame oil (no androgen support) and normal diet (no TCC supplement). Animals in Group Two were treated with testosterone propionate (TP) injection (0.2 mg/kg, sc in sesame oil) and received a normal diet. Animals in Group Three received vehicle control injections (no androgen support) and TCC supplemented diet (0.25% TCC (w/w) mixed in rat chow) for the 10 day treatment period. Group Four animals received testosterone propionate (TP) injection (0.2 mg/kg, sc in sesame oil) and TCC supplemented diet (0.25% TCC (w/w) mixed in rat chow). At the end of treatment, the animals were sacrificed by carbon dioxide asphyxiation and the liver, kidney, levator anibulbocavernosus muscle (LABC), glans penis, ventral prostate, seminal vesicles and Cowper's gland were surgically removed and weighed. All experiments were conducted in accordance with the regulations of the Animal Care and Use Committee of Yale University in facilities fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. [0132] Estrogen receptor (alpha) -mediated bioassay: Human ovarian carcinoma cells (BGl) that have been stably transfected with a luciferase reporter gene plasmid under the regulation of four estrogen-response elements was used to measure total bioactive estrogens. BGl cells were cultured in Alpha Minimum Essential Medium (Alpha-MEM) with 10% fetal bovine serum (FBS). When cells reached 80% confluence, cells were trypsinized (0.05% trypsin-EDTA) and well dispersed in phenol-red free Dulbecco's Minimum Essential Medium (DMEM) supplemented with 10% dextran charcoal treated FBS (DCC-FBS). Suspended cells (0.05 mL/well; density of 25,000 cells/0.05 mL) were added to 96-well tissue culture plates containing 0.15 mL/well of phenol-red free DMEM supplemented with 10% DCC-FBS. On each of the two subsequent days (Days 2 and 3), the media in each well is removed and replaced with 0.2 mL phenol-red free DMEM supplemented with 10% DCC- FBS. On Day 4, media was again removed and replaced with 0.2 mL phenol red-free DMEM supplemented with 10% DCC-FBS containing increasing concentrations of estradiol (E₂) standards or sera. Estradiol standards were disolved in ethyl alcohol and have a final alcohol content of 0.1% (v/v) to minimize any organic solvent effects. The plates were incubated for an additional 18 hours. The media was then removed and 0.1 mL cell lysis buffer was added to each well and allowed to incubate for 20 minutes. Cell lysates (0.04 mL) was then transferred to 96-well Microfluor II plates (Fisher Scientific, Santa Clara, CA). Luciferin substrate was injected into each well and the luciferase activity induced by the standards and/or test serum was measured by a Veritas Luminometer (Turner Biosystems, Sunnyvale, CA, USA). [0133] Glucocorticoid receptor (GR) -mediated bioassay: MDA-kb2 cells expressing GR were maintained in L- 15 media (Gibco BRL) supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin at 37⁰C, without CO₂. For experiments, cells were plated at 1 x 10⁴ cells per well in 100 μl of medium in 96-well tissue culture plates. When cells were attached (4-6 h), medium was removed and replaced with dosing medium prepared in phenol red free Dulbecco's Minimum Essential Medium (DMEM) containing 10% of dextran charcoal treated FBS (DCC-FBS). Cortisol standards were disolved in ethyl alcohol and have a final alcohol content of 0.1% (v/v) to minimize any organic solvent effects. The plates were incubated for an additional 18 hours. The media was then removed and 0.06 mL cell lysis buffer was added to each well and allowed to incubate for 20 minutes. Cell lysates (0.04 mL) was then transferred to 96-well Microfluor II plates (Fisher Scientific, Santa Clara, CA). Luciferin substrate was injected into each well and the luciferase activity induced by the standards and/or test serum was measured by a Veritas Luminometer (Turner Biosystems, Sunnyvale, CA, USA).

[0134] Statistical analysis: For in vitro studies, the values shown are mean ± SD from three independent experiments for each dose tested and for the in vivo study, the values shown are mean ± SD of each group. For both in vitro and in vivo data, one-way analysis of variance (ANOVA) was applied followed by multiple comparisons test when appropriate, using Sigmastat (Systat Software, San Jose, CA, USA). The level of significance was set at p < 0.05. For the in vitro study, treatments were compared to the negative control group containing vehicle only to test for agonist properties and for androgen antagonist properties, treatments were compared to the testosterone-positive control group. EXAMPLE 2: Triclocarban (TCC) Enhances Testosterone-Induced Signal Transduction [0135] This Example employed a cell-based andogen receptor-mediated reporting system directed to investigate the androgen/antiandrogenic activity of TCC. The results demonstrate little, if any androgenic activity by TCC alone, but when combined with testosterone, a 45% of increase of T induced signal was observed (P<0.05, Figure 2). This effect was not associated with any increased cell proliferation, as measured using the MTT assay described in Example 1 (see Figure 6).

EXAMPLE 3: Effects of TCC are Androgen Receptor-Dependent [0136] To further assess the mechanism by which TCC mediated this enhanced T signal, the antiandrogen, flutamide, which functions as a competitive inhibitor for androgen binding to hAR, was employed. Flutamide (10 μM) dramatically suppressed the amplification effect of 1 μM TCC (P<0.05, Figure 3) and these data support the concept that the amplification of transcriptional activity by TCC is hAR dependent. EXAMPLE 4: TCC Amplification of the Testosterone-induced Signal Occurs Through the Androgen Receptor

[0137] Numerous studies have indicated that AR mediated signaling is affected by an array of cytokines and growth factors that act through a web of complex signaling cascades. Of these, cAMP/PKA, as activated by forskolin, is particularly interesting because of its ability to phosphorylate AR in vivo and stimulate the expression of the AR-regulated gene expression. The concept that cAMP acts as an intracellular second messenger to a wide range of hormones, neurotransmitters, and other signaling substances has been well developed. The target for cAMP was identified as cAMP-dependent protein kinase (PKA). hi the absence of cAMP, PKA is an enzymatically inactive tetrameric holoenzyme. To investigate TCCs potential to active cAMP/PKA signaling, we investigated the ability of TCC to stimulate the transactivation of luciferase controlled by cAMP/PKA pathway in a cAMP/PKA mediated assay system. As shown in Figure 4, TCC alone did not active cAMP/PKA-mediated luciferase activity compared to control nor did it enhance the signal transduction induced by the presence of hCG, a strong stimulus for cAMP production in this system again suggesting that the amplification of the T-induced signal by TCC is upstream through the AR rather than the result of a post-translational modification of androgen receptor signaling through the cAMP/PKA pathway. EXAMPLE 5: Effect of TCC on Cell Proliferation and Cytotoxicity [0138] The structure of TCC (3,4,4'-trichlorocarbanilide) is shown in Figure 1. It is a polychlorinated diphenyl urea. Concentrations of TCC up to 1.0 μM did not result in cytotoxicity in 2933 Y cells when tested alone or in combination with 0.125 nM of T (Figure 6). Vehicle-treated or TCC-treated cells did not demonstrate statistically significant differences with respect to proliferation at the concentrations used in this study. EXAMPLE 6: Effect of TCC on AR-mediated Transcriptional Activity

[0139] At a concentration of 1.0 μM, TCC revealed little or no androgenicity when tested alone. In contrast, in the presence of a native androgen, such as testosterone (T, 0.125 nM), a 45% increase of the T induced signal was observed (PO.05, Figure 7), and this amplification of the T induced transcriptional activity by TCC was both time dependent (Figure 8a) and dose dependent (Figure 8b and 8c). This amplification of the T induced signal transcriptional activity was also detected in other urea compounds structurally similar to TCC (Figure 9). To further assess the mechanism by which TCC mediates the enhancement of the T signal, flutamide was employed. This known antiandrogen functions as a competitive inhibitor for androgen binding to the AR (Yin D et al., Molecular pharmacology, 63:211-223 (2003)) and at 10 μM, flutamide dramatically suppressed the amplification effect of 1.0 μM TCC (PO.05, Figure 7).

EXAMPLE 7: Competitive Binding of TCC for AR

[0140] To investigate the potential of TCC to mimic the native hormone by binding to the AR, a competitive binding assay was conducted. As shown in Figure 10, TCC did not compete for T binding to the AR at tested concentrations up to 200 μM. In contrast, the polarization value was reduced by 20% and 70% at DHT concentrations of 10 nM and 100 nM, respectively. EXAMPLE 8: The Effect of TCC Treatment on AR Protein

[0141] We investigated whether TCC increases the expression of the AR protein in cells that express endogenous AR. Western blot analysis indicated that compared to vehicle control, an increase of immunoreactive AR protein was detected in MD A-kb2 cells treated with T or T+TCC with the latter treatment yielding more AR protein (Figure 11). Similarly, T or T+TCC combination treatment increased immunoreactive AR expression in 293 cells compared to vehicle control, however, unlike the MDA-kb2 cells, there was no difference between the amount of protein observed in the T+TCC combination treatment and the T only treatment. EXAMPLE 9: Effect of TCC on cAMP/PKA-mediated Transcriptional Activity

[0142] Numerous studies have indicated that AR mediated signaling is affected by an array of cytokines and growth factors that act through a web of complex signaling cascades Kim J et al., Journal of molecular endocrinology, 34:107-118 (2005). Of these, cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) are particularly interesting because of the ability to phosphorylate AR in vivo and stimulate the expression of the AR-regulated gene expression (Kim J et al., Journal of molecular endocrinology, 34:107-118 (2005); Gioeli D et al., The Journal of biological chemistry, 277:29304-29314 (2002); Sadar MD et al., Endocrine-related cancer, 6:487-502 (1999)). The concept of cAMP as an intracellular second messenger to a wide range of hormones, neurotransmitters, and other signaling substances is well developed (Robinson GA and Sutherland EW, Advances in cytopharmacology, 1:263-272 (1971)). The target for cAMP has been identified as cAMP- dependent protein kinase (PKA). In the absence of cAMP, PKA is an enzymatically inactive tetrameric holoenzyme (Skalhegg BS and Tasken K, Front Biosci, 2:d331-342 (1997)). To investigate TCCs potential to activate cAMP/PKA signaling, the ability of TCC to stimulate the transactivation of luciferase controlled by the cAMP/PKA pathway in a cAMP/PKA mediated assay system was studied (Chen J et al., Reproductive toxicology (Elmsford, NY) 17:87-93 (2003); Jia XC et al., Biology of reproduction, 49:1310-1316 (1993)). As shown in Figure 12, TCC alone did not activate cAMP/PKA-mediated luciferase activity beyond control levels nor did it enhance the signal transduction induced by the presence of human chorionic gonadotropin (hCG), which is a strong stimulus for cAMP production in this system. EXAMPLE 9: The Effect of TCC Treatment on Organ Weight After 10 days of Treatment in Castrated Rats

[0143] To investigate TCCs potential amplification of native androgen ligands in vivo, we evaluated the effects of TCC in castrated male SD rats aged 48-52 (castrated at age 42-46 days). This model has been well established and widely used to study the androgenic/antiandro genie effects of AR ligands, EDS and/or AR modulators on accessory sex tissues (Ostrowski J et al., Endocrinology, 148:4-12 (2007)). In this model, the change in the weight of accessory sex organs following various treatments is used to indicate the amount of androgenic support. TP (0.2 mg/kg) was used as the positive control due to its superior pharmacokinetic properties and enhanced efficacy both in humans and animal models (Ostrowski J et al., Endocrinology, 148:4-12 (2007)). A suboptimal dose of 0.2 mg/kg TP was selected for use to ensure the ability to observe an amplification effect of TCC. No 7statistically significant differences were observed for total body, or kidney weights between any groups, however, there was a slight increase in the mean liver weight in the group of animals treated with TCC alone (Table 1). No significant differences were observed for the weights of the seminal vesicles, Cowper's gland, levator anibulbocavernosus (LABC) muscle and glans penis between sham-treated rats and rats receiving TCC only in the diet, however, an increase in ventral prostate weight was observed in rats treated with TCC only compared to sham-treated rats. In contrast, and as hypothesized, TP treatment alone significantly increased the weights of accessory sex organs compared to controls and TCC alone (Table 1). The co-treatment of TP with TCC revealed a substantial and significant increase in the weights of all accessory sex organs compared to TP treatment alone indicating a synergism between TP and TCC in vivo (Table 1).

Table 1. The in vivo effect of TCC treatment on organ weight (mean±SD) of male SD rats

Vehicle control TCC T T+TCC

Body weight (g) 217.75±8.62 222.75±11.07 217.08±6.42 217.00±8.15

Kidney (g) 2.07±0.26 1.98±0.26 1.96±0.27 1.91+0.21

Liver (g) 10.73±0.94 12.53±1.21 ^a 11.4±1.29 11.68±1.20

Seminal vesicles (mg) 105.42± 28.18 132.17±39.7 323.08±69.97 ^a 576.5±73.41 ^b

Ventral prostate (mg) 58.5±18.92 85.83±22.25^a 136.67±8.49 ^a 228.00±23.54 ^b

Glans penis (mg) 72.25±12.46 78.08±8.53 83.75±8.53 113.3±14.13 ^b

Cowper's gland (mg) 15.08±3.23 18.08±2.47 22.00±3.77 ^a 36.33±4.46 ^b

Levator anibulbocavernosus

129.25±5.99 133.92±7.35 323.92+7.28 ^a 366.92±12.23 ^b muscle (mg)

Values are mean± standard deviation of 12 rats in each group aP<0.05 compared to vehicle control. bP<0.05 compared to vehicle control and T treatment. EXAMPLE 10: Enhancement of Estradiol (E2) Induced Transcriptional Activity and Enhancement of Cross-reactivity of Cortisol by TCC in AR-mediated transcriptional activity by TCC

[0144] This example shows enhancement of estradiol (E2) induced transcriptional activity by TCC in an ER alpha-mediated bioassay (Rogers J et al, Li Vitro MoI Toxicol. 2000 13(l):67-82.), and enhancement of the cross-reactivity of Cortisol by TCC in the AR- mediated transcriptional activity in 2933Y cells. 2933Y cells also have low endogenous glucocorticoid receptor expression (Chen J et al, 2006 JCEM 2006 Nov;91(l l):4387-94). [0145] The effect of TCC on androgen signaling was initially discovered in an attempt to inventory EDS with (anti)androgenic properties using the 2933 Y cells. In that investigation, most EDS were identified as weak antagonists (Chen J et al., Toxicology and applied pharmacology, 221 :278-284 (2007)) but a small group of polychlorinated biphenyls were found to have "enhancing" properties and are further investigation herein. Figure 13 shows data which indicates that TCC induces similar effects on the estrogen receptor as well as enhancing the glucocorticoid induced signal transduction through the androgen and/or glucocorticoid receptor (Figure 13 A and B). Thus, our data indicates that TCC is likely to have a similar effect on all nuclear receptors.

[0146] hi the field of androgen therapy, the more potent androgens are hepatotoxic, and thus, oral delivery is difficult. It is for this reason, transdermal patches have been proposed. However, in cases where low-dose androgen therapies are safe, the addition of TCC-like compounds would enhance the androgen bioactivity while obviating concerns of the "first- pass" liver damage.

[0147] As concerns parturition and child birth, a large percent of deliveries are induce both for medical reasons and convenience. Current drugs that induce contractions are used with concerns that the cervix may not be appropriately prepared. The use of TCC to enhance the actions of endogenous estriol would provide a more physiologic induction of pregnancy by both maturing the cervix as well as overcoming the progesterone block to prostaglandin release. Thus, TCC could be used as an adjunct to oxytocin induction after appropriate cervical effacement. EXAMPLE 11 : Effect of TCC on Estrogen Receptor and Glucocorticoid Receptor Mediated Transcriptional Activity

[0148] At a concentration of 1.0 μM, TCC revealed little or no estrogenic or glucorticoid activity when tested alone. In contrast, in the presence of a native estrogen or glucocorticoids, such as estradiol or Cortisol, TCC significantly increased the estradiol or Cortisol induced signal (Figures 14 and 15). This amplification of the TCC amplified signal transcriptional activity was also detected when estrogens with low estrogenic potency such as estrone or estriol were tested (Figure 14).

[0149] The amplification effect of TCC on estrogens with weak potencies is expected to have important biological effects, for example, in inducing labor during child birth. First, late pregnancy in all higher primates is dominated by levels of estrogen that are never experienced in other mammalian species. In the great ape species and humans, estrone is a unique estrogen and considered a key hormone in late fetal development, in activities leading to parturition, and in a series of events that trigger labor. [0150] As for glucocorticoids, they are among the most successful therapies in the treatment of chronic inflammatory and autoimmune diseases. However, it is of concern that prolonged or high dose glucocorticoid exposure as a result of various glucocorticoid therapies will induce unwanted, adverse side effects such as immunosuppression, adrenal suppression, osteoporosis, and diabetes. The amplification effect of TCC on Cortisol may be used to lower the doses of glucocorticoids used and alleviated such unwanted side effects. Discussion

[0151] Recent reports relating to several non-steroidal compounds indicate that a number of compounds have the ability to modulate, activate and/or bind to the human AR (Bisson WH et al., Proc Natl Acad Sci USA (2007)). These compounds are of particular public concern because human exposures to many of these compounds are ubiquitous, can accumulate in the environment and human exposures to some of them are possibly constant (Bisson WH et al., Proc Natl Acad Sci USA (2007); Chen J et al., The Journal of pharmacology and experimental therapeutics, 312:546-553 (2005)). We, therefore, investigated the EDS properties of a subset of these compounds by employing a cell-based androgen receptor- mediated reporting system (Chen J et al., The Journal of clinical endocrinology and metabolism, 91 :4387-4394 (2006)) to determine if any of these compounds are able to interfere with the natural action of endogenous androgens. In that investigation, most EDS were identified as weak antagonists (Chen J et al., Toxicology and applied pharmacology, 221 :278-284 (2007)), but a small group of polychlorinated biphenyls were found to have "enhancing" properties and seemed worthy of further investigation.

[0152] TCC is an antimicrobial agent commonly added to personal care products. The present data indicate that TCC has little or no androgenic activity alone but has an amplification effect on strong native androgens such as T. This amplification effect is characterized by an increased transcriptional activity transduced through the AR as the co- treatment of flutamide significantly suppressed the signal in vitro (Figure 3). It has been reported that 0.39% of an average 138 mg of triclocarban (or 0.54 mg) applied to the entire body was absorbed after a typical "whole body" shower lather (Scharpf LG, Jr. et al., Archives of environmental health, 30:7-14 (1975)). Therefore, the actual systemic dose of TCC would be approximately 0.1 mg/L (or 0.1 μg/mL) for an adult of 60 kg with 5 L of blood. The concentration of TCC used in the in vitro study was 1.0 μM which is equal to approximately 0.3 μg/mL. Thus, this in vitro dose represents only a 3-fold increase above that of a typical human exposure after a whole body shower. Existing evidence also indicates that percutaneous penetration of similar compounds varies with the anatomic site of application. With chlorinated hydrocarbon pesticides for example, the forearm allowed relatively less penetration whereas the abdomen, scalp, and postauricular area and the scrotum allowed almost total absorption (Maibach HI et al., Pesticides. Archives of environmental health, 23:208-211 (1971)). [0153] One of the aims of the present study was to determine if the in vitro endocrine disrupting effects of TCC could be supported in vivo. The use of 0.25% (w/w) TCC in the study was based on reports of extended TCC exposure in the rat (Nolen GA and Dierckman TA, Toxicology and applied pharmacology, 51:417-425 (1979)). While many EDS seem to be less potent than the natural ligands in both in vitro and in vivo assays, comparable effects were observed when these compounds were administered at critical time points at doses that were several orders of magnitude lower (Soto AM et al., Best practice & research, 20:15-33 (2006)). Available data have demonstrated that TCC exposures by dermal or oral routes in rats and humans lead to similar metabolic profiles and that the administration of TCC in the diet is considered an appropriate way of assessing the toxicity of TCC (Hiles RA, Food and cosmetics toxicology, 15:205-211 (1977)). [0154] It is particularly noteworthy that in vivo, TCC in combination with TP resulted in a significant increase in accessory sex organ weights compared to TP treatment alone using the castrated male SD rat animal model (Table 1). Our data strongly suggest that TCC has a positive androgen receptor modulatory effect in tissues or cells that are androgen targets. These observations open the possibility that other nuclear receptor signal transduction systems could also be modulated by TCC in a similar fashion. This possibility was confirmed in vitro by demonstrating that TCC also potentiated the estrogen receptor (ER) alpha mediated signal transcriptional activity induced by estradiol as well as amplifying the Cortisol induced signal transduction in cells with endogenous expression of the glucocorticoid receptor (data not shown). [0155] It should also be noted that the seminal vesicle, ventral prostate and Cowper's gland weights increased additively with the TCC and testosterone combination treatment. In contrast, only a marginal increase in the levator anibulbocavernosus (LABC) muscle weight was observed with the combination treatment (Table 1). Further investigation will be required to determine whether or not TCC acts to preferentially enhance testosterone's ability to increase reproductive organ weight over that of muscle mass.

[0156] The concentration of 1.0 μM TCC tested in vitro was orders of magnitude in excess of the T concentration used. It is clear, therefore, that the relative binding efficiencies, if any, of TCC for the AR are orders of magnitude below that of the natural ligands. This conclusion is supported by the results of the AR competition assay in which TCC did not compete for T binding to the AR at concentrations up to 200 μM (Figure 10). These results also support the concept that TCC is not a typical hormone mimic because it shows minimal receptor activation in the absence of cognate ligand (Figure 7). [0157] Nuclear receptor mediated signaling is affected by an array of cytokines and growth factors that act through a web of complex signaling cascades (Robinson GA and Sutherland EW, Advances in cytopharmacology, 1:263-272 (1971)). We found TCC alone did not activate cAMP/PKA-mediated luciferase activity nor did it enhance the signal transduction induced by human chorionic gonadotropin (CG). These data indicate that the cAMP/PKA pathway may be involved in the amplification of the T induced transcriptional activity by TCC.

[0158] Recent evidence points to the potential role of MAPKS pathways in the nuclear receptor mediated signal augmentation for certain EDS (Jansen MS et al., Proc Natl Acad Sci USA, 101 :7199-7204 (2004)). The prolonged half life of the nuclear receptor, the recruitment of novel coactivators as well as the involvement of a secondary binding domain in the nuclear receptor may also contribute to the signal potentiation phenomenon of TCC (Chang CY and McDonnell DP, Trends in pharmacological sciences, 26:225-228 (2005); Gregory CW et al., Cancer research, 61:2892-2898 (2001); Heinlein CA and Chang C, Endocrine reviews, 23:175-200 (2002); Syms AJ et al., The Journal of biological chemistry, 260:455-461 (1985); Wang Y et al., Proc Natl Acad Sci USA, 103:9908-9911 (2006)). [0159] The synergistic increase of immunoreactive AR protein with T+TCC treatment in MDA-kb2 cells which express endogenous AR (Figure 11) could be the result of T and TCC on AR transcription and/or AR protein stability (Gregory CW et al., Cancer research, 61:2892-2898 (2001)). While the synergistic effect of TCC+T treatment on luciferase activity was observed in 2933 Y cells, no synergistic effect on the amount of immunoreactive AR was detected. This lack of a pronounced increase of AR in the T+TCC combination treatment in 2933 Y cells could be due to the inherent differences between the exogenous AR in 2933Y cells and endogenous AR in MDA-kb2 cells (Litvinov IV et al., The Prostate, 58:319-324 (2004)). Because much of the AR expression regulation is believed to occur at the post-transcriptional level, where untranslated regions (UTRs) play a central role, the lack of both 5 '-and 3'- UTRs in the exogenous AR transcripts in the 2933 Y cells could result in different patterns of post-transcriptional gene regulation (Litvinov FV et al., The Prostate, 58:319-324 (2004)). In addition, the synergistic effects of T+TCC on AR-mediated transcriptional activity in 2933 Y cells could arise from the altered DNA-binding activity of the receptor (Dai J et al., Clin Cancer Res, 8:2399-2405 (2002); Ikonen T et al.,

Endocrinology, 135:1359-1366 (1994)). Clearly, comprehensive investigations are required in order to identify the potential mechanisms of sex steroid amplification by TCC. [0160] Our studies identify a new category of EDS for androgens and other steroid hormones. The data presented herein indicate that TCC and its urea analogs should be categorized as steroid hormone amplifiers or enhancers rather than simple agonists or antagonists because these compounds demonstrate the novel EDS property of synergism with the native androgen hormone receptor ligand (Jansen MS et al., Proc Natl Acad Sd USA, 101 :7199-7204 (2004)). This represents the first report regarding the synergistic effect of TCC on native sex hormones in vitro and in vivo. Given the scarcity of toxicological data in humans and laboratory animal models with respect to TCC and related compounds, the properties exhibited here by TCC may have more significance than for previously identified EDS. In terms of modulating steroid hormone action, TCC and its analogs elicit a positive biological effect rather than an inhibitory or weakly agonistic effect and have the potential to act through multiple nuclear receptors. This effect would be more likely to induce hyperstimulation rather than the attenuation of normal stimulation. Furthermore, the amplification effect of TCC on endogenous sex steroids may have an array of widespread subtle physiological alterations in both males and females. For example, because the amplification of androgens by TCC occurs at the target cell, there is the likelihood that such exposures may be associated with idiopathic hyperandrogenism. Thus, despite seemingly "normal" native circulating androgen levels, virilization may occur. TCC exposure may also result in defects in development (i.e. cryptorchidism, hypospadias) or decreased reproductive function (decrease in sperm quality) in adults because compensation through the long-loop feedback would occur with the effect of lowering gonadotropin drive in response to TCC exposure. In females, increased androgenic feedback could disrupt the normal female- specific "positive" feedback loop associated with ovulation and derange ovarian function. The exposure to these EDS may also change the balance between estrogen signaling and androgen signaling in breast homeostasis. Depending on the level that hormone signaling pathways are disrupted (Savabieasfahani M et al., Endocrinology, 147:5956-5966 (2006); Moorman WJ et al., Andrologia, 32:285-293 (2000)), in utero exposure to TCC could also impair neurogenesis and sexually dimorphic neurobehavioral development. Since TCC has the potential to amplify synthetic steroidal compounds, further investigation of the interaction of TCC with oral contraceptives, hormone replacement therapy, synthetic androgens and glucocorticoid therapy is also warranted. [0161] We conclude that TCC and some related structures should be categorized as steroid hormone amplifiers or enhancers rather than simple agonist or antagonist as these compounds demonstrate novel EDCs properties of synergism with the native hormone receptor ligand. The recognition of a potential amplification effect of TCC-related compounds on endogenous androgens may have a widespread physiological/reproductive implications in both males and females. The enhancement of endogenous androgenic feedback on the pituitary in males could act to decrease gonadotropin drive and result hypogonadotropic hypogonadism despite seemingly "normal" circulating testosterone. In females, increased androgenic feedback could disrupt the normal female-specific "positive" feedback loop associated with ovulation and derange ovarian function. In both sexes abnormal sexual development and/or behavior could occur depending upon when, how severe and at what level the androgenic signally pathways were disrupted.

[0162] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications and changes in light thereof will be suggested to persons skilled in the art and are to be included within the purview of this application and are considered to be within the scope of the appended claims. All publications, patents, patent applications, and GenBank Accession Nos. cited herein are hereby incorporated by referenced in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS:

1. A method of enhancing steroid hormone signal transduction in a mammal, the method comprising contacting a cell with a composition comprising: (a) a therapeutically effective amount of a steroid; and (b) a therapeutically effective amount of a compound having the Formula I:

wherein R¹ is a member selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R³, CO₂H, NHC(O)R^a, OC(O)NHR³ and NHC(O)OR³; wherein R^a is C_1-6 alkyl and each R¹ group is optionally substituted with 1 to 2 R^m substituents, and wherein optionally any two R¹ group located on adjacent atoms can be combined to form a 5- to 6-membered saturated or unsaturated ring having from O to 2 heteroatoms selected from N, O and S; n is an integer from 0-4; R² and R³ are each members independently selected from the group consisting of hydrogen, Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy and Ci_-8 alkoxy; R⁴ is a member selected from the group consisting of Ci_-8 alkyl, C]_-8 haloalkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively, R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^b, CO₂H, NHC0R^b, 0C0NHR^b and NHC00R^b; wherein R^b is C_1-6 alkyl and each R⁰ group is optionally substituted with 1 to 2 R^p substituents, and wherein optionally any two R⁰ group located on adjacent atoms can be combined to form a 5- to 6- membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; and wherein R^m, Rⁿ and R^p are each independently selected from the group consisting of halogen, Ci_-8 alkyl, aryl, hydroxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino and Ci_-8 alkoxy.

2. The method according to claim 1, wherein in Formula I, R¹ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN, wherein each R¹ group is optionally substituted with 1 to 2 R^m substituents; n is an integer from 1 to 3; R² and R³ are each independently selected from the group consisting of hydrogen and Ci_-8 alkyl; R⁴ is Ci_-8 alkyl, and is optionally substituted with 1 to 2 R" substituents, or alternatively R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN; and each R⁰ group is optionally substituted with 1 to 2 R^p substituents.

3. The method according to claim 1, wherein in Formula I, R⁴ is selected from the group consisting of Ci_-8 alkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 R^π substituents.

4. The method according to claim 3, wherein R³ and R⁴ are each independently selected from the group consisting of methyl, ethyl, propyl and butyl.

5. The method according to claim 1, wherein in Formula I, R⁴ is selected from the group consisting of aryl and heteroaryl; and is optionally substituted with 1 to 4 R⁰ substituents.

6. The method according to claim δ, wherein the compound of Formula I has the Subformula Ia:

wherein m is an integer from 0 to 4.

7. The method according to claim 6, wherein R¹ and R⁰ are each independently selected from the group consisting of Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN; m and n are each an integer from 1 to 3; and R² and R³ are each independently hydrogen or C_1-8 alkyl.

8. The method according to claim 7, wherein R¹ and R⁰ are independently selected from the group consisting of fluoride, chloride, bromide and iodide; and m and n are each independently an integer from 1 to 3.

9. The method according to claim 6, wherein the compound of Formula I is selected from the group consisting of:

and

10. The method according to claim 1, wherein the steroid is testosterone.

11. The method according to claim 1, wherein the cell is in a mammal.

12. The method according to claim 11, wherein the mammal is a human.

13. The method according to claim 12, wherein the human has a disease or disorder associated with low levels of endogenous steroids.

14. The method according to claim 13, wherein the disease or disorder is selected from the group consisting of: menopause, peri-menopause, sexual dysfunction, delayed puberty, infertility, hypoandrogenism, and combinations thereof.

15. The method according to claim 1, wherein the steroid is estrogen.

16. The method according to claim 15, wherein the cell is in a mammal.

17. The method according to claim 16, wherein the mammal is a human.

18. The method according to claim 17, wherein the human has a disease, disorder, or condition treatable with estrogen.

19. The method according to claim 18, wherein the disease, disorder, or condition is selected from the group consisting of: symptoms resulting from post-menopause, child birth or parturition, and prostate cancer.

20. The method according to claim 18, wherein the symptoms resulting from post-menopause are selected from the group consisting of: osteoporosis, hot flashes, vaginal dryness, urinary stress incontinence, chilly sensations, dizziness, fatigue, irritability, and sweating.

21. The method according to claim 1, wherein the steroid is a Cortisol or glucocorticoid.

22. The method according to claim 21, wherein the cell is in a mammal.

23. The method according to claim 22, wherein the mammal is a human.

24. The method according to claim 23, wherein the human has a disease, disorder, or condition treatable with Cortisol or glucocorticoid.

25. The method according to claim 24, wherein the disease, disorder, or condition is selected from the group consisting of: allergic, inflammatory, and autoimmune disorders, acute transplant rejection, and graft-versus-host disease.

26. An in vitro method of identifying modulators of steroid hormone signal transduction, said method comprising: (a) contacting a cell expressing an androgen receptor with an androgen and a test compound suspected of having the ability to modulate steroid hormone signal transduction; and (b) determining whether the test compound modulates signal transduction through the androgen receptor, wherein a compound that increases signal transduction through the androgen receptor relative to a control is identified as a compound that enhances steroid hormone signal transduction and a compound that reduces signal transduction through the androgen receptor relative to a control is identified as a compound that represses steroid hormone signal transduction.

27. The method of claim 26, wherein the androgen is testosterone.

28. The method of claim 26, wherein the cell is transfected with the androgen receptor.

29. The method of claim 26, wherein step (b) comprises measuring the signal from a reporter gene following step (a).

30. The method of claim 29 wherein the reporter gene is luciferase.

31. A pharmaceutically acceptable composition comprising a therapeutically effective amount of a steroid and a therapeutically effective amount of a compound having Formula I as set forth in any of claims 1 to 9.

32. The composition according to claim 31, wherein said steroid is testosterone.

33. An in vitro method of identifying modulators of steroid hormone signal transduction, said method comprising: (a) contacting a cell expressing an estrogen receptor with an estrogen and a test compound suspected of having the ability to modulate steroid hormone signal transduction; and (b) determining whether the test compound modulates signal transduction through the estrogen receptor, wherein a compound that increases signal transduction through the estrogen receptor relative to a control is identified as a compound that enhances steroid hormone signal transduction and a compound that reduces signal transduction through the estrogen receptor relative to a control is identified as a compound that represses steroid hormone signal transduction.

34. An in vitro method of identifying modulators of steroid hormone signal transduction, said method comprising: (a) contacting a cell expressing a Cortisol or glucocorticoid receptor with a Cortisol or glucocorticoid and a test compound suspected of having the ability to modulate steroid hormone signal transduction; and (b) determining whether the test compound modulates signal transduction through the Cortisol or glucocorticoid receptor, wherein a compound that increases signal transduction through the Cortisol or glucocorticoid receptor relative to a control is identified as a compound that enhances steroid hormone signal transduction and a compound that reduces signal transduction through the Cortisol or glucocorticoid receptor relative to a control is identified as a compound that represses steroid hormone signal transduction.

35. A method of providing androgen replacement therapy with reduced attendant liver toxicity, the method comprising the step of administering to a subject in need thereof: (a) a therapeutically effective amount of an androgen replacement therapy agent; and (b) a therapeutically effective amount of a compound having the Formula I:

wherein R¹ is a member selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^a, CO₂H, NHC(O)R², OC(O)NHR³ and NHC(O)OR³; wherein R³ is Ci _-6 alkyl and each R¹ group is optionally substituted with 1 to 2 R^m substituents, and wherein optionally any two R¹ group located on adjacent atoms can be combined to form a 5- to 6-membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; n is an integer from 0-4; R and R are each members independently selected from the group consisting of hydrogen, Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy and Ci_-8 alkoxy; R⁴ is a member selected from the group consisting of C_1-8 alkyl, C_1-8 haloalkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively, R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, C_1-8 alkyl, C_1-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^b, CO₂H, NHCOR^b, OCONHR^b and NHCOOR^b; wherein R^b is Ci_-6 alkyl and each R⁰ group is optionally substituted with 1 to 2 R^p substituents, and wherein optionally any two R⁰ group located on adjacent atoms can be combined to form a 5- to 6- membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; and wherein R^m, Rⁿ and R^p are each independently selected from the group consisting of halogen, Ci_-8 alkyl, aryl, hydroxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino and Ci_-8 alkoxy, thereby providing androgen replacement therapy with reduced attendant liver toxicity.

36. The method according to claim 35, wherein in Formula I, R¹ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN, wherein each R¹ group is optionally substituted with 1 to 2 R^m substituents; n is an integer from 1 to 3; R² and R³ are each independently selected from the group consisting of hydrogen and Ci_-8 alkyl; R⁴ is Ci_-8 alkyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN; and each R⁰ group is optionally substituted with 1 to 2 R^p substituents.

37. The method according to claim 35, wherein in Formula I, R⁴ is selected from the group consisting Of Ci_-8 alkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents.

38. The method according to claim 37, wherein R³ and R⁴ are each independently selected from the group consisting of methyl, ethyl, propyl and butyl.

39. The method according to claim 1, wherein in Formula I, R is selected from the group consisting of aryl and heteroaryl; and is optionally substituted with 1 to 4 R⁰ substituents.

40. The method according to claim 39, wherein the compound of Formula I has the Subformula Ia:

(Ia) wherein m is an integer from 0 to 4.

41. The method according to claim 40, wherein R¹ and R⁰ are each independently selected from the group consisting of Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN; m and n are each an integer from 1 to 3; and R² and R³ are each independently hydrogen or Ci_-8 alkyl.

42. The method according to claim 41, wherein R¹ and R⁰ are independently selected from the group consisting of fluoride, chloride, bromide and iodide; and m and n are each independently an integer from 1 to 3.

43. The method according to claim 40, wherein the compound of Formula I is selected from the group consisting of:

44. The method of claim 35, wherein the androgen replacement therapy agent comprises testosterone.

45. A method of preparing the cervix for parturition or child birth, the method comprising the step of administering to a subject in need thereof: (a) a therapeutically effective amount of an estrogen; and (b) a therapeutically effective amount of a compound having the Formula I:

R² R³ (I) wherein R¹ is a member selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^a, CO₂H, NHC(O)R², OC(O)NHR³ and NHC(0)0R^a; wherein R^a is C_1-6 alkyl and each R¹ group is optionally substituted with 1 to 2 R^m substituents, and wherein optionally any two R¹ group located on adjacent atoms can be combined to form a 5- to 6-membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; n is an integer from 0-4; R² and R³ are each members independently selected from the group consisting of hydrogen, Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy and Ci_-8 alkoxy; R⁴ is a member selected from the group consisting OfCi_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively, R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, C_]-8 dialkylamino, NO₂, CN, CO₂R^b, CO₂H, NHC0R^b, 0C0NHR^b and NHC00R^b; wherein R^b is C_]-6 alkyl and each R⁰ group is optionally substituted with 1 to 2 R^p substituents, and wherein optionally any two R⁰ group located on adjacent atoms can be combined to form a 5- to 6- membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; and wherein R^m, Rⁿ and R^p are each independently selected from the group consisting of halogen, Ci_-8 alkyl, aryl, hydroxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino and Ci_-8 alkoxy, thereby preparing the cervix for parturition or child birth.

46. The method according to claim 45, wherein in Formula I, R¹ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN, wherein each R¹ group is optionally substituted with 1 to 2 R^m substituents; n is an integer from 1 to 3; R² and R³ are each independently selected from the group consisting of hydrogen and Ci_-8 alkyl; R⁴ is Ci_-8 alkyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN; and each R⁰ group is optionally substituted with 1 to 2 R^p substituents.

47. The method according to claim 45, wherein in Formula I, R⁴ is selected from the group consisting Of Ci_-8 alkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 R" substituents.

48. The method according to claim 47, wherein R³ and R⁴ are each independently selected from the group consisting of methyl, ethyl, propyl and butyl.

49. The method according to claim 45, wherein in Formula I, R⁴ is selected from the group consisting of aryl and heteroaryl; and is optionally substituted with 1 to 4 R⁰ substituents.

50. The method according to claim 49, wherein the compound of Formula I has the Subformula Ia:

wherein m is an integer from 0 to 4.

51. The method according to claim 50, wherein R¹ and R⁰ are each independently selected from the group consisting Of Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN; m and n are each an integer from 1 to 3; and R² and R³ are each independently hydrogen or Ci_-8 alkyl.

52. The method according to claim 51, wherein R¹ and R⁰ are independently selected from the group consisting of fluoride, chloride, bromide and iodide; and m and n are each independently an integer from 1 to 3.

53. The method according to claim 50, wherein the compound of Formula I is selected from the group consisting of:

54. The method of claim 45, wherein the estrogen is estradiol.

55. A method of inducing parturition or child birth, the method comprising the step of administering to a subject in need thereof:

(a) a therapeutically effective amount of: a compound having the Formula I for internal delivery:

(I) wherein

R is a member selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R³, CO₂H, NHC(O)R³, OC(O)NHR³ and NHC(0)0R^a; wherein R^a is C_]-6 alkyl and each R¹ group is optionally substituted with 1 to 2 R^m substituents, and wherein optionally any two R¹ group located on adjacent atoms can be combined to form a 5- to 6-membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; n is an integer from 0-4; R² and R³ are each members independently selected from the group consisting of hydrogen, Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy and C_]-8 alkoxy; R⁴ is a member selected from the group consisting of Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively, R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, C_1-8 dialkylamino, NO₂, CN, CO₂R^b, CO₂H, NHCOR^b, OCONHR^b and NHCOOR^b; wherein R^b is Ci_-6 alkyl and each R⁰ group is optionally substituted with 1 to 2 R^p substituents, and wherein optionally any two R⁰ group located on adjacent atoms can be combined to form a 5- to 6- membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; and wherein R^m, Rⁿ and R^p are each independently selected from the group consisting of halogen, Ci_-8 alkyl, aryl, hydroxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino and Ci_-8 alkoxy; and (b) a therapeutically effective amount of oxytocin, thereby inducing parturition or child birth.

56. The method according to claim 55, wherein in Formula I, R¹ is selected from the group consisting of halogen, Ci_-8 alkyl, C_1-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN, wherein each R¹ group is optionally substituted with 1 to 2 R^m substituents; n is an integer from 1 to 3; R² and R³ are each independently selected from the group consisting of hydrogen and Ci_-8 alkyl; R⁴ is Ci_-8 alkyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN; and each R⁰ group is optionally substituted with 1 to 2 R^p substituents.

57. The method according to claim 55, wherein in Formula I, R⁴ is selected from the group consisting of Ci_-8 alkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents.

58. The method according to claim 57, wherein R³ and R⁴ are each independently selected from the group consisting of methyl, ethyl, propyl and butyl.

59. The method according to claim 55, wherein in Formula I, R⁴ is selected from the group consisting of aryl and heteroaryl; and is optionally substituted with 1 to 4 R⁰ substituents.

60. The method according to claim 59, wherein the compound of Formula I has the Subformula Ia:

(Ia) wherein m is an integer from 0 to 4.

61. The method according to claim 60, wherein R¹ and R⁰ are each independently selected from the group consisting Of Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN; m and n are each an integer from 1 to 3; and R² and R³ are each independently hydrogen or Ci_-8 alkyl.

62. The method according to claim 61, wherein R¹ and R⁰ are independently selected from the group consisting of fluoride, chloride, bromide and iodide; and m and n are each independently an integer from 1 to 3.

63. The method according to claim 60, wherein the compound of Formula I is selected from the group consisting of:

64. A method of enhancing the efficacy of topical corticoid administration, the method comprising the step of administering to a subject in need thereof:

(a) a therapeutically effective amount of a topically administered corticoid; and

(b) a therapeutically effective amount of a compound having the Formula I:

wherein

R¹ is a member selected from the group consisting of halogen, Ci_-8 alkyl, C]_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^a, CO₂H, NHC(O)R³, OC(O)NHR³ and NHC(O)OR²; wherein R^a is Ci_-6 alkyl and each R¹ group is optionally substituted with 1 to 2 R^m substituents, and wherein optionally any two R¹ group located on adjacent atoms can be combined to form a 5- to 6-membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; n is an integer from 0-4;

R⁴ is a member selected from the group consisting of Ci_-8 alkyl, C]_-8 haloalkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 R" substituents, or alternatively, R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, C_1-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, C_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^b, CO₂H, NHCOR^b, OCONHR^b and NHCOOR^b; wherein R^b is Ci_-6 alkyl and each R⁰ group is optionally substituted with 1 to 2 R^p substituents, and wherein optionally any two R⁰ group located on adjacent atoms can be combined to form a 5- to 6- membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; and wherein R^m, Rⁿ and R^p are each independently selected from the group consisting of halogen, Ci_-8 alkyl, aryl, hydroxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino and Ci_-8 alkoxy, thereby enhancing the efficacy of topical corticoid administration.

65. The method according to claim 64, wherein in Formula I, R¹ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN, wherein each R¹ group is optionally substituted with 1 to 2 R^m substituents; n is an integer from 1 to 3; R² and R³ are each independently selected from the group consisting of hydrogen and Ci_-8 alkyl; R⁴ is Ci_-8 alkyl, and is optionally substituted with 1 to 2 R" substituents, or alternatively R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Cj_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN; and each R⁰ group is optionally substituted with 1 to 2 R^p substituents.

66. The method according to claim 64, wherein in Formula I, R⁴ is selected from the group consisting OfCi_-8 alkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents.

67. The method according to claim 66, wherein R³ and R⁴ are each independently selected from the group consisting of methyl, ethyl, propyl and butyl.

68. The method according to claim 64, wherein in Formula I, R⁴ is selected from the group consisting of aryl and heteroaryl; and is optionally substituted with 1 to 4 R⁰ substituents.

69. The method according to claim 68, wherein the compound of Formula I has the Subformula Ia:

(Ia) wherein m is an integer from 0 to 4.

70. The method according to claim 69, wherein R¹ and R⁰ are each independently selected from the group consisting of C_1-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN; m and n are each an integer from 1 to 3; and R² and R³ are each independently hydrogen or Ci_-8 alkyl.

71. The method according to claim 70, wherein R¹ and R⁰ are independently selected from the group consisting of fluoride, chloride, bromide and iodide; and m and n are each independently an integer from 1 to 3.

72. The method according to claim 69, wherein the compound of Formula I is selected from the group consisting of:

73. The method of claim 64, wherein the topical corticoid comprises Cortisol or hydrocortisone.

74. A method of treating delayed puberty, the method comprising the step of administering to a subject in need thereof:

(a) a therapeutically effective amount of a steroid hormone; and

(b) a therapeutically effective amount of a compound having the Formula I:

wherein R¹ is a member selected from the group consisting of halogen, C_1-8 alkyl, C_1-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R³, CO₂H, NHC(O)R⁸, 0C(0)NHR^a and NHC(0)0R^a; wherein R^a is C_]-6 alkyl and each R¹ group is optionally substituted with 1 to 2 R^m substituents, and wherein optionally any two R¹ group located on adjacent atoms can be combined to form a 5- to 6-membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; n is an integer from 0-4; R² and R³ are each members independently selected from the group consisting of hydrogen, Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy and Ci_-8 alkoxy; R⁴ is a member selected from the group consisting of Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively, R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, C_1-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^b, CO₂H, NHC0R^b, 0C0NHR^b and NHC00R^b; wherein R^b is Ci_-6 alkyl and each R⁰ group is optionally substituted with 1 to 2 R^p substituents, and wherein optionally any two R⁰ group located on adjacent atoms can be combined to form a 5- to 6- membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; and wherein R^m, R" and R^p are each independently selected from the group consisting of halogen, Ci_-8 alkyl, aryl, hydroxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino and Ci_-8 alkoxy, thereby treating delayed puberty.

75. The method according to claim 74, wherein in Formula I, R¹ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN, wherein each R¹ group is optionally substituted with 1 to 2 R^m substituents; n is an integer from 1 to 3; R² and R³ are each independently selected from the group consisting of hydrogen and Ci_-8 alkyl; R⁴ is Ci_-8 alkyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN; and each R⁰ group is optionally substituted with 1 to 2 R^p substituents.

76. The method according to claim 74, wherein in Formula I, R⁴ is selected from the group consisting Of Ci_-8 alkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents.

77. The method according to claim 76, wherein R³ and R⁴ are each independently selected from the group consisting of methyl, ethyl, propyl and butyl.

78. The method according to claim 74, wherein in Formula I, R⁴ is selected from the group consisting of aryl and heteroaryl; and is optionally substituted with 1 to 4 R⁰ substituents.

79. The method according to claim 78, wherein the compound of Formula I has the Subformula Ia:

wherein m is an integer from 0 to 4.

80. The method according to claim 79, wherein R¹ and R⁰ are each independently selected from the group consisting of Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, C]_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN; m and n are each an integer from 1 to 3; and R² and R³ are each independently hydrogen or Ci_-8 alkyl.

81. The method according to claim 80, wherein R¹ and R⁰ are independently selected from the group consisting of fluoride, chloride, bromide and iodide; and m and n are each independently an integer from 1 to 3.

82. The method according to claim 79, wherein the compound of Formula I is selected from the group consisting of:

83. The method of claim 74, wherein the steroid hormone comprises testosterone.

84. A method of treating of treating hypothyroidism, the method comprising the step of administering to a subject in need thereof: (a) a therapeutically effective amount of a thyroid hormone; and (b) a therapeutically effective amount of a compound having the Formula I:

R² R³ (I) wherein R¹ is a member selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^a, CO₂H, NHC(0)R^a, OC(O)NHR³ and NHC(O)OR³; wherein R^a is C, _-6 alkyl and each R¹ group is optionally substituted with 1 to 2 R^m substituents, and wherein optionally any two R¹ group located on adjacent atoms can be combined to form a 5- to 6-membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; n is an integer from 0-4; R² and R³ are each members independently selected from the group consisting of hydrogen, Ci_-8 alkyl, C_t-8 haloalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy and Ci_-8 alkoxy; R⁴ is a member selected from the group consisting of Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively, R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, C_1-8 dialkylamino, NO₂, CN, CO₂R^b, CO₂H, NHCOR^b, OCONHR^b and NHCOOR^b; wherein R^b is Ci_-6 alkyl and each R⁰ group is optionally substituted with 1 to 2 R^p substituents, and wherein optionally any two R⁰ group located on adjacent atoms can be combined to form a 5- to 6- membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; and wherein R^m, Rⁿ and R^p are each independently selected from the group consisting of halogen, Ci_-8 alkyl, aryl, hydroxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino and Ci_-8 alkoxy, thereby treating of treating hypothyroidism.

85. The method according to claim 84, wherein in Formula I, R¹ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN, wherein each R¹ group is optionally substituted with 1 to 2 R^m substituents; n is an integer from 1 to 3; R² and R³ are each independently selected from the group consisting of hydrogen and Ci_-8 alkyl; R⁴ is Ci_-8 alkyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN; and each R⁰ group is optionally substituted with 1 to 2 R^p substituents.

86. The method according to claim 84, wherein in Formula I, R⁴ is selected from the group consisting of Ci_-8 alkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents.

87. The method according to claim 86, wherein R³ and R⁴ are each independently selected from the group consisting of methyl, ethyl, propyl and butyl.

88. The method according to claim 84, wherein in Formula I, R⁴ is selected from the group consisting of aryl and heteroaryl; and is optionally substituted with 1 to 4 R⁰ substituents.

89. The method according to claim 88, wherein the compound of Formula I has the Subformula Ia:

wherein m is an integer from 0 to 4.

90. The method according to claim 89, wherein R¹ and R⁰ are each independently selected from the group consisting of Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN; m and n are each an integer from 1 to 3; and R² and R³ are each independently hydrogen or Ci_-8 alkyl.

91. The method according to claim 90, wherein R¹ and R⁰ are independently selected from the group consisting of fluoride, chloride, bromide and iodide; and m and n are each independently an integer from 1 to 3.

92. The method according to claim 89, wherein the compound of Formula I is selected from the group consisting of:

93. The method of claim 84, wherein the thyroid hormone comprises levothyroxine.

94. A method of enhancing the enhancing the efficacy of weak estrogen in hormone replacement therapy, the method comprising the step of administering to a subject in need thereof

(a) a therapeutically effective amount of a weak estrogen; and

(b) a therapeutically effective amount of a compound having the Formula I:

wherein

R¹ is a member selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, C_1-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^a, CO₂H, NHC(O)R², OC(O)NHR³ and NHC(O)OR³; wherein R³ is C_]-6 alkyl and each R¹ group is optionally substituted with 1 to 2 R^m substituents, and wherein optionally any two R¹ group located on adjacent atoms can be combined to form a 5- to 6-membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; n is an integer from 0-4;

R⁴ is a member selected from the group consisting Of Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively, R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, C_1-8 alkyl, C_1-8 haloalkyl, C_1-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Cj_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^b, CO₂H, NHCOR^b, OCONHR^b and NHCOOR^b; wherein R^b is Ci_-6 alkyl and each R⁰ group is optionally substituted with 1 to 2 R^p substituents, and wherein optionally any two R⁰ group located on adjacent atoms can be combined to form a 5- to 6- membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; and wherein R^m, Rⁿ and R^p are each independently selected from the group consisting of halogen, Ci_-8 alkyl, aryl, hydroxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino and Ci_-S alkoxy, thereby enhancing the efficacy of weak estrogen in hormone replacement therapy.

95. The method according to claim 94, wherein in Formula I, R¹ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci-s alkylamino, Ci_-8 dialkylamino, NO₂ and CN, wherein each R¹ group is optionally substituted with 1 to 2 R^m substituents; n is an integer from 1 to 3; R² and R³ are each independently selected from the group consisting of hydrogen and Ci_-8 alkyl; R⁴ is Ci_-8 alkyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN; and each R⁰ group is optionally substituted with 1 to 2 R^p substituents.

96. The method according to claim 94, wherein in Formula I, R⁴ is selected from the group consisting of Ci_-8 alkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents.

97. The method according to claim 96, wherein R³ and R⁴ are each independently selected from the group consisting of methyl, ethyl, propyl and butyl.

98. The method according to claim 94, wherein in Formula I, R is selected from the group consisting of aryl and heteroaryl; and is optionally substituted with 1 to 4 R⁰ substituents.

99. The method according to claim 98, wherein the compound of Formula I has the Subformula Ia: m

(Ia) wherein m is an integer from 0 to 4.

100. The method according to claim 99, wherein R¹ and R⁰ are each independently selected from the group consisting of Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN; m and n are each an integer from 1 to 3; and R² and R³ are each independently hydrogen or Ci_-8 alkyl.

101. The method according to claim 100, wherein R¹ and R⁰ are independently selected from the group consisting of fluoride, chloride, bromide and iodide; and m and n are each independently an integer from 1 to 3.

102. The method according to claim 99, wherein the compound of Formula

I is selected from the group consisting of:

103. The method of claim 94, wherein the weak estrogen comprises estrone or estriol.

104. A pharmaceutical composition comprising (a) a therapeutically effective amount of a steroid; and (b) a therapeutically effective amount of a compound having the Formula I:

wherein R¹ is a member selected from the group consisting of halogen, C_1-8 alkyl, C_1-8 haloalkyl, C_1-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, C_1-8 alkoxy, NH₂, C_1-8 alkylamino, C_]-8 dialkylamino, NO₂, CN, CO₂R³, CO₂H, NHC(O)R^a, OC(O)NHR^a and NHC(O)OR³; wherein R^a is Ci_-6 alkyl and each R¹ group is optionally substituted with 1 to 2 R^m substituents, and wherein optionally any two R¹ group located on adjacent atoms can be combined to form a 5- to 6-membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; n is an integer from 0-4; R² and R³ are each members independently selected from the group consisting of hydrogen, Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy and Ci_-8 alkoxy; R⁴ is a member selected from the group consisting of Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively, R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^b, CO₂H, NHCOR^b, OCONHR^b and NHCOOR^b; wherein R^b is C_]-6 alkyl and each R⁰ group is optionally substituted with 1 to 2 R^p substituents, and wherein optionally any two R⁰ group located on adjacent atoms can be combined to form a 5- to 6- membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; and wherein R^m, R" and R^p are each independently selected from the group consisting of halogen, C_1-8 alkyl, aryl, hydroxy, NH₂, C_1-8 alkylamino, C_1-8 dialkylamino and C_1-8 alkoxy.

105. The pharmaceutical composition according to claim 104, wherein in Formula I, R¹ is selected from the group consisting of halogen, C_1-8 alkyl, C_1-8 haloalkyl, hydroxy, Cj_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN, wherein each R¹ group is optionally substituted with 1 to 2 R^m substituents; n is an integer from 1 to 3; R² and R³ are each independently selected from the group consisting of hydrogen and Ci_-8 alkyl; R⁴ is Ci_-8 alkyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN; and each R⁰ group is optionally substituted with 1 to 2 R^p substituents.

106. The pharmaceutical composition according to claim 104, wherein in Formula I, R⁴ is selected from the group consisting of Ci_-8 alkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents.

107. The pharmaceutical composition according to claim 106, wherein R³ and R⁴ are each independently selected from the group consisting of methyl, ethyl, propyl and butyl.

108. The pharmaceutical composition according to claim 104, wherein in Formula I, R⁴ is selected from the group consisting of aryl and heteroaryl; and is optionally substituted with 1 to 4 R⁰ substituents.

109. The pharmaceutical composition according to claim 108, wherein the compound of Formula I has the Sub formula Ia:

(Ia) wherein m is an integer from 0 to 4.

110. The pharmaceutical composition according to claim 109, wherein R¹ and R⁰ are each independently selected from the group consisting Of Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, C_1-8 alkylamino, C]_-8 dialkylamino, NO₂ and CN; m and n are each an integer from 1 to 3; and R² and R³ are each independently hydrogen or Ci_-8 alkyl.

111. The pharmaceutical composition according to claim 110, wherein R¹ and R⁰ are independently selected from the group consisting of fluoride, chloride, bromide and iodide; and m and n are each independently an integer from 1 to 3.

112. The pharmaceutical composition according to claim 109, wherein the compound of Formula I is selected from the group consisting of:

113. The pharmaceutical composition according to claim 104, wherein the steroid is selected from the group consisting of: an androgen, an estrogen, a glucocorticoid, and thyroid hormone.

114. A pharmaceutical composition for use as a systemic anti-inflammatory agent comprising (a) a therapeutically effective amount of a corticoid; and (b) a therapeutically effective amount of a compound having the Formula I:

wherein

R is a member selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^a, CO₂H, NHC(O)R^a, OC(O)NHR^a and NHC(O)OR³; wherein R^a is Ci_-6 alkyl and each R¹ group is optionally substituted with 1 to 2 R^m substituents, and wherein optionally any two R¹ group located on adjacent atoms can be combined to form a 5- to 6-membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; n is an integer from 0-4; R² and R³ are each members independently selected from the group consisting of hydrogen, Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy and Ci_-8 alkoxy; R⁴ is a member selected from the group consisting OfCi_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively, R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, C_1-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^b, CO₂H, NHCOR^b, OCONHR^b and NHCOOR^b; wherein R^b is Ci_-6 alkyl and each R⁰ group is optionally substituted with 1 to 2 R^p substituents, and wherein optionally any two R⁰ group located on adjacent atoms can be combined to form a 5- to 6- membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; and wherein R^m, Rⁿ and R^p are each independently selected from the group consisting of halogen, Ci_-8 alkyl, aryl, hydroxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino and Ci_-8 alkoxy.

115. The pharmaceutical composition according to claim 114, wherein in Formula I, R¹ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN, wherein each R¹ group is optionally substituted with 1 to 2 R^m substituents; n is an integer from 1 to 3; R² and R³ are each independently selected from the group consisting of hydrogen and Ci_-8 alkyl; R⁴ is Ci_-8 alkyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, C_]-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN; and each R⁰ group is optionally substituted with 1 to 2 R^p substituents.

116. The pharmaceutical composition according to claim 114, wherein in Formula I, R⁴ is selected from the group consisting OfC_1-8 alkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 R" substituents.

117. The pharmaceutical composition according to claim 116, wherein R³ and R⁴ are each independently selected from the group consisting of methyl, ethyl, propyl and butyl.

118. The pharmaceutical composition according to claim 114, wherein in Formula I, R⁴ is selected from the group consisting of aryl and heteroaryl; and is optionally substituted with 1 to 4 R⁰ substituents.

119. The pharmaceutical composition according to claim 118, wherein the compound of Formula I has the Sub formula Ia:

R² R³ (Ia) wherein m is an integer from 0 to 4.

120. The pharmaceutical composition according to claim 119, wherein R¹ and R⁰ are each independently selected from the group consisting of C_1-8 alkyl, C_1-8 haloalkyl, hydroxy, C_1-8 alkoxy, NH₂, C_1-8 alkylamino, C_1-8 dialkylamino, NO₂ and CN; m and n are each an integer from 1 to 3; and R² and R³ are each independently hydrogen or C_1-8 alkyl.

121. The pharmaceutical composition according to claim 120, wherein R¹ and R⁰ are independently selected from the group consisting of fluoride, chloride, bromide and iodide; and m and n are each independently an integer from 1 to 3.

122. The pharmaceutical composition according to claim 119, wherein the compound of Formula I is selected from the group consisting of:

123. The pharmaceutical composition of claim 114, wherein the corticoid comprises beclometasone, prednisone, or dexamethasone.

124. A method of enhancing steroid hormone signal transduction in a mammal, the method comprising administering to said mammal a composition for internal delivery comprising: a therapeutically effective amount of a compound having the Formula I:

(I) wherein

R¹ is a member selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R³, CO₂H, NHC(O)R³, OC(O)NHR³ and NHC(O)OR³; wherein R^a is Ci_-6 alkyl and each R¹ group is optionally substituted with 1 to 2 R^m substituents, and wherein optionally any two R¹ group located on adjacent atoms can be combined to form a 5- to 6-membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; n is an integer from 0-4;

R⁴ is a member selected from the group consisting of Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively, R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, C_1-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^b, CO₂H, NHCOR^b, OCONHR^b and NHCOOR^b; wherein R^b is C_]-6 alkyl and each R⁰ group is optionally substituted with 1 to 2 R^p substituents, and wherein optionally any two R⁰ group located on adjacent atoms can be combined to form a 5- to 6- membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; and

wherein R^m, R" and R^p are each independently selected from the group consisting of halogen, Ci_-8 alkyl, aryl, hydroxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino and Ci_-8 alkoxy.

125. The method according to claim 124, wherein in Formula I, R¹ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN, wherein each R¹ group is optionally substituted with 1 to 2 R^m substituents; n is an integer from 1 to 3; R² and R³ are each independently selected from the group consisting of hydrogen and Ci_-8 alkyl; R⁴ is Ci_-8 alkyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN; and each R⁰ group is optionally substituted with 1 to 2 R^p substituents.

126. The method according to claim 124, wherein in Formula I, R⁴ is selected from the group consisting of Ci_-8 alkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents.

127. The method according to claim 126, wherein R³ and R⁴ are each independently selected from the group consisting of methyl, ethyl, propyl and butyl.

128. The method according to claim 124, wherein in Formula I, R⁴ is selected from the group consisting of aryl and heteroaryl; and is optionally substituted with 1 to 4 R⁰ substituents.

129. The method according to claim 128, wherein the compound of Formula I has the Subformula Ia:

(Ia) wherein m is an integer from 0 to 4.

130. The method according to claim 129, wherein R¹ and R⁰ are each independently selected from the group consisting of Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, C_1-8 dialkylamino, NO₂ and CN; m and n are each an integer from 1 to 3; and R² and R³ are each independently hydrogen or Ci_-8 alkyl.

131. The method according to claim 130, wherein R¹ and R⁰ are independently selected from the group consisting of fluoride, chloride, bromide and iodide; and m and n are each independently an integer from 1 to 3.

132. The method according to claim 129, wherein the compound of Formula I is selected from the group consisting of:

133. The method according to claim 124, wherein the mammal is a human.

134. The method according to claim 133, wherein the human has a disease or disorder associated with low levels of endogenous steroids.

135. The method according to claim 134, wherein the disease or disorder is selected from the group consisting of: menopause, peri-menopause, sexual dysfunction, delayed puberty, infertility, hypoandrogenism, and combinations thereof.

136. The method according to claim 134, wherein the endogenous steroid is selected from the group consisting of an androgen, an estrogen, a glucocorticoid, Cortisol, and a thyroid hormone..

137. The method according to claim 133, wherein the human has a disease, disorder, or condition treatable with estrogen.

138. The method according to claim 137, wherein the disease, disorder, or condition is selected from the group consisting of: symptoms resulting from post-menopause, child birth or parturition, and prostate cancer.

139. The method according to claim 138, wherein the symptoms resulting from post-menopause are selected from the group consisting of: osteoporosis, hot flashes, vaginal dryness, urinary stress incontinence, chilly sensations, dizziness, fatigue, irritability, and sweating.

140. The method according to claim 133, wherein the human has a disease, disorder, or condition treatable with Cortisol or glucocorticoid.

141. The method according to claim 140, wherein the disease, disorder, or condition is selected from the group consisting of: allergic, inflammatory, and autoimmune disorders, acute transplant rejection, and graft-versus-host disease.

142. The method according to claim 133, wherein the human has a disease, disorder, or condition treatable with thyroid hormone.

143. The method according to claim 142, wherein the disease, disorder, or condition is hypothyroidism.

144. The method of claim 124, wherein said internal delivery is selected from the group consisting of oral delivery, injectable delivery, nasal delivery, and transmucosal delivery.

145. A pharmaceutical composition for internal delivery comprising a therapeutically effective amount of a compound having the Formula I:

wherein R¹ is a member selected from the group consisting of halogen, C_1-8 alkyl, C_1-8 haloalkyl, C_1-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, C_1-8 alkoxy, NH₂, Cj_-8 alkylamino, C_1-8 dialkylamino, NO₂, CN, CO₂R³, CO₂H, NHC(O)R³, OC(O)NHR³ and NHC(O)OR³; wherein R^a is Ci_-6 alkyl and each R¹ group is optionally substituted with 1 to 2 R^m substituents, and wherein optionally any two R¹ group located on adjacent atoms can be combined to form a 5- to 6-membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; n is an integer from 0-4; R² and R³ are each members independently selected from the group consisting of hydrogen, Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy and Ci_-8 alkoxy; R⁴ is a member selected from the group consisting Of Ci_-8 alkyl, Ci_-8 haloalkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively, R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, Ci_-8 heteroalkyl, C_2-8 alkenyl, C_2-8 alkynyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂, CN, CO₂R^b, CO₂H, NHC0R^b, 0C0NHR^b and NHC00R^b; wherein R^b is Ci_-6 alkyl and each R⁰ group is optionally substituted with 1 to 2 R^p substituents, and wherein optionally any two R⁰ group located on adjacent atoms can be combined to form a 5- to 6- membered saturated or unsaturated ring having from 0 to 2 heteroatoms selected from N, O and S; and

wherein R^m, Rⁿ and R^p are each independently selected from the group consisting of halogen, Ci_-8 alkyl, aryl, hydroxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino and Ci_-8 alkoxy.

146. The pharmaceutical composition according to claim 145, wherein in Formula I, R¹ is selected from the group consisting of halogen, C_1-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, C_1-8 alkylamino, C_1-8 dialkylamino, NO₂ and CN, wherein each R¹ group is optionally substituted with 1 to 2 R^m substituents; n is an integer from 1 to 3; R² and R³ are each independently selected from the group consisting of hydrogen and Ci_-8 alkyl; R⁴ is Ci_-8 alkyl, and is optionally substituted with 1 to 2 Rⁿ substituents, or alternatively R⁴ is selected from the group consisting of aryl and heteroaryl, and is optionally substituted with from 1 to 4 R⁰ substituents, wherein R⁰ is selected from the group consisting of halogen, Ci_-8 alkyl, Ci_-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN; and each R⁰ group is optionally substituted with 1 to 2 R^p substituents.

147. The pharmaceutical composition according to claim 146, wherein in Formula I, R⁴ is selected from the group consisting OfC_1-8 alkyl, C_2-8 alkenyl and C_2-8 alkynyl, and is optionally substituted with 1 to 2 Rⁿ substituents.

148. The pharmaceutical composition according to claim 147, wherein R³ and R⁴ are each independently selected from the group consisting of methyl, ethyl, propyl and butyl.

149. The pharmaceutical composition according to claim 148, wherein in Formula I, R⁴ is selected from the group consisting of aryl and heteroaryl; and is optionally substituted with 1 to 4 R⁰ substituents.

150. The pharmaceutical composition according to claim 149, wherein the compound of Formula I has the Sub formula Ia:

R² R³ (Ia) wherein m is an integer from 0 to 4.

151. The pharmaceutical composition according to claim 150, wherein R¹ and R⁰ are each independently selected from the group consisting of C_1-8 alkyl, C_1-8 haloalkyl, hydroxy, Ci_-8 alkoxy, NH₂, Ci_-8 alkylamino, Ci_-8 dialkylamino, NO₂ and CN; m and n are each an integer from 1 to 3; and R² and R³ are each independently hydrogen or Ci_-8 alkyl.

152. The pharmaceutical composition according to claim 151, wherein R and R⁰ are independently selected from the group consisting of fluoride, chloride, bromide and iodide; and m and n are each independently an integer from 1 to 3.

153. The pharmaceutical composition according to claim 150, wherein the compound of Formula I is selected from the group consisting of:

154. The pharmaceutical composition according to claim 145, wherein said internal delivery is selected from the group consisting of oral delivery, injectable delivery, nasal delivery, and transmucosal delivery.