METHODS FOR INHIBITING MUSCLE ATROPHY

Abstract

In one aspect, the invention relates methods for inhibiting or preventing muscle atrophy or increasing muscle mass by providing to a subject in need thereof an effective amount of ursolic acid, a derivative thereof, or an analog of the ursane scaffold. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 13/698,645 (having a 371(c) date of Apr. 22, 2013), which is a national phase application of International Application No. PCT/US11/37238, filed May 19, 2011, which claims the benefit of U.S. Provisional Application Nos. 61/346,813, filed on May 20, 2010, and 61/445,488, filed on Feb. 22, 2011. The entire contents of each of the prior applications are hereby incorporated by reference in their entereity herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant VA Career Development Award-2 to Christopher M. Adams, and support from a VA Research Enhancement Award Program to Steven D. Kunkel. The United States government has certain rights in the invention.

BACKGROUND

Skeletal muscle atrophy is characteristic of starvation and a common effect of aging. It is also a nearly universal consequence of severe human illnesses, including cancer, chronic renal failure, congestive heart failure, chronic respiratory disease, insulin deficiency, acute critical illness, chronic infections such as HIV/AIDS, muscle denervation, and many other medical and surgical conditions that limit muscle use. However, medical therapies to prevent or reverse skeletal muscle atrophy in human patients do not exist. As a result, millions of individuals suffer sequelae of muscle atrophy, including weakness, falls, fractures, opportunistic respiratory infections, and loss of independence. The burden that skeletal muscle atrophy places on individuals, their families, and society in general, is tremendous.

The pathogenesis of skeletal muscle atrophy is not well understood. Nevertheless, important advances have been made. For example, it has been been described previously that insulin/IGF1 signaling promotes muscle hypertrophy and inhibits muscle atrophy, but is reduced by atrophy-inducing stresses such as fasting or muscle denervation (Bodine S C, et al. (2001) Nat Cell Biol 3(11):1014-1019; Sandri M, et al. (2004) Cell 117(3):399-4121; Stitt T N, et al. (2004) Mol Cell 14(3):395-403; Hu Z, et al. (2009) The Journal of clinical investigation 119(10):3059-3069; Dobrowolny G, et al. (2005) The Journal of cell biology 168(2):193-199; Kandarian S C & Jackman R W (2006) Muscle & nerve 33(2):155-165; Hirose M, et al. (2001) Metabolism: clinical and experimental 50(2):216-222; Pallafacchina G, et al. (2002) Proceedings of the National Academy of Sciences of the United States of America 99(14):9213-9218). The hypertrophic and anti-atrophic effects of insulin/IGF1 signaling are mediated at least in part through increased activity of phosphoinositide 3-kinase (PI3K) and its downstream effectors, including Akt and mammalian target of rapamycin complex 1 (mTORC1) Sandri M (2008) Physiology (Bethesda) 23:160-170; Glass D J (2005) The international journal of biochemistry & cell biology 37(10): 1974-1984).

Another important advance came from microarray studies of atrophying rodent muscle (Lecker S H, et al. (2004) Faseb J 18(1):39-51; Sacheck J M, et al. (2007) Faseb J 21(1):140-155; Jagoe R T, et al. Faseb J 16(13):1697-1712). Those studies showed that several seemingly disparate atrophy-inducing stresses (including fasting, muscle denervation and severe systemic illness) generated many common changes in skeletal muscle mRNA expression. Some of those atrophy-associated changes promote muscle atrophy in mice; these include induction of the mRNAs encoding atroginI/MAFbx and MuRF1 (two E3 ubiquitin ligases that catalyze proteolytic events), and repression of the mRNA encoding PGC-1 α (a transcriptional co-activator that inhibits muscle atrophy) (Sandri M, et al. (2006) Proceedings of the National Academy of Sciences of the United States of America 103(44):16260-16265; Wenz T, et al. Proceedings of the National Academy of Sciences of the United States of America 106(48):20405-20410; Bodine S C, et al. (2001) Science (New York, N.Y. 294(5547):1704-1708; Lagirand-Cantaloube J, et al. (2008) The EMBO journal 27(8):1266-1276; Cohen S, et al. (2009) The Journal of cell biology 185(6):1083-1095; Adams V, et al. (2008) Journal of molecular biology 384(1):48-59). However, the roles of many other mRNAs that are increased or decreased in atrophying rodent muscle are not yet defined. Data on the mechanisms of human muscle atrophy are even more limited, although atrogin-1 and MuRF1 are likely to be involved (Leger B, et al. (2006) Faseb J20(3):583-585; Doucet M, et al. (2007) American journal of respiratory and critical care medicine 176(3):261-269; Levine S, et al. (2008) The New England journal of medicine 358(13): 1327-1335).

Despite advances in understanding the physiology and pathophysiology of muscle atrophy, there is still a scarcity of compounds that are both potent, efficacious, and selective modulators of muscle growth and also effective in the treatment of muscle atrophy associated and diseases in which the muscle atrophy or the need to increase muscle mass is involved. These needs and other needs are satisfied by the present invention.

SUMMARY

In accordance with the purpose(s) of the invention, as embodied and broadly described herein, the invention, in one aspect, relates to compounds useful in methods to inhibit muscle atrophy and to increase muscle mass by providing to a subject in need thereof an effective amount of ursolic acid or a derivative thereof, and pharmaceutical compositions comprising compounds used in the methods.

In further aspects, the the purpose(s) of the invention, as embodied and broadly described herein, the invention, in one aspect, relates to compounds useful in methods to modulate muscle growth, methods to inhibit muscle atrophy and to increase muscle mass, methods to induce skeletal muscle hypertrophy, methods to enhance tissue growth, and pharmaceutical compositions comprising compounds used in the methods.

Disclosed are methods for preventing or treating muscle atrophy in an animal, the method comprising administering to the animal a compound of the formula:

wherein each is an optional covalent bond, and R⁰ is optionally present; wherein n is 0 or 1; wherein R⁰, when present, is hydrogen; wherein R^1a is selected from C1-C6 alkyl and —C(O)ZR¹⁰; wherein R^1b is selected from C1-C6 alkyl; or wherein R^1a and R^1b are covalently bonded and, along with the intermediate carbon, together comprise optionally substituted C3-C5 cycloalkyl or optionally substituted C2-C5 heterocycloalkyl; wherein R^2a and R^2b are independently selected from hydrogen and —OR¹¹, provided that at least one of R^2a and R^2b is —OR¹¹; or wherein R^2a and R^2b together comprise ═O; wherein each of R^3a and R^3b is independently selected from hydrogen, hydroxyl, C1-C6 alkyl, and C1-C6 alkoxyl, provided that R^3a and R^3b are not simultaneously hydroxyl; or wherein R^3a and R^3b are covalently bonded and, along with the intermediate carbon, together comprise optionally substituted C3-C5 cycloalkyl or optionally substituted C2-C5 heterocycloalkyl; wherein each of R⁴, R⁵, and R⁶ is independently selected from C1-C6 alkyl; wherein R⁷ is selected from C1-C6 alkyl, —CH₂OR¹², and —C(O)ZR¹²; wherein R⁸ is selected from hydrogen and C1-C6 alkyl; wherein each of R^9a and R^9b is independently selected from hydrogen and C1-C6 alkyl, provided that R^9a and R^9b are not simultaneously hydrogen; or wherein R^9a and R^9b are covalently bonded and, along with the intermediate carbon, together comprise optionally substituted C3-C5 cycloalkyl or optionally substituted C2-C5 heterocycloalkyl; wherein R¹⁰ is selected from hydrogen and C1-C6 alkyl; wherein each R¹¹ is independently selected from hydrogen, C1-C6 alkyl, C1-C5 heteroalkyl, C3-C6 cycloalkyl, C4-C6 heterocycloalkyl, phenyl, heteroaryl, and —C(O)R¹⁴; wherein R¹¹, where permitted, is substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; wherein R¹² is selected from hydrogen and optionally substituted organic residue having from 1 to 20 carbons; wherein Z is selected from —O— and —NR¹³—; wherein R¹³ is selected from hydrogen and C1-C4 alkyl; or, wherein Z is N, R¹² and R¹³ are covalently bonded and —NR¹²R¹³ comprises a moiety of the formula:

wherein Y is selected from —O—, —S—, —SO—, —SO₂—, —NH—, —NCH₃—; and wherein R¹⁴ is C1-C6 alkyl and substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof, in an amount effective to prevent or treat muscle atrophy in the animal, wherein the amount is greater than 1000 per day when the compound is ursolic acid, boswellic acid, corosolic acid, betulinic acid, or UA0713.

Also disclosed are methods for increasing muscle mass and/or muscular strength in an animal, the method comprising administering to the animal a compound of the formula:

wherein each is an optional covalent bond, and R⁰ is optionally present; wherein n is 0 or 1; wherein R⁰, when present, is hydrogen; wherein R^1a is selected from C1-C6 alkyl and —C(O)ZR¹⁰; wherein R^1b is selected from C1-C6 alkyl; or wherein R^1a and R^1b are covalently bonded and, along with the intermediate carbon, together comprise optionally substituted C3-C5 cycloalkyl or optionally substituted C2-C5 heterocycloalkyl; wherein R^2a and R^2b are independently selected from hydrogen and —OR¹¹, provided that at least one of R^3a and R^3b is —OR¹¹; or wherein R^2a and R^2b together comprise ═O; wherein each of R^3a and R^3b is independently selected from hydrogen, hydroxyl, C1-C6 alkyl, and C1-C6 alkoxyl, provided that R^3a and R^3b are not simultaneously hydroxyl; or wherein R^3a and R^3b are covalently bonded and, along with the intermediate carbon, together comprise optionally substituted C3-C5 cycloalkyl or optionally substituted C2-C5 heterocycloalkyl; wherein each of R⁴, R⁵, and R⁶ is independently selected from C1-C6 alkyl; wherein R⁷ is selected from C1-C6 alkyl, —CH₂OR¹², and —C(O)ZR¹²; wherein R⁸ is selected from hydrogen and C1-C6 alkyl; wherein each of R^9a and R^9b is independently selected from hydrogen and C1-C6 alkyl, provided that R^9a and R^9b are not simultaneously hydrogen; or wherein R^9a and R^9b are covalently bonded and, along with the intermediate carbon, together comprise optionally substituted C3-C5 cycloalkyl or optionally substituted C2-C5 heterocycloalkyl; wherein R¹⁰ is selected from hydrogen and C1-C6 alkyl; wherein each R¹¹ is independently selected from hydrogen, C1-C6 alkyl, C1-C5 heteroalkyl, C3-C6 cycloalkyl, C4-C6 heterocycloalkyl, phenyl, heteroaryl, and —C(O)R¹⁴; wherein R¹¹, where permitted, is substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; wherein R¹² is selected from hydrogen and optionally substituted organic residue having from 1 to 20 carbons; wherein Z is selected from —O— and —NR¹³—; wherein R¹³ is selected from hydrogen and C1-C4 alkyl; or, wherein Z is N, R¹² and R¹³ are covalently bonded and —NR¹²R¹³ comprises a moiety of the formula:

wherein Y is selected from —O—, —S—, —SO—, —SO₂—, —NH—, —NCH₃—; and wherein R¹⁴ is C₁-C6 alkyl and substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof, in an amount effective to increase muscle mass and/or muscular strength in the animal, wherein the amount is greater than about 1000 mg per day when the compound is ursolic acid, boswellic acid, corosolic acid, betulinic acid, or UA0713.

Also disclosed are methods for enhancing tissue growth in vitro, the method comprising administering to the tissue a compound of the formula:

wherein each is an optional covalent bond, and R⁰ is optionally present; wherein n is 0 or 1; wherein R⁰, when present, is hydrogen; wherein R^1a is selected from C1-C6 alkyl and —C(O)ZR¹⁰; wherein R^1b is selected from C1-C6 alkyl; or wherein R^1a and R^1b are covalently bonded and, along with the intermediate carbon, together comprise optionally substituted C3-C5 cycloalkyl or optionally substituted C2-C5 heterocycloalkyl; wherein R^2a and R^2b are independently selected from hydrogen and —OR¹¹, provided that at least one of R^2a and R^2b is —OR¹¹; or wherein R^2a and R^2b together comprise ═O; wherein each of R^3a and R^3b is independently selected from hydrogen, hydroxyl, C1-C6 alkyl, and C1-C6 alkoxyl, provided that R^3a and R^3b are not simultaneously hydroxyl; or wherein R^3a and R^3b are covalently bonded and, along with the intermediate carbon, together comprise optionally substituted C3-C5 cycloalkyl or optionally substituted C2-C5 heterocycloalkyl; wherein each of R⁴, R⁵, and R⁶ is independently selected from C1-C6 alkyl; wherein R⁷ is selected from C1-C6 alkyl, —CH₂OR¹², and —C(O)ZR¹²; wherein R⁸ is selected from hydrogen and C1-C6 alkyl; wherein each of R^9a and R^9b is independently selected from hydrogen and C1-C6 alkyl, provided that R^9a and R^9b are not simultaneously hydrogen; or wherein R^9a and R^9b are covalently bonded and, along with the intermediate carbon, together comprise optionally substituted C3-C5 cycloalkyl or optionally substituted C2-C5 heterocycloalkyl; wherein R¹⁰ is selected from hydrogen and C1-C6 alkyl; wherein each R¹¹ is independently selected from hydrogen, C1-C6 alkyl, C1-C5 heteroalkyl, C3-C6 cycloalkyl, C4-C6 heterocycloalkyl, phenyl, heteroaryl, and —C(O)R¹⁴; wherein R¹¹, where permitted, is substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; wherein R¹² is selected from hydrogen and optionally substituted organic residue having from 1 to 20 carbons; wherein Z is selected from —O— and —NR¹³—; wherein R¹³ is selected from hydrogen and C1-C4 alkyl; or, wherein Z is N, R¹² and R¹³ are covalently bonded and —NR¹²R¹³ comprises a moiety of the formula:

wherein Y is selected from —O—, —S—, —SO—, —SO₂—, —NH—, —NCH₃—; and wherein R¹⁴ is C1-C6 alkyl and substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof, in an amount effective to enhance growth of the tissue.

Also disclosed are methods for enhancing muscle formation in a mammal, the method comprising administering to the mammal a compound of the formula:

wherein R¹¹ is selected from hydrogen, C1-C6 alkyl, C1-C5 heteroalkyl, C3-C6 cycloalkyl, C4-C6 heterocycloalkyl, phenyl, heteroaryl, and —C(O)R¹⁴; wherein R¹¹, where permitted, is substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; wherein R¹² is selected from hydrogen and optionally substituted organic residue having from 1 to 20 carbons; wherein Z is selected from —O— and —NR¹³—; wherein R¹³ is selected from hydrogen and C1-C4 alkyl; or, wherein Z is N, R¹² and R¹³ are covalently bonded and —NR¹²R¹³ comprises a moiety of the formula:

wherein Y is selected from —O—, —S—, —SO—, —SO₂—, —NH—, —NCH₃—; and wherein R¹⁴ is C1-C6 alkyl and substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof, in an amount greater than about 1000 mg per day and effective to enhance muscle formation in the mammal.

Also disclosed are methods for testing for performance enhancing use of a ursolic acid analog in an animal, the method comprising: (a) obtaining a biological test sample from the animal; and (b) measuring the amount of a compound of formula:

wherein each is an optional covalent bond, and R⁰ is optionally present; wherein n is 0 or 1; wherein R⁰, when present, is hydrogen; wherein R^1a is selected from C1-C6 alkyl and —C(O)ZR¹⁰; wherein R^1b is selected from C1-C6 alkyl; wherein one of R^2a and R^2b is —OR¹¹, and the other is hydrogen; wherein each of R^3a and R^3b is independently selected from hydrogen, hydroxyl, C1-C6 alkyl, and C1-C6 alkoxyl, provided that R^3a and R^3b are not simultaneously hydroxyl; wherein each of R⁴, R⁵, and R⁶ is independently selected from C1-C6 alkyl; wherein R⁷ is selected from C1-C6 alkyl and —C(O)ZR¹²; wherein R⁸ is selected from hydrogen and C1-C6 alkyl; wherein each of R^9a and R^9b is independently selected from hydrogen and C1-C6 alkyl, provided that R^9a and R^9b are not simultaneously hydrogen; wherein R¹⁰ is selected from hydrogen and C1-C6 alkyl; wherein R¹¹ is selected from hydrogen, C1-C6 alkyl, C1-C5 heteroalkyl, C3-C6 cycloalkyl, C4-C6 heterocycloalkyl, phenyl, heteroaryl, and —C(O)R¹⁴; wherein R¹¹, where permitted, is substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; wherein R¹² is selected from hydrogen and optionally substituted organic residue having from 1 to 20 carbons; wherein Z is selected from —O— and —NR¹³—; wherein R¹³ is selected from hydrogen and C1-C4 alkyl; and wherein R¹⁴ is C1-C6 alkyl and substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof, in the test sample to determine whether a superphysiological amount of the compound is present in the test sample; wherein the superphysiological amount of the compound in the test sample is indicative of performance enhancing use of the compound.

Also disclosed are pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an effective amount of a compound of the formula:

wherein each is an optional covalent bond, and R⁰ is optionally present; wherein n is 0 or 1; wherein R⁰, when present, is hydrogen; wherein R^1a is selected from C1-C6 alkyl and —C(O)ZR¹⁰; wherein R^1b is selected from C1-C6 alkyl; or wherein R^1a and R^1b are covalently bonded and, along with the intermediate carbon, together comprise optionally substituted C3-C5 cycloalkyl or optionally substituted C2-C5 heterocycloalkyl; wherein R^2a and R^2b are independently selected from hydrogen and —OR¹¹, provided that at least one of R^2a and R^2b is —OR¹¹; or wherein R^2a and R^2b together comprise ═O; wherein each of r^3a and R^3b is independently selected from hydrogen, hydroxyl, C1-C6 alkyl, and C1-C6 alkoxyl, provided that R^3a and R^3b are not simultaneously hydroxyl; or wherein R^3a and R^3b are covalently bonded and, along with the intermediate carbon, together comprise optionally substituted C3-C5 cycloalkyl or optionally substituted C2-C5 heterocycloalkyl; wherein each of R⁴, R⁵, and R⁶ is independently selected from C1-C6 alkyl; wherein R⁷ is selected from C1-C6 alkyl, —CH₂OR¹², and —C(O)ZR¹²; wherein R⁸ is selected from hydrogen and C1-C6 alkyl; wherein each of R^9a and R^9b is independently selected from hydrogen and C1-C6 alkyl, provided that R^9a and R^9b are not simultaneously hydrogen; or wherein R^9a and R^9b are covalently bonded and, along with the intermediate carbon, together comprise optionally substituted C3-C5 cycloalkyl or optionally substituted C2-C5 heterocycloalkyl; wherein R¹⁰ is selected from hydrogen and C1-C6 alkyl; wherein each R¹¹ is independently selected from hydrogen, C1-C6 alkyl, C1-C5 heteroalkyl, C3-C6 cycloalkyl, C4-C6 heterocycloalkyl, phenyl, heteroaryl, and —C(O)R¹⁴; wherein R¹¹, where permitted, is substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; wherein R¹² is selected from hydrogen and optionally substituted organic residue having from 1 to 20 carbons; wherein Z is selected from —O— and —NR¹³—; wherein R¹³ is selected from hydrogen and C1-C4 alkyl; or, wherein Z is N, R¹² and R¹³ are covalently bonded and —NR¹²R¹³ comprises a moiety of the formula:

wherein Y is selected from —O—, —S—, —SO—, —SO₂—, —NH—, —NCH₃—; and wherein R¹⁴ is C1-C6 alkyl and substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof, in an amount effective to prevent or treat muscle atrophy in the animal, wherein the amount is greater than about 1000 mg per day when the compound is ursolic acid, boswellic acid, corosolic acid, betulinic acid, or UA0713.

Also disclosed are kits comprising at least one compound having a structure represented by a formula:

wherein each is an optional covalent bond, and R⁰ is optionally present; wherein n is 0 or 1; wherein R⁰, when present, is hydrogen; wherein R^1a is selected from C1-C6 alkyl and —C(O)ZR¹⁰; wherein R^1b is selected from C1-C6 alkyl; or wherein R^1a and R^1b are covalently bonded and, along with the intermediate carbon, together comprise optionally substituted C3-C5 cycloalkyl or optionally substituted C2-C5 heterocycloalkyl; wherein R^2a and R^2b are independently selected from hydrogen and —OR¹¹, provided that at least one of R^2a and R^2b is —OR¹¹; or wherein R^2a and R^2b together comprise ═O; wherein each of R^2a and R^3b is independently selected from hydrogen, hydroxyl, C1-C6 alkyl, and C1-C6 alkoxyl, provided that R^3a and R^3b are not simultaneously hydroxyl; or wherein R^3a and R^3b are covalently bonded and, along with the intermediate carbon, together comprise optionally substituted C3-C5 cycloalkyl or optionally substituted C2-C5 heterocycloalkyl; wherein each of R⁴, R⁵, and R⁶ is independently selected from C1-C6 alkyl; wherein R⁷ is selected from C1-C6 alkyl, —CH₂OR¹², and —C(O)ZR¹²; wherein R⁸ is selected from hydrogen and C1-C6 alkyl; wherein each of R^9a and R^9b is independently selected from hydrogen and C1-C6 alkyl, provided that R^9a and R^9b are not simultaneously hydrogen; or wherein R^9a and R^9b are covalently bonded and, along with the intermediate carbon, together comprise optionally substituted C3-C5 cycloalkyl or optionally substituted C2-C5 heterocycloalkyl; wherein R¹⁰ is selected from hydrogen and C1-C6 alkyl; wherein each R¹¹ is independently selected from hydrogen, C1-C6 alkyl, C1-C5 heteroalkyl, C3-C6 cycloalkyl, C4-C6 heterocycloalkyl, phenyl, heteroaryl, and —C(O)R¹⁴; wherein R¹¹, where permitted, is substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; wherein R¹² is selected from hydrogen and optionally substituted organic residue having from 1 to 20 carbons; wherein Z is selected from —O— and —NR¹³—; wherein R¹³ is selected from hydrogen and C1-C4 alkyl; or, wherein Z is N, R¹² and R¹³ are covalently bonded and —NR¹²R¹³ comprises a moiety of the formula:

wherein Y is selected from —O—, —S—, —SO—, —SO₂—, —NH—, —NCH₃—; and wherein R¹⁴ is C1-C6 alkyl and substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof, and one or more of: (a) a protein supplement; (b) an anabolic agent; (c) a catabolic agent; (d) a dietary supplement; (e) at least one agent known to treat a disorder associated with muscle wasting; (f) instructions for treating a disorder associated with cholinergic activity; or (g) instructions for using the compound to increase muscle mass and/or muscular strength.

Also disclosed are methods for manufacturing a medicament associated with muscle atrophy or the need to increase muscle mass comprising combining at least one disclosed compound or at least one disclosed product with a pharmaceutically acceptable carrier or diluent.

Also disclosed are uses of a disclosed compound or a disclosed product in the manufacture of a medicament for the treatment of a disorder associated with muscle atrophy or the need to increase muscle mass.

While aspects of the present invention can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present invention can be described and claimed in any statutory class.

Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects and together with the description serve to explain the principles of the invention.

FIG. 1 shows a schematic overview of the discovery process leading to a pharmacological compound that promotes skeletal muscle growth and inhibits skeletal muscle atrophy.

FIG. 2 shows representative data on the effect of fasting on skeletal muscle mRNA expression in healthy human adults.

FIG. 3 shows qPCR analysis of representative fasting-responsive mRNAs from human skeletal muscle.

FIG. 4 shows representative data on the identification of ursolic acid as an inhibitor of fasting-induced skeletal muscle atrophy.

FIG. 5 shows representative data on the identification of ursolic acid as an inhibitor of denervation-induced muscle atrophy.

FIG. 6 shows representative data on ursolic acid-mediated induction of muscle hypertrophy.

FIG. 7 shows representative data on the effect of ursolic acid on mouse skeletal muscle specific tetanic force.

FIG. 8 shows representative data on the effect of ursolic acid on muscle growth, atrophic gene expression, trophic gene expression, and skeletal muscle IGF-I signaling.

FIG. 9 shows representative data on the effect of ursolic acid on skeletal muscle expression of IGF1 gene exons, adipose IGF1 mRNA expression, and skeletal muscle insulin signaling.

FIG. 10 shows representative data on the effect of ursolic acid on adiposity.

FIG. 11 shows representative data on the effect of ursolic acid on food consumption, liver weight, kidney weight, and plasma ALT, bilirubin, and creatinine concentrations.

FIG. 12 shows representative data on the effect of ursolic acid on weight gain, white adipose tissue weight, skeletal muscle weight, brown adipose tissue weight and energy expenditure in a mouse model of obesity and metabolic syndrome.

FIG. 13 shows representative data on the effect of ursolic acid on obesity-related pre-diabetes, diabetes, fatty liver disease and hyperlipidemia in a mouse model of obesity and metabolic syndrome.

FIG. 14 shows representative data on the effect of oleanolic acid on skeletal muscle mass.

FIG. 15 shows representative data on the effect of targeted inhibition of PTP1B on skeletal muscle growth.

FIG. 16 shows representative data on the effect of ursolic acid serum concentration on muscle mass and adiposity.

Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

DESCRIPTION

The present invention can be understood more readily by reference to the following detailed description of the invention and the Examples included therein.

Before the present compounds, compositions, articles, systems, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

A. DEFINITIONS

As used herein, nomenclature for compounds, including organic compounds, can be given using common names, IUPAC, IUBMB, or CAS recommendations for nomenclature. When one or more stereochemical features are present, Cahn-Ingold-Prelog rules for stereochemistry can be employed to designate stereochemical priority, E/Z specification, and the like. One of skill in the art can readily ascertain the structure of a compound if given a name, either by systemic reduction of the compound structure using naming conventions, or by commercially available software, such as CHEMDRAW™ (Cambridgesoft Corporation, U.S.A.).

As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a functional group,” “an alkyl,” or “a residue” includes mixtures of two or more such functional groups, alkyls, or residues, and the like.

Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

A weight percent (wt. %) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.

As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

As used herein, “ursolic acid” refers to ursolic acid, or extracts containing ursolic acid from plants such as apples, holy basil, bilberries, cranberries, elder flower, peppermint, lavender, oregano, thyme, sage, hawthorn, bearberry or prunes.

As used herein, “ursolic acid derivatives” refers to corosolic acid, betulinic acid, hederagenin, boswellic acids, UA0713, a substituted ursolic acid analog, an ursane compound or any other pentacyclic triterpene acids that prevents muscle atrophy, reduces muscle atrophy, increases muscle mass, increases muscle strength in an animal, including in humans, increases Akt phosphorylation, increases S6K phosphorylation, or stimulates biochemical events known to precede or follow Akt phosphorylation or S6K phosphorylation. For example, and not to be limiting, biochemical events known to precede or follow Akt phosphorylation or S6K phosphorylation can be events such as insulin receptor phosphorylation, IGF-I receptor phosphorylation, insulin receptor substrate (IRS) protein phosphorylation, phosphoinositide-3 kinase phosphorylation, phosphoinositide-3 kinase activation, phosphoinositide dependent kinase 1 activation, mammalian target of rapamycin complex 2 activation, adrenergic receptor activation, heterotrimeric G protein activation, adenylate cyclase activation, increased intracellular cyclic AMP, AMP kinase activation, protein kinase A activation, protein kinase C activation, CREB activation, mitogen activated protein kinase pathway activation, mammalian target of rapamycin complex 1 activation, 4E-BP1 phosphorylation, 4E-BP1 inactivation, GSK3β phosphorylation, GSK3β inactivation, increased protein synthesis, increased glucose uptake, Foxo transcription factor phosphorylation, Foxo transcription factor inactivation, Cdkn1a phosphorylation, Cdkn1a inactivation, reduced atrogin-1 mRNA, reduced MuRF1 mRNA, increased VEGFA mRNA, or increased IGF1 mRNA.

As used herein, the term “subject” refers to the target of administration, e.g. an animal. Thus the subject of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Alternatively, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. In one aspect, the subject is a mammal. A patient refers to a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects. In some aspects of the disclosed methods, the subject has been diagnosed with a need for treatment of one or more muscle disorders prior to the administering step. In some aspects of the disclosed method, the subject has been diagnosed with a need for increasing muscle mass prior to the administering step. In some aspects of the disclosed method, the subject has been diagnosed with a need for increasing muscle mass prior to the administering step.

As used herein, the term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In various aspects, the term covers any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the disease from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the disease, i.e., arresting its development; or (iii) relieving the disease, i.e., causing regression of the disease. In one aspect, the subject is a mammal such as a primate, and, in a further aspect, the subject is a human. The term “subject” also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.).

As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.

As used herein, the term “diagnosed” means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein. For example, “diagnosed with a muscle atrophy disorder” means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by a compound or composition that can increase muscle mass. As a further example, “diagnosed with a need for increasing muscle mass” refers to having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition characterized by muscle atrophy or other disease wherein increasing muscle mass would be beneficial to the subject. Such a diagnosis can be in reference to a disorder, such as muscle atrophy, and the like, as discussed herein.

As used herein, the phrase “identified to be in need of treatment for a disorder,” or the like, refers to selection of a subject based upon need for treatment of the disorder. For example, a subject can be identified as having a need for treatment of a disorder (e.g., a disorder related to muscle atrophy) based upon an earlier diagnosis by a person of skill and thereafter subjected to treatment for the disorder. It is contemplated that the identification can, in one aspect, be performed by a person different from the person making the diagnosis. It is also contemplated, in a further aspect, that the administration can be performed by one who subsequently performed the administration.

As used herein, the terms “administering” and “administration” refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.

The term “contacting” as used herein refers to bringing a disclosed compound and a cell, target receptor, or other biological entity together in such a manner that the compound can affect the activity of the target (e.g., receptor, transcription factor, cell, etc.), either directly; i.e., by interacting with the target itself, or indirectly; i.e., by interacting with another molecule, co-factor, factor, or protein on which the activity of the target is dependent.

As used herein, the terms “effective amount” and “amount effective” refer to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition. For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side affects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a disease or condition.

As used herein, “EC₅₀,” is intended to refer to the concentration or dose of a substance (e.g., a compound or a drug) that is required for 50% enhancement or activation of a biological process, or component of a process, including a protein, subunit, organelle, ribonucleoprotein, etc. EC₅₀ also refers to the concentration or dose of a substance that is required for 50% enhancement or activation in vivo, as further defined elsewhere herein. Alternatively, EC₅₀ can refer to the concentration or dose of compound that provokes a response halfway between the baseline and maximum response. The response can be measusred in a in vitro or in vivo system as is convenient and appropriate for the biological response of interest. For example, the response can be measured in vitro using cultured muscle cells or in an ex vivo organ culture system with isolated muscle fibers. Alternatively, the response can be measured in vivo using an appropriate research model such as rodent, including mice and rats. The mouse or rat can be an inbred strain with phenotypic characteristics of interest such as obesity or diabetes. As appropriate, the response can be measured in a transgenic or knockout mouse or rat wherein the a gene or genes has been introduced or knocked-out, as appropriate, to replicate a disease process.

As used herein, “IC₅₀,” is intended to refer to the concentration or dose of a substance (e.g., a compound or a drug) that is required for 50% inhibition or diminuation of a biological process, or component of a process, including a protein, subunit, organelle, ribonucleoprotein, etc. IC₅₀ also refers to the concentration or dose of a substance that is required for 50% inhibition or diminuation in vivo, as further defined elsewhere herein. Alternatively, IC₅₀ also refers refers to the half maximal (50%) inhibitory concentration (IC) or inhibitory dose of a substance. The response can be measusred in a in vitro or in vivo system as is convenient and appropriate for the biological response of interest. For example, the response can be measured in vitro using cultured muscle cells or in an ex vivo organ culture system with isolated muscle fibers. Alternatively, the response can be measured in vivo using an appropriate research model such as rodent, including mice and rats. The mouse or rat can be an inbred strain with phenotypic characteristics of interest such as obesity or diabetes. As appropriate, the response can be measured in a transgenic or knockout mouse or rat wherein the a gene or genes has been introduced or knocked-out, as appropriate, to replicate a disease process.

The term “pharmaceutically acceptable” describes a material that is not biologically or otherwise undesirable, i.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner.

As used herein, the term “derivative” refers to a compound having a structure derived from the structure of a parent compound (e.g., a compound disclosed herein) and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compounds. Exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of a parent compound.

As used herein, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

A residue of a chemical species, as used in the specification and concluding claims, refers to the moiety that is the resulting product of the chemical species in a particular reaction scheme or subsequent formulation or chemical product, regardless of whether the moiety is actually obtained from the chemical species. Thus, an ethylene glycol residue in a polyester refers to one or more —OCH₂CH₂O— units in the polyester, regardless of whether ethylene glycol was used to prepare the polyester. Similarly, a sebacic acid residue in a polyester refers to one or more —CO(CH₂)₈CO— moieties in the polyester, regardless of whether the residue is obtained by reacting sebacic acid or an ester thereof to obtain the polyester.

As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms “substitution” or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. It is also contemplated that, in certain aspects, unless expressly indicated to the contrary, individual substituents can be further optionally substituted (i.e., further substituted or unsubstituted).

In defining various terms, “A¹,” “A²,” “A³,” and “A⁴” are used herein as generic symbols to represent various specific substituents. These symbols can be any substituent, not limited to those disclosed herein, and when they are defined to be certain substituents in one instance, they can, in another instance, be defined as some other substituents.

The term “alkyl” as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dode cyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. The alkyl group can be cyclic or acyclic. The alkyl group can be branched or unbranched. The alkyl group can also be substituted or unsubstituted. For example, the alkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein. A “lower alkyl” group is an alkyl group containing from one to six (e.g., from one to four) carbon atoms.

Throughout the specification “alkyl” is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituent(s) on the alkyl group. For example, the term “halogenated alkyl” or “haloalkyl” specifically refers to an alkyl group that is substituted with one or more halide, e.g., fluorine, chlorine, bromine, or iodine. The term “alkoxyalkyl” specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below. The term “alkylamino” specifically refers to an alkyl group that is substituted with one or more amino groups, as described below, and the like. When “alkyl” is used in one instance and a specific term such as “alkylalcohol” is used in another, it is not meant to imply that the term “alkyl” does not also refer to specific terms such as “alkylalcohol” and the like.

This practice is also used for other groups described herein. That is, while a term such as “cycloalkyl” refers to both unsubstituted and substituted cycloalkyl moieties, the substituted moieties can, in addition, be specifically identified herein; for example, a particular substituted cycloalkyl can be referred to as, e.g., an “alkylcycloalkyl.” Similarly, a substituted alkoxy can be specifically referred to as, e.g., a “halogenated alkoxy,” a particular substituted alkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, the practice of using a general term, such as “cycloalkyl,” and a specific term, such as “alkylcycloalkyl,” is not meant to imply that the general term does not also include the specific term.

The term “cycloalkyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and the like. The term “heterocycloalkyl” is a type of cycloalkyl group as defined above, and is included within the meaning of the term “cycloalkyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted. The cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol as described herein.

The term “polyalkylene group” as used herein is a group having two or more CH₂ groups linked to one another. The polyalkylene group can be represented by the formula —(CH₂)_a—, where “a” is an integer of from 2 to 500.

The terms “alkoxy” and “alkoxyl” as used herein to refer to an alkyl or cycloalkyl group bonded through an ether linkage; that is, an “alkoxy” group can be defined as —OA¹ where A¹ is alkyl or cycloalkyl as defined above. “Alkoxy” also includes polymers of alkoxy groups as just described; that is, an alkoxy can be a polyether such as —OA¹-OA² or —OA¹-(OA²)_a-OA³, where “a” is an integer of from 1 to 200 and A¹, A², and A³ are alkyl and/or cycloalkyl groups.

The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon double bond.

Asymmetric structures such as (A¹A²)C═C(A³A⁴) are intended to include both the E and Z isomers. This can be presumed in structural formulae herein wherein an asymmetric alkene is present, or it can be explicitly indicated by the bond symbol C═C. The alkenyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.

The term “cycloalkenyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one carbon-carbon double bound, i.e., C═C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, norbornenyl, and the like. The term “heterocycloalkenyl” is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkenyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group and heterocycloalkenyl group can be substituted or unsubstituted. The cycloalkenyl group and heterocycloalkenyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

The term “alkynyl” as used herein is a hydrocarbon group of 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon triple bond. The alkynyl group can be unsubstituted or substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.

The term “cycloalkynyl” as used herein is a non-aromatic carbon-based ring composed of at least seven carbon atoms and containing at least one carbon-carbon triple bound. Examples of cycloalkynyl groups include, but are not limited to, cycloheptynyl, cyclooctynyl, cyclononynyl, and the like. The term “heterocycloalkynyl” is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkynyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkynyl group and heterocycloalkynyl group can be substituted or unsubstituted. The cycloalkynyl group and heterocycloalkynyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

The term “aryl” as used herein is a group that contains any carbon-based aromatic group including, but not limited to, benzene, naphthalene, phenyl, biphenyl, phenoxybenzene, and the like. The term “aryl” also includes “heteroaryl,” which is defined as a group that contains an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. Likewise, the term “non-heteroaryl,” which is also included in the term “aryl,” defines a group that contains an aromatic group that does not contain a heteroatom. The aryl group can be substituted or unsubstituted. The aryl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein. The term “biaryl” is a specific type of aryl group and is included in the definition of “aryl.” Biaryl refers to two aryl groups that are bound together via a fused ring structure, as in naphthalene, or are attached via one or more carbon-carbon bonds, as in biphenyl.

The term “aldehyde” as used herein is represented by the formula —C(O)H. Throughout this specification “C(O)” is a short hand notation for a carbonyl group, i.e., C═O.

The terms “amine” or “amino” as used herein are represented by the formula —NA¹A², where A¹ and A² can be, independently, hydrogen or alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

The term “alkylamino” as used herein is represented by the formula —NH(-alkyl) where alkyl is a described herein. Representative examples include, but are not limited to, methylamino group, ethylamino group, propylamino group, isopropylamino group, butylamino group, isobutylamino group, (sec-butyl)amino group, (tert-butyl)amino group, pentylamino group, isopentylamino group, (tert-pentyl)amino group, hexylamino group, and the like.

The term “dialkylamino” as used herein is represented by the formula —N(-alkyl)₂ where alkyl is a described herein. Representative examples include, but are not limited to, dimethylamino group, diethylamino group, dipropylamino group, diisopropylamino group, dibutylamino group, diisobutylamino group, di(sec-butyl)amino group, di(tert-butyl)amino group, dipentylamino group, diisopentylamino group, di(tert-pentyl)amino group, dihexylamino group, N-ethyl-N-methylamino group, N-methyl-N-propylamino group, N-ethyl-N-propylamino group and the like.

The term “carboxylic acid” as used herein is represented by the formula —C(O)OH.

The term “ester” as used herein is represented by the formula —OC(O)A¹ or —C(O)OA¹, where A¹ can be alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “polyester” as used herein is represented by the formula -(A¹O(O)C-A²-C(O)O)_a— or -(A¹O(O)C-A²-OC(O))_a—, where A¹ and A² can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and “a” is an interger from 1 to 500. “Polyester” is as the term used to describe a group that is produced by the reaction between a compound having at least two carboxylic acid groups with a compound having at least two hydroxyl groups.

The term “ether” as used herein is represented by the formula A¹OA², where A¹ and A² can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein. The term “polyether” as used herein is represented by the formula —(A¹O-A²O)_a—, where A¹ and A² can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and “a” is an integer of from 1 to 500. Examples of polyether groups include polyethylene oxide, polypropylene oxide, and polybutylene oxide.

The term “halide” as used herein refers to the halogens fluorine, chlorine, bromine, and iodine.

The term “heterocycle,” as used herein refers to single and multi-cyclic aromatic or non-aromatic ring systems in which at least one of the ring members is other than carbon. Heterocycle includes azetidine, dioxane, furan, imidazole, isothiazole, isoxazole, morpholine, oxazole, oxazole, including, 1,2,3-oxadiazole, 1,2,5-oxadiazole and 1,3,4-oxadiazole, piperazine, piperidine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolidine, tetrahydrofuran, tetrahydropyran, tetrazine, including 1,2,4,5-tetrazine, tetrazole, including 1,2,3,4-tetrazole and 1,2,4,5-tetrazole, thiadiazole, including, 1,2,3-thiadiazole, 1,2,5-thiadiazole, and 1,3,4-thiadiazole, thiazole, thiophene, triazine, including 1,3,5-triazine and 1,2,4-triazine, triazole, including, 1,2,3-triazole, 1,3,4-triazole, and the like.

The term “hydroxyl” as used herein is represented by the formula —OH.

The term “ketone” as used herein is represented by the formula A¹C(O)A², where A¹ and A² can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

The term “azide” as used herein is represented by the formula —N₃.

The term “nitro” as used herein is represented by the formula —NO₂.

The term “nitrile” as used herein is represented by the formula —CN.

The term “silyl” as used herein is represented by the formula —SiA¹A²A³, where A¹, A², and A³ can be, independently, hydrogen or an alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

The term “sulfo-oxo” as used herein is represented by the formulas —S(O)A¹, —S(O)₂A¹, —OS(O)₂A¹, or —OS(O)₂OA¹, where A¹ can be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. Throughout this specification “S(O)” is a short hand notation for S═O. The term “sulfonyl” is used herein to refer to the sulfo-oxo group represented by the formula —S(O)₂A¹, where A¹ can be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “sulfone” as used herein is represented by the formula A¹S(O)₂A², where A¹ and A² can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “sulfoxide” as used herein is represented by the formula A¹S(O)A², where A¹ and A² can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

The term “thiol” as used herein is represented by the formula —SH.

“R¹,” “R²,” “R³,” “Rⁿ,” where n is an integer, as used herein can, independently, possess one or more of the groups listed above. For example, if R¹ is a straight chain alkyl group, one of the hydrogen atoms of the alkyl group can optionally be substituted with a hydroxyl group, an alkoxy group, an alkyl group, a halide, and the like. Depending upon the groups that are selected, a first group can be incorporated within second group or, alternatively, the first group can be pendant (i.e., attached) to the second group. For example, with the phrase “an alkyl group comprising an amino group,” the amino group can be incorporated within the backbone of the alkyl group. Alternatively, the amino group can be attached to the backbone of the alkyl group. The nature of the group(s) that is (are) selected will determine if the first group is embedded or attached to the second group.

As described herein, compounds of the invention may contain “optionally substituted” moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. In is also contemplated that, in certain aspects, unless expressly indicated to the contrary, individual substituents can be further optionally substituted (i.e., further substituted or unsubstituted).

The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain aspects, their recovery, purification, and use for one or more of the purposes disclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; —(CH₂)_0-4R^∘; —(CH₂)_0-4OR^∘; —O(CH₂)_0-4R^∘, —O—(CH₂)_0-4C(O)OR^∘; —(CH₂)_0-4CH(OR^∘)₂; —(CH₂)_0-4SR^∘; —(CH₂)_0-4Ph, which may be substituted with R^∘; —(CH₂)_0-4O(CH₂)_0-1Ph which may be substituted with R^∘; —CH═CHPh, which may be substituted with R^∘; —(CH₂)_0-4O(CH₂)_0-1-pyridyl which may be substituted with R^∘; —NO₂; —CN; —N₃; —(CH₂)_0-4N(R^∘)₂; —(CH₂)_0-4N(R^∘)C(O)R^∘; —N(R^∘)C(S)R^∘; —(CH₂)_{0- 4}N(R^∘)C(O)NR^∘₂; —N(R^∘)C(S)NR^∘₂; —(CH₂)_0-4N(R^∘)C(O)OR^∘; —N(R^∘)N(R^∘)C(O)R^∘; —N(R^∘)N(R^∘)C(O)NR^∘₂; —N(R^∘)N(R^∘)C(O)OR^∘; —(CH₂)_0-4C(O)R^∘; —C(S)R^∘; —(CH₂)_0-4C(O)OR^∘; —(CH₂)_0-4C(O)SR^∘; —(CH₂)_0-4C(O)OSiR^∘₃; —(CH₂)_0-4OC(O)R^∘; —OC(O)(CH₂)_0-4SR—, SC(S)SR^∘; —(CH₂)_0-4SC(O)R^∘; —(CH₂)_0-4C(O)NR^∘₂; —C(S)NR^∘₂; —C(S)SR^∘; —SC(S)SR^∘, —(CH₂)_0-4OC(O)NR^∘₂; —C(O)N(OR^∘)R^∘; —C(O)C(O)R^∘; —C(O)CH₂C(O)R^∘; —C(NOR^∘)R^∘; —(CH₂)_0-4SSR^∘; —(CH₂)_0-4S(O)₂R^∘; —(CH₂)_0-4S(O)₂OR^∘; —(CH₂)_0-4OS(O)₂R^∘; —S(O)₂NR^∘₂; —(CH₂)_0-4S(O)R^∘; —N(R^∘S(O)₂NR^∘₂; —N(R^∘)S(O)₂R^∘; —N(OR^∘)R^∘; —C(NH)NR^∘₂; —P(O)₂R^∘; —P(O)R^∘₂; —OP(O)R^∘₂; —OP(O)(OR^∘)₂; SiR^∘₃; —(C_1-4 straight or branched)alkylene)O—N(R^∘)₂; or —(C_1-4 straight or branched alkylene)C(O)O—N(R^∘)₂, wherein each R^∘ may be substituted as defined below and is independently hydrogen, C_1-6 aliphatic, —CH₂Ph, —O(CH₂)_0-1Ph, —CH₂-(5-6 membered heteroaryl ring), or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R^∘, taken together with their intervening atom(s), form a 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R^∘ (or the ring formed by taking two independent occurrences of R^∘ together with their intervening atoms), are independently halogen, —(CH₂)_0-2R^●, -(haloR^●), —(CH₂)_0-2OH, —(CH₂)_0-2OR^●, —(CH₂)_0-2CH(OR^●)₂; —O(haloR^●), —CN, —N₃, —(CH₂)_0-2C(O)R^●, —(CH₂)_0-2C(O)OH, —(CH₂)_0-2C(O)OR^●, —(CH₂)_0-2SR^●, —(CH₂)_0-2SH, —(CH₂)_0-2NH₂, —(CH₂)_0-2NHR^●, —(CH₂)_0-2NR^●₂, —NO₂, —SiR^●₃, —OSiR^●₃, —C(O)SR^●, —(C_{1- 4} straight or branched alkylene)C(O)OR^●, or —SSR^● wherein each R^● is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from C_1-4 aliphatic, —CH₂Ph, —O(CH₂)_0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R^∘ include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: ═O, ═S, ═NNR*₂, ═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))_2-3O—, or —S(C(R*₂))_2-3S—, wherein each independent occurrence of R* is selected from hydrogen, C_1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: —O(CR*₂)_2-3O—, wherein each independent occurrence of R* is selected from hydrogen, C_1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R* include halogen, —R^●, -(haloR^●), —OH, —OR^●, —O(haloR^●), —CN, —C(O)OH, —C(O)OR^●, —NH₂, —NHR^●, —NR^●₂, or —NO₂, wherein each r^● is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C_1-4 aliphatic, —CH₂Ph, —O(CH₂)_0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include —R^†, —NR^†₂, —C(O)R^†, —C(O)OR^†, —C(O)C(O)R^†, —C(O)CH₂C(O)R^†, —S(O)₂R^†, —S(O)₂NR^†₂, —C(S)NR^†₂, —C(NH)NR^†₂, or —N(R^†)S(O)₂R^†; wherein each R^† is independently hydrogen, C_1-6 aliphatic which may be substituted as defined below, unsubstituted —OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R^†, taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^† are independently halogen, —R^●, -(haloR^●), —OH, —OR^●, —O(haloR^●), —CN, —C(O)OH, —C(O)OR^●, —NH₂, —NHR^●, —NR^●₂, or —NO₂, wherein each R^● is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C_1-4 aliphatic, —CH₂Ph, —O(CH₂)_0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

The term “leaving group” refers to an atom (or a group of atoms) with electron withdrawing ability that can be displaced as a stable species, taking with it the bonding electrons. Examples of suitable leaving groups include halides and sulfonate esters, including, but not limited to, triflate, mesylate, tosylate, brosylate, and halides.

The terms “hydrolysable group” and “hydrolysable moiety” refer to a functional group capable of undergoing hydrolysis, e.g., under basic or acidic conditions. Examples of hydrolysable residues include, without limitatation, acid halides, activated carboxylic acids, and various protecting groups known in the art (see, for example, “Protective Groups in Organic Synthesis,” T. W. Greene, P. G. M. Wuts, Wiley-Interscience, 1999).

The term “organic residue” defines a carbon containing residue, i.e., a residue comprising at least one carbon atom, and includes but is not limited to the carbon-containing groups, residues, or radicals defined hereinabove. Organic residues can contain various heteroatoms, or be bonded to another molecule through a heteroatom, including oxygen, nitrogen, sulfur, phosphorus, or the like. Examples of organic residues include but are not limited alkyl or substituted alkyls, alkoxy or substituted alkoxy, mono or di-substituted amino, amide groups, etc. Organic residues can preferably comprise 1 to 18 carbon atoms, 1 to 15, carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. In a further aspect, an organic residue can comprise 2 to 18 carbon atoms, 2 to 15, carbon atoms, 2 to 12 carbon atoms, 2 to 8 carbon atoms, 2 to 4 carbon atoms, or 2 to 4 carbon atoms.

A very close synonym of the term “residue” is the term “radical,” which as used in the specification and concluding claims, refers to a fragment, group, or substructure of a molecule described herein, regardless of how the molecule is prepared. For example, a 2,4-thiazolidinedione radical in a particular compound has the structure

regardless of whether thiazolidinedione is used to prepare the compound. In some embodiments the radical (for example an alkyl) can be further modified (i.e., substituted alkyl) by having bonded thereto one or more “substituent radicals.” The number of atoms in a given radical is not critical to the present invention unless it is indicated to the contrary elsewhere herein.

“Organic radicals,” as the term is defined and used herein, contain one or more carbon atoms. An organic radical can have, for example, 1-26 carbon atoms, 1-18 carbon atoms, 1-12 carbon atoms, 1-8 carbon atoms, 1-6 carbon atoms, or 1-4 carbon atoms. In a further aspect, an organic radical can have 2-26 carbon atoms, 2-18 carbon atoms, 2-12 carbon atoms, 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. Organic radicals often have hydrogen bound to at least some of the carbon atoms of the organic radical. One example, of an organic radical that comprises no inorganic atoms is a 5, 6, 7, 8-tetrahydro-2-naphthyl radical. In some embodiments, an organic radical can contain 1-10 inorganic heteroatoms bound thereto or therein, including halogens, oxygen, sulfur, nitrogen, phosphorus, and the like. Examples of organic radicals include but are not limited to an alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, mono-substituted amino, di-substituted amino, acyloxy, cyano, carboxy, carboalkoxy, alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide, substituted dialkylcarboxamide, alkylsulfonyl, alkylsulfinyl, thioalkyl, thiohaloalkyl, alkoxy, substituted alkoxy, haloalkyl, haloalkoxy, aryl, substituted aryl, heteroaryl, heterocyclic, or substituted heterocyclic radicals, wherein the terms are defined elsewhere herein. A few non-limiting examples of organic radicals that include heteroatoms include alkoxy radicals, trifluoromethoxy radicals, acetoxy radicals, dimethylamino radicals and the like.

“Inorganic radicals,” as the term is defined and used herein, contain no carbon atoms and therefore comprise only atoms other than carbon. Inorganic radicals comprise bonded combinations of atoms selected from hydrogen, nitrogen, oxygen, silicon, phosphorus, sulfur, selenium, and halogens such as fluorine, chlorine, bromine, and iodine, which can be present individually or bonded together in their chemically stable combinations. Inorganic radicals have 10 or fewer, or preferably one to six or one to four inorganic atoms as listed above bonded together. Examples of inorganic radicals include, but not limited to, amino, hydroxy, halogens, nitro, thiol, sulfate, phosphate, and like commonly known inorganic radicals. The inorganic radicals do not have bonded therein the metallic elements of the periodic table (such as the alkali metals, alkaline earth metals, transition metals, lanthanide metals, or actinide metals), although such metal ions can sometimes serve as a pharmaceutically acceptable cation for anionic inorganic radicals such as a sulfate, phosphate, or like anionic inorganic radical. Inorganic radicals do not comprise metalloids elements such as boron, aluminum, gallium, germanium, arsenic, tin, lead, or tellurium, or the noble gas elements, unless otherwise specifically indicated elsewhere herein.

Compounds described herein can contain one or more double bonds and, thus, potentially give rise to cis/trans (E/Z) isomers, as well as other conformational isomers. Unless stated to the contrary, the invention includes all such possible isomers, as well as mixtures of such isomers.

Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g., each enantiomer and diastereomer, and a mixture of isomers, such as a racemic or scalemic mixture. Compounds described herein can contain one or more asymmetric centers and, thus, potentially give rise to diastereomers and optical isomers. Unless stated to the contrary, the present invention includes all such possible diastereomers as well as their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof. Mixtures of stereoisomers, as well as isolated specific stereoisomers, are also included. During the course of the synthetic procedures used to prepare such compounds, or in using racemization or epimerization procedures known to those skilled in the art, the products of such procedures can be a mixture of stereoisomers.

Many organic compounds exist in optically active forms having the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (−) are employed to designate the sign of rotation of plane-polarized light by the compound, with (−) or meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these compounds, called stereoisomers, are identical except that they are non-superimposable mirror images of one another. A specific stereoisomer can also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture. Many of the compounds described herein can have one or more chiral centers and therefore can exist in different enantiomeric forms. If desired, a chiral carbon can be designated with an asterisk (*). When bonds to the chiral carbon are depicted as straight lines in the disclosed formulas, it is understood that both the (R) and (S) configurations of the chiral carbon, and hence both enantiomers and mixtures thereof, are embraced within the formula. As is used in the art, when it is desired to specify the absolute configuration about a chiral carbon, one of the bonds to the chiral carbon can be depicted as a wedge (bonds to atoms above the plane) and the other can be depicted as a series or wedge of short parallel lines is (bonds to atoms below the plane). The Cahn-Inglod-Prelog system can be used to assign the (R) or (S) configuration to a chiral carbon.

Compounds described herein comprise atoms in both their natural isotopic abundance and in non-natural abundance. The disclosed compounds can be isotopically-labelled or isotopically-substituted compounds identical to those described, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ¹⁸F and ³⁶Cl, respectively. Compounds further comprise prodrugs thereof, and pharmaceutically acceptable salts of said compounds or of said prodrugs which contain the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention. Certain isotopically-labelled compounds of the present invention, for example those into which radioactive isotopes such as ³H and ¹⁴C are incorporated, are useful in drug and/or substrate tissue distribution assays. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C, isotopes are particularly preferred for their ease of preparation and detectability.

Further, substitution with heavier isotopes such as deuterium, i.e., ²H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically labelled compounds of the present invention and prodrugs thereof can generally be prepared by carrying out the procedures below, by substituting a readily available isotopically labelled reagent for a non-isotopically labelled reagent.

The compounds described in the invention can be present as a solvate. In some cases, the solvent used to prepare the solvate is an aqueous solution, and the solvate is then often referred to as a hydrate. The compounds can be present as a hydrate, which can be obtained, for example, by crystallization from a solvent or from aqueous solution. In this connection, one, two, three or any arbitrary number of solvate or water molecules can combine with the compounds according to the invention to form solvates and hydrates. Unless stated to the contrary, the invention includes all such possible solvates.

The term “co-crystal” means a physical association of two or more molecules which owe their stability through non-covalent interaction. One or more components of this molecular complex provide a stable framework in the crystalline lattice. In certain instances, the guest molecules are incorporated in the crystalline lattice as anhydrates or solvates, see e.g. “Crystal Engineering of the Composition of Pharmaceutical Phases. Do Pharmaceutical Co-crystals Represent a New Path to Improved Medicines?” Almarasson, O., et. al., The Royal Society of Chemistry, 1889-1896, 2004. Examples of co-crystals include p-toluenesulfonic acid and benzenesulfonic acid.

It is also appreciated that certain compounds described herein can be present as an equilibrium of tautomers. For example, ketones with an a-hydrogen can exist in an equilibrium of the keto form and the enol form.

Likewise, amides with an N-hydrogen can exist in an equilibrium of the amide form and the imidic acid form. Unless stated to the contrary, the invention includes all such possible tautomers.

It is known that chemical substances form solids which are present in different states of order which are termed polymorphic forms or modifications. The different modifications of a polymorphic substance can differ greatly in their physical properties. The compounds according to the invention can be present in different polymorphic forms, with it being possible for particular modifications to be metastable. Unless stated to the contrary, the invention includes all such possible polymorphic forms.

In some aspects, a structure of a compound can be represented by a formula:

which is understood to be equivalent to a formula:

wherein n is typically an integer. That is, Rⁿ is understood to represent five independent substituents, R^n(a), R^n(b), R^n(c), R^n(d), R^n(e). By “independent substituents,” it is meant that each R substituent can be independently defined. For example, if in one instance R^n(a) is halogen, then R^n(b) is not necessarily halogen in that instance.

Certain materials, compounds, compositions, and components disclosed herein can be obtained commercially or readily synthesized using techniques generally known to those of skill in the art. For example, the starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.) or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989).

Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including: matters of logic with respect to arrangement of steps or operational flow; plain meaning derived from grammatical organization or punctuation; and the number or type of embodiments described in the specification.

Disclosed are the components to be used to prepare the compositions of the invention as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds can not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods of the invention.

It is understood that the compositions disclosed herein have certain functions. Disclosed herein are certain structural requirements for performing the disclosed functions, and it is understood that there are a variety of structures that can perform the same function that are related to the disclosed structures, and that these structures will typically achieve the same result.

B. COMPOUNDS

In one aspect, the invention relates to compounds useful in methods to inhibit muscle atrophy and to increase muscle mass by providing to a subject in need thereof an effective amount of ursolic acid or a derivative thereof, and pharmaceutical compositions comprising compounds used in the methods. In a further aspect, the invention relates to compounds useful in methods to modulate muscle growth, methods to inhibit muscle atrophy and to increase muscle mass, methods to induce skeletal muscle hypertrophy, methods to enhance tissue growth, and pharmaceutical compositions comprising compounds used in the methods.

In one aspect, the compounds of the invention are useful in the treatment of muscle disorders. In a further aspect, the muscle disorder can be skeletal muscle atrophy secondary to malnutrition, muscle disuse (secondary to voluntary or involuntary bedrest), neurologic disease (including multiple sclerosis, amyotrophic lateral sclerosis, spinal muscular atrophy, critical illness neuropathy, spinal cord injury or peripheral nerve injury), orthopedic injury, casting, and other post-surgical forms of limb immobilization, chronic disease (including cancer, congestive heart failure, chronic pulmonary disease, chronic renal failure, chronic liver disease, diabetes mellitus, Cushing syndrome and chronic infections such as HIV/AIDS or tuberculosis), burns, sepsis, other illnesses requiring mechanical ventiliation, drug-induced muscle disease (such as glucorticoid-induced myopathy and statin-induced myopathy), genetic diseases that primarily affect skeletal muscle (such as muscular dystrophy and myotonic dystrophy), autoimmune diseases that affect skeletal muscle (such as polymyositis and dermatomyositis), spaceflight, or age-related sarcopenia.

It is contemplated that each disclosed derivative can be optionally further substituted. It is also contemplated that any one or more derivative can be optionally omitted from the invention. It is understood that a disclosed compound can be provided by the disclosed methods. It is also understood that the disclosed compounds can be employed in the disclosed methods of using.

1. Ursolic Acid Derivatives

Ursolic acid is a highly water-insoluble pentacyclic triterpene acid that possesses a wide range of biological effects, including anti-cancer, anti-oxidant, anti-inflammatory, anti-allergic, hepatoprotective, gastroprotective, hypolipidemic, hypoglycemic, lipolytic anti-obesity, anti-atherogenic and immunomodulatory effects (Liu J (1995) Journal of ethnopharmacology 49(2):57-68; Liu J (2005) Journal of ethnopharmacology 100(1-2):92-94; Wang Z H, et al. (2010) European journal of pharmacology 628(1-3):255-260; Jang S M, et al. (2009) Int Immunopharmacol 9(1):113-119). However, its effects on skeletal muscle were not known previously. At the molecular level, ursolic acid inhibits the STAT3 activation pathway, reduces matrix metalloproteinase-9 expression via the glucocorticoid receptor, inhibits protein tyrosine phosphatases, acts as an insulin mimetic, activates PPARα, inhibits NF-kB transcription factors, translocates hormone-senstive lipase to stimulate lipolysis and inhibits the hepatic polyol pathway, among many other described effects. Its effects on skeletal muscle and IGF-I signaling were not previously known.

As medicine, ursolic acid is well tolerated and can be used topically and orally. Ursolic acid is present in many plants, including apples, basil, bilberries, cranberries, elder flower, peppermint, rosemary, lavender, oregano, thyme, hawthorn, prunes. Apple peels contain high quantity of ursolic acid and related compounds which are responsible for the anti-cancer activity of apple. Ursolic acid can also serve as a starting material for synthesis of more potent bioactive derivatives, such as anti-tumor agents.

Other names for ursolic acid include 3-β-hydroxy-urs-12-en-28-oic acid, urson, prunol, micromerol, and malol. The structure is shown below:

Other closely related pentacyclic triterpene acids with insulin sensitizing actions include oleanolic acid (Wang et at, 2010), corosolic acid (Sivakumar et at, 2009) and UA0713 (Zhang et at, 2006).

In one aspect, the invention relates to compounds of the formula:

wherein each is an optional covalent bond, and R⁰ is optionally present; wherein n is 0 or 1; wherein R⁰, when present, is hydrogen; wherein R^1a is selected from C1-C6 alkyl and —C(O)ZR¹⁰; wherein R^1b is selected from C1-C6 alkyl, or wherein R^1a and R^1b are optionally covalently bonded and, together with the intermediate carbon, comprise an optionally substituted C3-C5 cycloalkyl or C2-C5 heterocycloalkyl; wherein one of R^2a and R^2b is —OR¹¹, and the other is hydrogen, or R^2a and R^2b together comprise ═O; wherein each of R^3a and R^3b is independently selected from hydrogen, hydroxyl, C1-C6 alkyl, and C1-C6 alkoxyl, provided that R^3a and R^3b are not simultaneously hydroxyl, wherein R^3a and R^3b are optionally covalently bonded and, together with the intermediate carbon, comprise an optionally substituted C3-C5 cycloalkyl or C2-C5 heterocycloalkyl; wherein each of R⁴, R⁵, and R⁶ is independently selected from C1-C6 alkyl; wherein R⁷ is selected from C1-C6 alkyl, —CH₂OR¹² and —C(O)ZR¹²; wherein R⁸ is selected from hydrogen and C1-C6 alkyl; wherein each of R^9a and R^9b is independently selected from hydrogen and C1-C6 alkyl, provided that R^9a and R^9b are not simultaneously hydrogen; or wherein R^9a and R^9b are covalently bonded and, along with the intermediate carbon, together comprise C3-C5 cycloalkyl or C2-C5 heterocycloalkyl; wherein R¹⁰ is selected from hydrogen and C1-C6 alkyl; wherein R¹¹ is selected from hydrogen, C1-C6 alkyl, C1-C5 heteroalkyl, C3-C6 cycloalkyl, C4-C6 heterocycloalkyl, phenyl, heteroaryl, and —C(O)R¹⁴; wherein R¹¹, where permitted, is substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; wherein R¹² is selected from hydrogen and optionally substituted organic residue having from 1 to 20 carbons; wherein Z is selected from —O— and —NR¹³—; wherein R¹³ is selected from hydrogen and C1-C4 alkyl; or, wherein Z is N, R¹² and R¹³ are covalently bonded and —NR¹²R¹³ comprises a moiety of the formula:

wherein Y is selected from —O—, —S—, —SO—, —SO₂—, —NH—, —NCH₃—; and wherein R¹⁴ is C1-C6 alkyl and substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof

In a further aspect, the invention relates to compounds of a formula:

wherein each of R^1a and R^1b is C1-C6 alkyl; wherein one of R^2a and R^2b is —OR¹¹, and the other is hydrogen; wherein each of R⁴, R⁵, and R⁶ is independently C1-C6 alkyl; wherein R⁸ is selected from hydrogen and C1-C6 alkyl; wherein R^9b is C1-C6 alkyl; wherein R¹¹ is selected from hydrogen, C1-C6 alkyl, C1-C5 heteroalkyl, C3-C6 cycloalkyl, C4-C6 heterocycloalkyl, phenyl, heteroaryl, and —C(O)R¹⁴; wherein R¹¹, where permitted, is substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; wherein R¹² is selected from hydrogen and optionally substituted organic residue having from 1 to 20 carbons; wherein Z is selected from —O— and —NR¹³—; wherein R¹³ is selected from hydrogen and C1-C4 alkyl; or, wherein Z is N, R¹² and R¹³ are covalently bonded and —NR¹²R¹³ comprises a moiety of the formula:

wherein Y is selected from —O—, —S—, —SO—, —SO₂—, —NH—, —NCH₃—; and wherein R¹⁴ is C1-C6 alkyl and substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl.

In a further aspect, the invention relates to compounds of a formula:

wherein R^1a is selected from C1-C6 alkyl and —C(O)ZR¹⁰; wherein R^1b is selected from C1-C6 alkyl, or R^1a and R^1b are covalently bonded and, together with the intermediate carbon, comprise an optionally substituted 3- to 7-membered spirocycloalkyl; wherein R⁸ is C1-C6 alkyl; wherein R^9a is C1-C6 alkyl; wherein R¹¹ is selected from hydrogen, C1-C6 alkyl, C1-C5 heteroalkyl, C3-C6 cycloalkyl, C4-C6 heterocycloalkyl, phenyl, heteroaryl, and —C(O)R¹⁴; wherein R¹¹, where permitted, is substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; wherein R¹² is selected from hydrogen and optionally substituted organic residue having from 1 to 20 carbons; wherein Z is selected from —O— and —NR¹³—; wherein R¹³ is selected from hydrogen and C1-C4 alkyl; or, wherein Z is N, R¹² and R¹³ are covalently bonded and —NR¹²R¹³ comprises a moiety of the formula:

wherein Y is selected from —O—, —S—, —SO—, —SO₂—, —NH—, —NCH₃—; and wherein R¹⁴ is C1-C6 alkyl and substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl.

In a further aspect, the invention relates to compounds of a formula:

In a further aspect, the invention relates to compounds of a formula:

wherein R^1a is —C(O)ZR¹⁰; wherein R^1b is C1-C6 alkyl; wherein one of R^2a and R^2b is —OR¹¹, and the other is hydrogen; wherein each of R⁴, R⁵, and R⁶ is independently selected from C1-C6 alkyl; wherein R⁷ is selected from C1-C6 alkyl; wherein R⁸ is selected from hydrogen and C1-C6 alkyl; wherein R^9a is selected from hydrogen and C1-C6 alkyl; wherein Z is selected from —O— and —NR¹³—; wherein R¹⁰ is selected from hydrogen and C1-C6 alkyl; wherein R¹¹ is selected from hydrogen, C1-C6 alkyl, C1-C5 heteroalkyl, C3-C6 cycloalkyl, C4-C6 heterocycloalkyl, phenyl, heteroaryl, and —C(O)R¹⁴; wherein R¹¹, where permitted, is substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; wherein R¹³ is selected from hydrogen and C1-C4 alkyl; or, wherein Z is N, R¹² and R¹³ are covalently bonded and —NR¹²R¹³ comprises a moiety of the formula:

wherein Y is selected from —O—, —S—, —SO—, —SO₂—, —NH—, —NCH₃—; and wherein R¹⁴ is C1-C6 alkyl and substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl.

In a further aspect, the invention relates to compounds of a formula:

wherein each of R^1a and R^1b is independently C1-C6 alkyl; wherein one of R^2a and R^2b is —OR¹¹, and the other is hydrogen; wherein one of R^3a and R^3b is —OR¹¹, and the other is hydrogen; wherein each of R⁴, R⁵, and R⁶ is independently selected from C1-C6 alkyl; wherein R⁸ is C1-C6 alkyl; wherein R^9a is C1-C6 alkyl; wherein each R¹¹ is independently selected from hydrogen, C1-C6 alkyl, C1-C5 heteroalkyl, C3-C6 cycloalkyl, C4-C6 heterocycloalkyl, phenyl, heteroaryl, and —C(O)R¹⁴; wherein R¹¹, where permitted, is substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; wherein R¹² is selected from hydrogen and optionally substituted organic residue having from 1 to 20 carbons; wherein Z is selected from —O— and —NR¹³—; wherein R¹³ is selected from hydrogen and C1-C4 alkyl; or, wherein Z is N, R¹² and R¹³ are covalently bonded and —NR¹²R¹³ comprises a moiety of the formula:

wherein Y is selected from —O—, —S—, —SO—, —SO₂—, —NH—, —NCH₃—; and wherein R¹⁴ is C1-C6 alkyl and substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl.

In a further aspect, the invention relates to compounds of a formula:

wherein R¹¹ is selected from hydrogen, C1-C6 alkyl, C1-C5 heteroalkyl, C3-C6 cycloalkyl, C4-C6 heterocycloalkyl, phenyl, heteroaryl, and —C(O)R¹⁴; wherein R¹¹, where permitted, is substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; wherein R¹² is selected from hydrogen and optionally substituted organic residue having from 1 to 20 carbons; wherein Z is selected from —O— and —NR¹³—; wherein R¹³ is selected from hydrogen and C1-C4 alkyl; or, wherein Z is N, R¹² and R¹³ are covalently bonded and —NR¹²R¹³ comprises a moiety of the formula:

wherein Y is selected from —O—, —S—, —SO—, —SO₂—, —NH—, —NCH₃—; and wherein R¹⁴ is C1-C6 alkyl and substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof.

In a further aspect, the compound is administered in an amount effective to prevent or treat muscle atrophy in the animal. In a still further aspect, the compound is administered in amount is greater than about 1000 mg per day when the compound is ursolic acid, beta-boswellic acid, corosolic acid, betulinic acid, or UA0713. In a yet further aspect, the compound is administered in an amount greater than about 1000 mg per day and effective to enhance muscle formation in the mammal.

In a further aspect, the invention relates to compounds of a formula selected from:

a. R⁰ Groups and Optional Bonds

In one aspect, an optional covalent bond can be represented by —. Thus, in certain aspects, a particular bond is present, thereby providing a single covalent bond. In further aspects, a particular bond is present, thereby providing a double covalent bond. In further aspects, a particular bond is absent, thereby providing a double covalent bond.

In one aspect, R⁰ is optionally present. That is, in certain aspects, R⁰ is present. In further aspects, R⁰ is absent. In a further aspect, R⁰, when present, is hydrogen. It is understood that the presence and/or absence of R⁰ Groups and optional bonds serve to satisfy valence of the adjacent chemical moieties.

b. R¹ Groups

In one aspect, R^1a is selected from C1-C6 alkyl and —C(O)ZR¹⁰; wherein R^1b is selected from C1-C6 alkyl; or wherein R^1a and R^1b are covalently bonded and, along with the intermediate carbon, together comprise optionally substituted C3-C5 cycloalkyl or optionally substituted C2-C5 heterocycloalkyl. In a further aspect, R^1a is —CO₂H. In a further aspect, R^1b is methyl. In a further aspect, R^1a and R^1b are both methyl.

In one aspect, R^1a is —C(O)ZR¹⁰. In a further aspect, R^1a is selected from C1-C6 alkyl, for example, methyl, ethyl, propyl, butyl, pentyl, or hexyl. In a further aspect, R^1b is selected from C1-C6 alkyl, for example, methyl, ethyl, propyl, butyl, pentyl, or hexyl.

In a further aspect, R^1a and R^1b are covalently bonded and, along with the intermediate carbon, together comprise optionally substituted C3-C5 cycloalkyl or optionally substituted C2-C5 heterocycloalkyl.

c. R² Groups

In one aspect, R^2a and R^2b are independently selected from hydrogen and —OR¹¹, provided that at least one of R^2a and R^2b is —OR¹¹; or wherein R^2a and R^2b together comprise ═O. In a further aspect, R^2a is hydrogen, and R^2b is —OR¹¹. In a further aspect, R^2a is —OR¹¹, and R^2b is hydrogen. In a further aspect, R^2a and R^2b together comprise ═O.

In a further aspect, R^2a is hydrogen. In a further aspect, R^2a is —OR¹¹; wherein R¹¹ is selected from hydrogen, C1-C6 alkyl, and —C(O)R¹⁴; wherein R¹⁴ is C1-C6 alkyl. In a further aspect, R^2b is —OR¹¹; wherein R¹¹ is selected from hydrogen, C1-C6 alkyl, and —C(O)R¹⁴; and wherein R¹⁴ is C1-C6 alkyl. In a further aspect, R^2b is —OR¹¹; wherein R¹¹ is hydrogen.

In a further aspect, R^2b is hydrogen. In a further aspect, R^2a is —OR¹¹; wherein R¹¹ is selected from hydrogen, C1-C6 alkyl, and —C(O)R¹⁴; wherein R¹⁴ is C1-C6 alkyl. In a further aspect, R^2a is —OR¹¹; wherein R¹¹ is hydrogen.

d. R³ Groups

In one aspect, each of R^3a and R^3b is independently selected from hydrogen, hydroxyl, C1-C6 alkyl, and C1-C6 alkoxyl, provided that R^3a and R^3b are not simultaneously hydroxyl; or wherein R^3a and R^3b are covalently bonded and, along with the intermediate carbon, together comprise optionally substituted C3-C5 cycloalkyl or optionally substituted C2-C5 heterocycloalkyl.

In a further aspect, R^3a is hydrogen. In a further aspect, R^3b is —OR¹¹; wherein R¹¹ is selected from hydrogen, C1-C6 alkyl, and —C(O)R¹⁴; wherein R¹⁴ is C1-C6 alkyl.

e. R⁴ Groups

In one aspect, R⁴ is independently selected from C1-C6 alkyl, for example, methyl, ethyl, propyl, butyl, pentyl, or hexyl. In a further aspect, R⁴ is methyl. In a further aspect, R⁴, R⁵, and R⁶ are all methyl.

f. R⁵ Groups

In one aspect, R⁵ is independently selected from C1-C6 alkyl, for example, methyl, ethyl, propyl, butyl, pentyl, or hexyl. In a further aspect, R⁵ is methyl.

g. R⁶ Groups

In one aspect, R⁶ is independently selected from C1-C6 alkyl, for example, methyl, ethyl, propyl, butyl, pentyl, or hexyl. In a further aspect, R⁶ is methyl.

h. R⁷ Groups

In one aspect, R⁷ is selected from C1-C6 alkyl, —CH₂OR¹², and —C(O)ZR¹². In a further aspect, R⁷ is C1-C6 alkyl, for example, methyl, ethyl, propyl, butyl, pentyl, or hexyl. In a further aspect, R⁷ is —CH₂OR¹². In a further aspect, R⁷ is and —C(O)ZR¹².

i. R⁸ Groups

In one aspect, R⁸ is selected from hydrogen and C1-C6 alkyl. In a further aspect, R⁸ is hydrogen. In a further aspect, R⁸ is C1-C6 alkyl, for example, methyl, ethyl, propyl, butyl, pentyl, or hexyl.

j. R⁹ Groups

In one aspect, each of R^9a and R^9b is independently selected from hydrogen and C1-C6 alkyl, provided that R^9a and R^9b are not simultaneously hydrogen; or wherein R^9a and R^9b are covalently bonded and, along with the intermediate carbon, together comprise optionally substituted C3-C5 cycloalkyl or optionally substituted C2-C5 heterocycloalkyl.

In a further aspect, R^9a is hydrogen. In a further aspect, R^9a is C1-C6 alkyl, for example, methyl, ethyl, propyl, butyl, pentyl, or hexyl. In a further aspect, R^9b is hydrogen. In a further aspect, R^9b is C1-C6 alkyl, for example, methyl, ethyl, propyl, butyl, pentyl, or hexyl. In a further aspect, R^9b is selected from methyl, ethyl, vinyl, n-propyl, propen-2-yl, propyl, 2-propenyl, n-butyl, 1-buten-2-yl, 1-buten-3-yl, i-butyl, 1-buten-2-yl, 1-buten-3-yl, s-butyl, 2-buten-1-yl, 2-buten-2-yl, 2-buten-3-yl, and t-butyl.

In a further aspect, R^9a and R^9b are covalently bonded and, along with the intermediate carbon, together comprise optionally substituted C3-C5 cycloalkyl or optionally substituted C2-C5 heterocycloalkyl.

k. R¹⁰ Groups

In one aspect, R¹⁰ is selected from hydrogen and C1-C6 alkyl. In a further aspect, R¹⁰ is hydrogen. In a further aspect, R¹⁰ is C1-C6 alkyl, for example, methyl, ethyl, propyl, butyl, pentyl, or hexyl.

l. R¹¹ Groups

In one aspect, each R¹¹ is independently selected from hydrogen, C1-C6 alkyl, C1-C5 heteroalkyl, C3-C6 cycloalkyl, C4-C6 heterocycloalkyl, phenyl, heteroaryl, and —C(O)R¹⁴; wherein R¹¹, where permitted, is substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl.

In a further aspect, R¹¹ is hydrogen. In a further aspect, R¹¹ is selected from C1-C6 alkyl, C1-C5 heteroalkyl, C3-C6 cycloalkyl, C4-C6 heterocycloalkyl, phenyl, heteroaryl, and —C(O)R¹⁴. In a further aspect, R¹¹ is C1-C6 alkyl. In a further aspect, R¹¹ is C1-C5 heteroalkyl. In a further aspect, R¹¹ is C3-C6 cycloalkyl. In a further aspect, R¹¹ is C4-C6 heterocycloalkyl. In a further aspect, R¹¹ is phenyl. In a further aspect, R¹¹ is heteroaryl. In a further aspect, R¹¹ is —C(O)R¹⁴.

In a further aspect, R¹¹ is unsubstituted. In a further aspect, R¹¹, where permitted, is substituted with 0-2 groups. In a further aspect, R¹¹, where permitted, is substituted with 1 group. In a further aspect, R¹¹, where permitted, is substituted with 2 groups.

m. R¹² Groups

In one aspect, R¹² is selected from hydrogen and optionally substituted organic residue having from 1 to 20 carbons. In a further aspect, R¹² is hydrogen. In a further aspect, R¹² is optionally substituted organic residue having from 1 to 20 carbons. In a further aspect, R¹² is optionally substituted organic residue having from 3 to 12 carbons.

In a further aspect, R¹² is hydrogen. In a further aspect, R¹² is alkyl. In a further aspect, R¹² is heteroalkyl. In a further aspect, R¹² is cycloalkyl. In a further aspect, R¹² is heterocycloalkyl. In a further aspect, R¹² is aryl. In a further aspect, R¹² is heteroaryl. In a further aspect, R¹² is substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl. In a further aspect, R¹² comprises a group having a formula:

wherein m is an integer from 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); and wherein AA represents an amino acid residue. In a further aspect, R¹² is AA is a phenylalanine residue. In a further aspect, R¹² comprises a group having a formula:

n. R¹³ Groups

In one aspect, R¹³ is selected from hydrogen and C1-C4 alkyl; or, wherein Z is N, R¹² and R¹³ are covalently bonded and —NR¹²R¹³ comprises a moiety of the formula:

wherein Y is selected from —O—, —S—, —SO—, —SO₂—, —NH—, and —NCH₃—.

In a further aspect, R¹³ is hydrogen. In a further aspect, R¹³ is C1-C4 alkyl, for example, methyl, ethyl, propyl, or butyl. In a further aspect, Z is N, and —NR¹²R¹³ comprises a moiety of the formula:

o. R¹⁴ Groups

In one aspect, R¹⁴ is C1-C6 alkyl and substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl.

In a further aspect, R¹⁴ is C1-C6 alkyl, for example, methyl, ethyl, propyl, butyl, pentyl, or hexyl. In a further aspect, R¹⁴ is unsubstituted. In a further aspect, R¹⁴, where permitted, is substituted with 0-2 groups. In a further aspect, R¹⁴, where permitted, is substituted with 1 group. In a further aspect, R¹⁴, where permitted, is substituted with 2 groups.

p. AA Groups

In one aspect, AA represents an amino acid residue, for example, phenylalanine.

q. Y Groups

In one aspect, Y is selected from —O—, —S—, —SO—, —SO₂—, —NH—, and —NCH₃—.

r. Z Groups

In one aspect, Z is selected from —O— and —NR¹³—. In a further aspect, Z is —O—. In a further aspect, Z is —NR¹³—; wherein R¹³ is hydrogen. In a further aspect, Z is —NR₁₃—; wherein R¹³ is C1-C4 alkyl.

2. Example Compounds

In one aspect, a compound can be present as one or more of the following structures:

In a further aspect, a compound can be present as one or more of the following structures:

3. Inhibtion of Muscle Atrophy and Induction of Muscle Hypertrophy

In one aspect, the disclosed compounds inhibit muscle atrophy. In a further aspect, the disclosed compounds increase muscle mass. In a still further aspect, the disclosed compounds induce muscle hypertrophy. In a yet further aspect, the disclosed compounds inhibit of muscle atrophy and increase muscle mass. In an even further aspect, the disclosed compounds inhibit of muscle atrophy and induce muscle hypertrophy. In a further aspect, the inhibition of muscle atrophy is in an animal. In an even further aspect, the increase in muscle mass is in an animal. In a still further aspect, the animal is a mammal, In a yet further aspect, the mammal is a human. In a further aspect, the mammal is a mouse. In a yet further aspect, the mammal is a rodent.

In a further aspect, the disclosed compounds inhibit muscle atrophy when administered at an oral dose of greater than about 200 mg per day in a human. In a yet further aspect, the disclosed compounds inhibit muscle atrophy when administered at an oral dose of greater than about 300 mg per day in a human. In a still further aspect, the disclosed compounds inhibit muscle atrophy when administered at an oral dose of greater than about 400 mg per day in a human. In an even further aspect, the disclosed compounds inhibit muscle atrophy when administered at an oral dose of greater than about 500 mg per day in a human. In a further aspect, the disclosed compounds inhibit muscle atrophy when administered at an oral dose of greater than about 750 mg per day in a human. In a yet further aspect, the disclosed compounds inhibit muscle atrophy when administered at an oral dose of greater than about 1000 mg per day in a human. In a still further aspect, the disclosed compounds inhibit muscle atrophy when administered at an oral dose of greater than about mg per day in a human. In an even further aspect, the disclosed compounds inhibit muscle atrophy when administered at an oral dose of greater than about 2000 mg per day in a human.

In a further aspect, the disclosed compounds increase muscle mass when administered at an oral dose of greater than about 200 mg per day in a human. In a yet further aspect, the disclosed compounds increase muscle mass when administered at an oral dose of greater than about 300 mg per day in a human. In a still further aspect, the disclosed compounds increase muscle mass when administered at an oral dose of greater than about 400 mg per day in a human. In an even further aspect, the disclosed compounds increase muscle mass when administered at an oral dose of greater than about 500 mg per day in a human. In a further aspect, the disclosed compounds increase muscle mass when administered at an oral dose of greater than about 750 mg per day in a human. In a yet further aspect, the disclosed compounds increase muscle mass when administered at an oral dose of greater than about 1000 mg per day in a human. In a still further aspect, the disclosed compounds increase muscle mass when administered at an oral dose of greater than about mg per day in a human. In an even further aspect, the disclosed compounds increase muscle mass when administered at an oral dose of greater than about 2000 mg per day in a human.

It is contemplated that one or more compounds can optionally be omitted from the disclosed invention.

C. METHODS OF MAKING THE COMPOUNDS

In one aspect, the disclosed compounds comprise the products of the synthetic methods described herein. In a further aspect, the disclosed compounds comprise a compound produced by a synthetic method described herein. In a still further aspect, the invention comprises a pharmaceutical composition comprising a therapeutically effective amount of the product of the disclosed methods and a pharmaceutically acceptable carrier. In a still further aspect, the invention comprises a method for manufacturing a medicament comprising combining at least one compound of any of disclosed compounds or at least one product of the disclosed methods with a pharmaceutically acceptable carrier or diluent.

In one aspect, the invention relates to methods of making functionalized ursane compounds useful in methods of inhibiting muscle atrophy and increasing muscle mass. Such compounds can be useful in the treatment of various maladies associated with muscle wasting, useful for increasing muscle mass and/or muscle strength, as well as in enhancing muscle formation and/or muscular performance. The compounds of the invention can be prepared by employing reactions as shown in the following schemes, in addition to other standard manipulations that are known in the literature, exemplified in the experimental sections or clear to one skilled in the art. For clarity, examples having a single substituent are shown where multiple substituents are allowed under the definitions disclosed herein. The following examples are provided so that the invention might be more fully understood, are illustrative only, and should not be construed as limiting.

1. Route 1: Alkyl Etherification

In one aspect, functionalized ursane compounds of the present invention can be prepared generically as shown below.

Compounds are represented in generic form, with substituents as noted in compound descriptions elsewhere herein. A more specific example is set forth below.

In one aspect, Route 1 step 1 begins with a free acid. In an appropriate solvent, a base (e.g., K₂CO₃, NaOH) strong enough to deprotonate the carboxylic acid, but not the alcohol, is added, and the reaction is conducted at a temperature effective and for a time effective to insure carboxylic acid deprotonation. An appropriate alkyl halide or halide equivalent is added to the reaction mixture, and the reaction is conducted at a temperature effective and for a time effective to insure alkylation of the carboxyl group. In a further aspect, an alternate Route 1 step 1 also begins with the free carboxylic acid. Diazomethane is added, and the reaction is conducted at a temperature effective and for a time effective to insure reaction.

In a further aspect, Route 1 step 2 the alkyl ester is dissolved in an appropriate dry solvent under anhydrous reaction conditions. A base is added, and the reaction is conducted at a temperature effective and for a time effective to insure deprotonation. Then, an appropriate alkyl, heteroalkyl, cycloalkyl, or heterocycloalkyl halide or halide equivalent (i.e., R¹¹X) is added to the reaction mixture. The reaction is conducted at a temperature effective and for a time effective insure complete reaction.

In a further aspect, in Route 1 step 3, the O-alkylated ursane compound alkyl ester is hydrolyzed with an appropriate base, such as LiOH, in an appropriate organic-aqueous mixed solvent system at a temperature effective and for a time effective to insure reaction. Then the reaction mixture can be acidified to a suitable pH with an appropriate aqueous acid of an sufficient concentration and at a temperature effective and for a time effective to insure reaction.

2. Route 2: Aryl Etherification

In one aspect, functionalized ursane compounds of the present invention can be prepared generically as shown below.

Compounds are represented in generic form, with substituents as noted in compound descriptions elsewhere herein. A more specific example is set forth below.

In one aspect, Route 2 step 1 begins with the ursane compound free carboxylic acid. In an appropriate solvent, a base (e.g., K₂CO₃, NaOH) strong enough to deprotonate the carboxylic acid, but not the alcohol group, is added, and the reaction is conducted at a temperature effective and for a time effective to insure deprotonation. Then, an appropriate alkyl halide or halide equivalent is added to the reaction mixture, and the reaction is conducted at a temperature effective and for a time effective to insure alkylation of the carboxyl group. In a further aspect, an alternate Route 2 step 1 begins with the ursane compound free carboxylic acid in an appropriate solvent. Diazomethane is added, and the reaction is conducted at a temperature effective and for a time effective to insure reaction.

In a further apect, Route 2 step 2, the ursane compound alkyl ester is dissolved in an appropriate, dry solvent, along with phenol, an aryl alcohol, or appropriate heteroaryl alcohol, under anhydrous reaction conditions, followed by the addition of triphenylphosphine. The reaction is conducted at a effective temperature and for an effective time period. Then, an appropriate coupling agent, such as DIAD or DEAD, is added, and the reaction is conducted at a temperature effective and for a time effective to insure reaction. In a further aspect, in Route 2 step 3, the O-arylated or heteroarylated ursane compound alkyl ester can be treated with an appropriate base, such as LiOH, in an appropriate organic-aqueous mixed solvent system at a temperature effective and for a time effective to insure complete reaction. The reaction mixture can then be acidified to a suitable pH.

3. Route 3: Acylation

In one aspect, functionalized ursane compounds of the present invention can be prepared generically as shown below.

Compounds are represented in generic form, with substituents as noted in compound descriptions elsewhere herein. A more specific example is set forth below.

In one aspect, Route 3 step 1 begins with the the ursane compound free carboxylic acid. In an appropriate solvent, a base (e.g., K₂CO₃, NaOH) strong enough to deprotonate the carboxylic acid, but not the alcohol group, is added, and the reaction is allowed to progress at a temperature effective and for a time effective to insure carboxylic acid deprotonation. Then, an appropriate benzyl halide or halide equivalent is added to the reaction mixture, and the reaction is conducted at a temperature effective and for a time effective to insure protection of the carboxyl group.

In Route 3 step 2, the ursane compound benzyl ester is dissolved in an appropriate, dry solvent under anhydrous reaction conditions, followed by the addition of an appropriate acid scavenger (weak base, e.g., K₂CO₃ or DIEA). The acyl halide (e.g., R¹⁴COX) or equivalent acylating reagent is then added. The reaction is conducted at a temperature effective and for a time effective to insure reaction. In a further aspect, in an alternate Route 3 step 2, the the ursane compound benzyl ester and a suitable carboxylic acid (e.g., R¹⁴CO₂H) are dissolved in an appropriate, dry solvent under anhydrous reaction conditions. Ethyl-(N′,N′-dimethylamino)propylcarbodiimide hydrochloride (EDC), 1-hydroxybenzotriazole (HOBt), and a trialkylamine (R₃N) are then added, and the reaction is conducted at a temperature effective and for a time effective to insure reaction.

In Route 3 step 3, the acylated ursane compound benzyl ester is reduced under standard conditions (e.g., hydrogenation with hydrogen gas in the presence of a suitable palladium catalyst), thereby liberating the ursane compound free carboxlic acid.

4. Route 4: Esterification

In one aspect, functionalized ursane compounds of the present invention can be prepared generically as shown below.

Compounds are represented in generic form, with substituents as noted in compound descriptions elsewhere herein. A more specific example is set forth below.

In one aspect, Route 4 step 1 begins with the ursane compound free carboxylic acid. An appropriate alcohol (e.g., R¹²OH) is added, and the reaction is conducted at a temperature effective and for a time effective to time to insure reaction.

In a further aspect in an alternate syntheis, Route 4 step 1 begins with the ursane compound free carboxylic acid in a dry solvent under dry reaction conditions.

Tetrahydropyran (THP) is added, along with an acid catalyst (e.g., pTsOH). The reaction is conducted at a temperature effective and for a time effective to insure protection of the hydroxyl group. A base (e.g., NaOH or NaH) is then added to the THP-protected ursane compound free carboxylic acid, in a dry solvent under anhydrous reaction conditions. The reaction is conducted at a temperature effective and for a time effective to insure carboxylic acid deprotonation. Then, an appropriate alkyl halide (i.e., R¹²X) or equivalent is added to the reaction mixture, and the reaction is conducted at a temperature effective and for a time effective to insure alkylation of the carboxyl group. Route 4 step 3 begins with the THP-protected ursane compound alkyl ester in an alcohol solvent. An acid catalyst (e.g., pTsOH) is added, and the reaction is conducted at a temperature effective and for a time effective to insure deprotection.

5. Route 5: Amide Formation

In one aspect, functionalized ursane compounds of the present invention can be prepared generically as shown below.

Compounds are represented in generic form, with substituents as noted in compound descriptions elsewhere herein. A more specific example is set forth below.

In one aspect, Route 5 step 1 begins with the ursane compound free carboxylic acid in a dry solvent. Under dry reaction conditions, tetrahydropyran (THP) and an acid catalyst (e.g., pTsOH) are added. The reaction is then conducted at a temperature effective and for a time effective to insure protection of the hydroxyl group. In Route 5 step 2, the THP-protected ursane compound free carboxylic acid is dissolved in an appropriate, dry solvent. Under anhydrous reaction conditions, a suitable amine (e.g., R¹²R¹³NH) is added, along with ethyl-(N′,N′-dimethylamino)propylcarbodiimide hydrochloride (EDC), 1-hydroxybenzotriazole (HOBt), and a trialkylamine (R3N), and the reaction is conducted at a temperature effective and for a time effective to time to insure complete reaction. In Route 5 step 3, the THP-protected ursane compound amide can then be deprotected by addition of an acid catalyst (e.g., pTsOH), and the reaction is conducted at a temperature effective and for a time effective to insure reaction.

6. Route 6: Reduction to Alcohol

In one aspect, functionalized ursane compounds of the present invention can be prepared generically as shown below.

Compounds are represented in generic form, with substituents as noted in compound descriptions elsewhere herein. A more specific example is set forth below.

In one aspect, the ursane compound free carboxylic acid, in a dry solvent, can be reacted with lithium aluminum hydride (LiAlH₄) under dry reaction conditions to provide the corresponding primary alcohol. Alternatively, the ursane compound free carboxylic acid, in a dry solvent, can be reacted with diborane (B₂H₆) under dry reaction conditions to provide the corresponding primary alcohol. It is understood that protecting group chemistry, if needed, can also be used to protect sensitive remote functionality during these reaction steps.

7. Route 7: Hydroxyl Inversion

In one aspect, functionalized ursane compounds of the present invention can be prepared generically as shown below.

Compounds are represented in generic form, with substituents as noted in compound descriptions elsewhere herein. A more specific example is set forth below.

In one aspect, a hydroxyl functionality can be substituted with another group (e.g., alkoxyl, acyl, amino, etc.), while inverting the stereochemistry at the adjacent carbon, by reaction with an appropriate protic nucleophile in the presence of diethylazodicarboxylate (DEAD) and triphenylphosphine under Mitsunobu reaction conditions. While —OR¹¹ is shown, it is understood that additional moieties (e.g., acetoxyl, amino, etc.) can be substituted at that position by appropriate selection of protic nucleophile (e.g., acetic acid, ammonia, etc.).

8. Plant Sources of Ursolic Acid Derivatives

Many pentacyclic acid triterpenes useful as synthetic precursors to the ursolic acid derivatives in the synthetic methods described above may be isolated and purified from a natural source such as plants or materials derived from plants. Alternatively, certain known synthetic precursors useful in the preparation of ursolic acid derivatives can often be obtained from commercial sources. Ursolic Acid is a useful known synthetic precursor to ursolic acid derivatives that can be used as a synthetic precursor to prepare certain disclosed compounds. For example, ursolic acid can be isolated from plants such as Holy Basil (Ocimum sanctum L.), peppermint leaves (Mentha piperita L.), lavender (Lavandula augustifolia Mill.), oregano (Origanum vulgare L.), thyme (Thymus vulgaris L.), hawthorn (Crataegus laevigata (Poir) DC), cherry laurel leaves (Prunus laurocerasus L.), loquat leaves (Eriobotrya japonica L.), glossy privet leaves (Ligustrum lucidum Ait. L.), bilberry (Vacciunum myrtillus L.), Devil's Claw (Harpagophytum procumbens DC), Elder Flowers (European var.; Sambucus nigra L.), and periwinkle (Vinca minor L.).

A variety of methods that are generally applicable to purifying ursolic acid and ursolic acid derivatives. For example, Nishimura, et al. (J. Nat. Prod. 1999, 62, 1061-1064) described the identification of 2,3-dihydroxy-24-nor-urs-4(23),12-dien-28-oic acid and 23-hydroxyursolic acid. Nishimura described procedures to isolate these compounds. Procedures described herein demonstrate these compounds will be contained in flash chromatography fraction 3 (FCF3) as described in the examples. Similar HPLC procedures described herein can be used to further purify these compounds including using a gradient with water with 0.05% TFA and acetonitrile with 0.05% TFA, mobile phase A and B respectively, with a C18 BetaMax Neutral column (250×8 mm; 5 um). The gradient may consist of 40% β isocratic for 5 min, then from approximately 40% to 70% B in 30 min. A skilled artisan would recognize the general applicability of the methods described in Nishimura et al to efficiently isolate either the ursolic acid, ursolic acid derivatives or structurally related pentacyclic acid triterpenes from various plants.

Other illustrative methods that are generally applicable to purifying ursolic acid and ursolic acid derivatives are also known. For example, Chaturvedula, et al. (J. Nat. Prod. 2004, 67, p. 899-901) described the isolation of 3-acetoxy-2-hydroxy ursolic acid, 3-(p-coumaroyl)ursolic acid, and 2,3-diacetoxyursolic acid. Adnyana, et al. (J. Nat. Prod. 2001, 64, p. 360-363) described the isolation of 2,3,6,19-tetrahydroxyoleanolic acid, 2,3,19-trihydroxyoleanolic acid, 2,3,19,23-tetrahydroxyursolic acid, and 2,3,23-trihydroxyoleanolic acid. Ikuta, et al. (J. Nat. Prod. 2003, 66, p. 1051-1054) described the isolation of 2,3-dihydroxyurs-12-en-11-on-28-oic acid and 2,3-dihydroxy-11-methoxyurs-12-en-28-oic acid. For example, similar HPLC procedures such as those described in U.S. Pat. No. 7,612,045 can be used to further purify these compounds including using a gradient with water with 0.05% TFA and acetonitrile with 0.05% TFA, mobile phase A and B respectively, with a C18 BetaMax Neutral column (250×8 mm; 5 um). The gradient may consist of 40% β isocratic for 5 min, then from approximately 40% to 70% B in 30 min.

Finally, another source of the known synthetic precursors useful in the synthetic methods described above to prepare ursolic acid derivatives are commercial sources or vendors. Purified forms of corosolic acid, ursolic acid, oleanolic acid, madecassic acid, asiatic acid, pygenic acid (A, B or C), caulophyllogenin and echinocystic acid may be obtained from a commercial source. For example, ursolic acid and oleanolic acid may be purchased from Sigma-Aldrich Chemical Company (St. Louis, Mo., USA) and corosolic acid, asiatic acid, madecassic acid, pygenic acid (A, B, or C), caulophyllogenin and echinocystic acid may be purchased from Chromadex (Santa Ana, Calif., USA). The compounds obtained from commercial sources may be furthered separated and purified as needed using methods such as column chromatography, high pressure liquid chromatography (HPLC), and/or recrystallization described herein. Additional methods of isolation of precursors are described in U.S. Pat. No. 7,612,045, U.S. patent application Ser. No. 10/355,201, and U.S. patent application Ser. No. 10/445,943.

It is further anticipated that the compounds of the invention can be obtained by direct synthesis. Direct synthesis may include either total synthesis or semi-synthesis. Exemplary synthetic methods for obtaining these compounds are described above. Additional synethetic procedures useful in the preparation of ursolic acid derivatives are described in U.S. Pat. No. 3,903,089, U.S. Pat. No. 7,612,045, and U.S. patent application Ser. No. 10/445,943, U.S. patent application Ser. No. 10/355,201. Further synthetic methods useful in the preparation of ursolic acid derivatives are Meng, Y., et al. (2010) Molecules 15:4033-4040; Gao, Y., et al. (2010) Molecules 15:4439-4449; Sporn, M. B., et al. (2011) Journal of Natural Products 74:537-545; Chadalapaka, G., et al. (2008) Biorganic and Medicinal Chemistry Letters 18(8):2633-2639; and, Sun, H., et al. (2006) Botanical Studies 47:339-368.

It is contemplated that each disclosed methods can further comprise additional steps, manipulations, and/or components. It is also contemplated that any one or more step, manipulation, and/or component can be optionally omitted from the invention. It is understood that a disclosed methods can be used to provide the disclosed compounds. It is also understood that the products of the disclosed methods can be employed in the disclosed methods of using.

D. PHARMACEUTICAL COMPOSITIONS

In one aspect, the invention relates to pharmaceutical compositions comprising the disclosed compounds. That is, a pharmaceutical composition can be provided comprising a therapeutically effective amount of at least one disclosed compound or at least one product of a disclosed method and a pharmaceutically acceptable carrier.

In one aspect, the invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a compound having a structure represented by a formula:

wherein each is an optional covalent bond, and R⁰ is optionally present; wherein n is 0 or 1; wherein R⁰, when present, is hydrogen; wherein R^1a is selected from C1-C6 alkyl and —C(O)ZR¹⁰; wherein R^1b is selected from C1-C6 alkyl, or wherein R^1a and R^1b are optionally covalently bonded and, together with the intermediate carbon, comprise an optionally substituted C3-C5 cycloalkyl or C2-C5 heterocycloalkyl; wherein one of R^2a and R^2b is —OR¹¹, and the other is hydrogen, or R^2a and R^2b together comprise ═O; wherein each of R^3a and R^3b is independently selected from hydrogen, hydroxyl, C1-C6 alkyl, and C1-C6 alkoxyl, provided that R^3a and R^3b are not simultaneously hydroxyl, wherein R^3a and R^3b are optionally covalently bonded and, together with the intermediate carbon, comprise an optionally substituted C3-C5 cycloalkyl or C2-C5 heterocycloalkyl; wherein each of R⁴, R⁵, and R⁶ is independently selected from C1-C6 alkyl; wherein R⁷ is selected from C1-C6 alkyl, —CH₂OR¹² and —C(O)ZR¹²; wherein R⁸ is selected from hydrogen and C1-C6 alkyl; wherein each of R^9a and R^9b is independently selected from hydrogen and C1-C6 alkyl, provided that R^9a and R^9b are not simultaneously hydrogen; or wherein R^9a and R^9b are covalently bonded and, along with the intermediate carbon, together comprise C3-C5 cycloalkyl or C2-C5 heterocycloalkyl; wherein R¹⁹ is selected from hydrogen and C1-C6 alkyl; wherein R¹¹ is selected from hydrogen, C1-C6 alkyl, C1-C5 heteroalkyl, C3-C6 cycloalkyl, C4-C6 heterocycloalkyl, phenyl, heteroaryl, and —C(O)R¹⁴; wherein R¹¹, where permitted, is substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; wherein R¹² is selected from hydrogen and optionally substituted organic residue having from 1 to 20 carbons; wherein Z is selected from —O— and —NR¹³—; wherein R¹³ is selected from hydrogen and C1-C4 alkyl; or, wherein Z is N, R¹² and R¹³ are covalently bonded and —NR¹²R¹³ comprises a moiety of the formula:

wherein Y is selected from —O—, —S—, —SO—, —SO₂—, —NH—, —NCH₃—; and wherein R¹⁴ is C1-C6 alkyl and substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof, in an amount effective to prevent or treat muscle atrophy in the animal, wherein the amount is greater than about 1000 mg per day when the compound is ursolic acid, boswellic acid, corosolic acid, betulinic acid, or UA0713.

In one aspect, the animal is an animal. In a further aspect, the animal is a mammal. In a yet further aspect, the mammal is a primate. In a still further aspect, the mammal is a human. In an even further aspect, the human is a patient.

In a further aspect, the animal is a domesticated animal. In a still further aspect, the domesticated animal is a domesticated fish, domesticated crustacean, or domesticated mollusk. In a yet further aspect, the domesticated animal is poultry. In an even further aspect, the poultry is selected from chicken, turkey, duck, and goose. In a still further aspect, the domesticated animal is livestock. In a yet further aspect, the livestock animal is selected from pig, cow, horse, goat, bison, and sheep.

In a further aspect, the effective amount is a therapeutically effective amount. In a still further aspect, the effective amount is a prophylactically effective amount. In a yet further aspect, the muscle disorder is muscle atrophy . In an even further aspect, the muscle disorder is a condition in need of increasing muscle mass. In an even further aspect, the effective amount is greater than about 1000 mg per day when the compound is ursolic acid, beta-boswellic acid, corosolic acid, betulinic acid, or UA0713.

In a further aspect, the pharmaceutical composition is administered following identification of the mammal in need of treatment of muscle atrophy. In a still further aspect, the pharmaceutical composition is administered following identification of the mammal in need of prevention of muscle atrophy. In an even further aspect, the mammal has been diagnosed with a need for treatment of muscle atrophy prior to the administering step.

In a further aspect, the compound is not ursolic acid, beta-boswellic acid, corosolic acid, betulinic acid, or UA0713. In a yet further aspect, the compound is ursolic acid, beta-boswellic acid, corosolic acid, betulinic acid, or UA0713.

In certain aspects, the disclosed pharmaceutical compositions comprise the disclosed compounds (including pharmaceutically acceptable salt(s) thereof) as an active ingredient, a pharmaceutically acceptable carrier, and, optionally, other therapeutic ingredients or adjuvants. The instant compositions include those suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

As used herein, the term “pharmaceutically acceptable salts” refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids. When the compound of the present invention is acidic, its corresponding salt can be conveniently prepared from pharmaceutically acceptable non-toxic bases, including inorganic bases and organic bases. Salts derived from such inorganic bases include aluminum, ammonium, calcium, copper (-ic and -ous), ferric, ferrous, lithium, magnesium, manganese (-ic and -ous), potassium, sodium, zinc and the like salts. Particularly preferred are the ammonium, calcium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, as well as cyclic amines and substituted amines such as naturally occurring and synthesized substituted amines. Other pharmaceutically acceptable organic non-toxic bases from which salts can be formed include ion exchange resins such as, for example, arginine, betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.

As used herein, the term “pharmaceutically acceptable non-toxic acids”, includes inorganic acids, organic acids, and salts prepared therefrom, for example, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid and the like. Preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids.

In practice, the compounds of the invention, or pharmaceutically acceptable salts thereof, of this invention can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous). Thus, the pharmaceutical compositions of the present invention can be presented as discrete units suitable for oral administration such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient. Further, the compositions can be presented as a powder, as granules, as a solution, as a suspension in an aqueous liquid, as a non-aqueous liquid, as an oil-in-water emulsion or as a water-in-oil liquid emulsion. In addition to the common dosage forms set out above, the compounds of the invention, and/or pharmaceutically acceptable salt(s) thereof, can also be administered by controlled release means and/or delivery devices. The compositions can be prepared by any of the methods of pharmacy. In general, such methods include a step of bringing into association the active ingredient with the carrier that constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both. The product can then be conveniently shaped into the desired presentation.

Thus, the pharmaceutical compositions of this invention can include a pharmaceutically acceptable carrier and a compound or a pharmaceutically acceptable salt of the compounds of the invention. The compounds of the invention, or pharmaceutically acceptable salts thereof, can also be included in pharmaceutical compositions in combination with one or more other therapeutically active compounds.

The pharmaceutical carrier employed can be, for example, a solid, liquid, or gas. Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen.

In preparing the compositions for oral dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated by standard aqueous or nonaqueous techniques

A tablet containing the composition of this invention can be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants. Compressed tablets can be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.

The pharmaceutical compositions of the present invention comprise a compound of the invention (or pharmaceutically acceptable salts thereof) as an active ingredient, a pharmaceutically acceptable carrier, and optionally one or more additional therapeutic agents or adjuvants. The instant compositions include compositions suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

Pharmaceutical compositions of the present invention suitable for parenteral administration can be prepared as solutions or suspensions of the active compounds in water. A suitable surfactant can be included such as, for example, hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Further, a preservative can be included to prevent the detrimental growth of microorganisms.

Pharmaceutical compositions of the present invention suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In all cases, the final injectable form must be sterile and must be effectively fluid for easy syringability. The pharmaceutical compositions must be stable under the conditions of manufacture and storage; thus, preferably should 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), vegetable oils, and suitable mixtures thereof.

Pharmaceutical compositions of the present invention can be in a form suitable for topical use such as, for example, an aerosol, cream, ointment, lotion, dusting powder, mouth washes, gargles, and the like. Further, the compositions can be in a form suitable for use in transdermal devices. These formulations can be prepared, utilizing a compound of the invention, or pharmaceutically acceptable salts thereof, via conventional processing methods. As an example, a cream or ointment is prepared by mixing hydrophilic material and water, together with about 5 wt % to about 10 wt % of the compound, to produce a cream or ointment having a desired consistency.

Pharmaceutical compositions of this invention can be in a form suitable for rectal administration wherein the carrier is a solid. It is preferable that the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories can be conveniently formed by first admixing the composition with the softened or melted carrier(s) followed by chilling and shaping in moulds.

In addition to the aforementioned carrier ingredients, the pharmaceutical formulations described above can include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like. Furthermore, other adjuvants can be included to render the formulation isotonic with the blood of the intended recipient. Compositions containing a compound of the invention, and/or pharmaceutically acceptable salts thereof, can also be prepared in powder or liquid concentrate form.

In the treatment conditions which require modulation of cellular function related to muscle growth an appropriate dosage level will generally be about 0.01 to 500 mg per kg patient body weight per day and can be administered in single or multiple doses. Preferably, the dosage level will be about 0.1 to about 250 mg/kg per day; more preferably 0.5 to 100 mg/kg per day. A suitable dosage level can be about 0.01 to 250 mg/kg per day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg per day. Within this range the dosage can be 0.05 to 0.5, 0.5 to 5.0 or 5.0 to 50 mg/kg per day. For oral administration, the compositions are preferably provided in the from of tablets containing 1.0 to 1000 miligrams of the active ingredient, particularly 1.0, 5.0, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900 and 1000 milligrams of the active ingredient for the symptomatic adjustment of the dosage of the patient to be treated. The compound can be administered on a regimen of 1 to 4 times per day, preferably once or twice per day. This dosing regimen can be adjusted to provide the optimal therapeutic response.

It is understood, however, that the specific dose level for any particular patient will depend upon a variety of factors. Such factors include the age, body weight, general health, sex, and diet of the patient. Other factors include the time and route of administration, rate of excretion, drug combination, and the type and severity of the particular disease undergoing therapy.

The present invention is further directed to a method for the manufacture of a medicament for modulating cellular activity related to muscle growth (e.g., treatment of one or more disorders associated with muscle dysfunction or atrophy) in mammals (e.g., humans) comprising combining one or more disclosed compounds, products, or compositions with a pharmaceutically acceptable carrier or diluent. Thus, in one aspect, the invention relates to a method for manufacturing a medicament comprising combining at least one disclosed compound or at least one disclosed product with a pharmaceutically acceptable carrier or diluent.

The disclosed pharmaceutical compositions can further comprise other therapeutically active compounds, which are usually applied in the treatment of the above mentioned pathological conditions.

It is understood that the disclosed compositions can be prepared from the disclosed compounds. It is also understood that the disclosed compositions can be employed in the disclosed methods of using.

E. METHODS OF USING THE COMPOUNDS AND COMPOSITIONS

1. Muscle Atrophy

Muscle atrophy is defined as a decrease in the mass of the muscle; it can be a partial or complete wasting away of muscle. When a muscle atrophies, this leads to muscle weakness, since the ability to exert force is related to mass. Muscle atrophy is a co-morbidity of several common diseases, and patients who have “cachexia” in these disease settings have a poor prognosis.

Muscle atrophy can also be skeletal muscle loss or weakness caused by malnutrition, aging, muscle disuse (such as voluntary and involuntary bedrest, neurologic disease (such as multiple sclerosis, amyotrophic lateral sclerosis, spinal muscular atrophy, critical illness neuropathy, spinal cord injury, peripheral neuropathy, or peripheral nerve injury), injury to the limbs or joints, casting, other post-surgical forms of limb immobilization, or spaceflight), chronic disease (such as cancer, congestive heart failure, chronic pulmonary disease, chronic renal failure, chronic liver disease, diabetes mellitus, glucocorticoid hypersecretion, and chronic infections such as HIV/AIDS or tuberculosis), burn injuries, sepsis, other illnesses requiring mechanical ventiliation, drug-induced muscle disease (such as glucocorticoid-induced myopathy and statin-induced myopathy), genetic diseases that primarily affect skeletal muscle (such as muscular dystrophy, myotonic dystrophy and inclusion body myositis), or autoimmune diseases that affect skeletal muscle (such as polymyositis and dermatomyositis).

There are many diseases and conditions which cause muscle atrophy, including malnutrition, muscle disuse (secondary to voluntary or involuntary bedrest, neurologic disease (including multiple sclerosis, amyotrophic lateral sclerosis, spinal muscular atrophy, critical illness neuropathy, spinal cord injury or peripheral nerve injury), orthopedic injury, casting, and other post-surgical forms of limb immobilization), chronic disease (including cancer, congestive heart failure, chronic pulmonary disease, chronic renal failure, chronic liver disease, diabetes mellitus, Cushing syndrome and chronic infections such as HIV/AIDS or tuberculosis), burns, sepsis, other illnesses requiring mechanical ventilation, drug-induced muscle disease (such as glucorticoid-induced myopathy and statin-induced myopathy), genetic diseases that primarily affect skeletal muscle (such as muscular dystrophy and myotonic dystrophy), autoimmune diseases that affect skeletal muscle (such as polymyositis and dermatomyositis), spaceflight, and aging.

Muscle atrophy occurs by a change in the normal balance between protein synthesis and protein degradation. During atrophy, there is a down-regulation of protein synthesis pathways, and an activation of protein breakdown pathways. The particular protein degradation pathway which seems to be responsible for much of the muscle loss seen in a muscle undergoing atrophy is the ATP-dependent, ubiquitin/proteasome pathway. In this system, particular proteins are targeted for destruction by the ligation of at least four copies of a small peptide called ubiquitin onto a substrate protein. When a substrate is thus “poly-ubiquitinated,” it is targeted for destruction by the proteasome. Particular enzymes in the ubiquitin/proteasome pathway allow ubiquitination to be directed to some proteins but not others—specificity is gained by coupling targeted proteins to an “E3 ubiquitin ligase.” Each E3 ubiquitin ligase binds to a particular set of substrates, causing their ubiquitination. For example, in skeletal muscle, the E3 ubiquitin ligases atrogin-1 and MuRF1 are known to play essential roles protein degradation and muscle atrophy.

Muscle atrophy can be opposed by the signaling pathways which induce muscle hypertrophy, or an increase in muscle size. Therefore one way in which exercise induces an increase in muscle mass is to downregulate the pathways which have the opposite effect. One important rehabilitation tool for muscle atrophy includes the use of functional electrical stimulation to stimulate the muscles which has had limited success in the rehabilitation of paraplegic patients.

Ursolic acid or ursolic acid derivatives can be used as a therapy for illness- and age-related muscle atrophy. It can be useful as a monotherapy or in combination with other strategies that have been considered, such as myostatin inhibition (Zhou, X., et al. (2010) Cell 142(4): 531-543). Given its capacity to reduce adiposity, fasting blood glucose and plasma lipid levels, ursolic acid or ursolic acid derivatives can also be used as a therapy for obesity, metabolic syndrome and type 2 diabetes.

The disclosed compounds can be used as single agents or in combination with one or more other drugs in the treatment, prevention, control, amelioration or reduction of risk of the aforementioned diseases, disorders and conditions for which compounds of formula I or the other drugs have utility, where the combination of drugs together are safer or more effective than either drug alone. The other drug(s) can be administered by a route and in an amount commonly used therefore, contemporaneously or sequentially with a disclosed compound. When a disclosed compound is used contemporaneously with one or more other drugs, a pharmaceutical composition in unit dosage form containing such drugs and the disclosed compound is preferred. However, the combination therapy can also be administered on overlapping schedules. It is also envisioned that the combination of one or more active ingredients and a disclosed compound will be more efficacious than either as a single agent.

Systemic administration of ursolic acid (by parenteral injection or by oral consumption) can be used to promote muscle growth and reduce muscle atrophy in all muscles, including those of the limbs and the diaphragm. Local administration of ursolic acid (by a topical route or localized injection) can be used to promote local muscle growth, as can be required following a localized injury or surgery.

In one aspect, the subject compounds can be coadministered with agents that stimulate insulin signaling, IGF1 signaling and/or muscle growth including insulin, insulin analogs, insulin-like growth factor 1, metformin, thiazoladinediones, sulfonylureas, meglitinides, leptin, dipeptidyl peptidase-4 inhibitors, glucagon-like peptide-1 agonists, tyrosine-protein phosphatase non-receptor type 1 (PTPN1 a.k.a. PTP1B) inhibitors, myostatin signaling inhibitors, clenbuterol, and androgens including testosterone and 5-dehydroepiandrosterone. The derivative can be corosolic acid, UA0713, or other pentacyclic triterpene acids. The ursolic acid, derivative or salt thereof can be administered orally, intramuscularly, intravenously or intraarterially. The ursolic acid, derivative or salt thereof can be substantially pure. The ursolic acid, derivative or salt thereof can be administered at about 10 mg/day to 10 g/day.

In another aspect, the subject compounds can be administered in combination with agents that stimulate insulin signaling, IGF1 signaling and/or muscle growth including insulin, insulin analogs, insulin-like growth factor 1, metformin, thiazoladinediones, sulfonylureas, meglitinides, leptin, dipeptidyl peptidase-4 inhibitors, glucagon-like peptide-1 agonists, tyrosine-protein phosphatase non-receptor type 1 (PTPN1, which is also commonly referred to as PTP1B) inhibitors, myostatin signaling inhibitors, clenbuterol, and androgens including testosterone and 5-dehydroepiandrosterone. The derivative can be corosolic acid, UA0713, or other pentacyclic triterpene acids. The ursolic acid, derivative or salt thereof can be administered orally, intramuscularly, intravenously or intraarterially. The ursolic acid, derivative or salt thereof can be substantially pure. The ursolic acid, derivative or salt thereof can be administered at about 10 mg/day to 10 g/day.

The pharmaceutical compositions and methods of the present invention can further comprise other therapeutically active compounds as noted herein which are usually applied in the treatment of the above mentioned pathological conditions.

2. Treatment Methods

The compounds disclosed herein are useful for treating, preventing, ameliorating, controlling or reducing the risk of a variety of muscle disorders. Examples of such muscle disorders include, but are not limited to, skeletal muscle atrophy secondary to malnutrition, muscle disuse (secondary to voluntary or involuntary bedrest), neurologic disease (including multiple sclerosis, amyotrophic lateral sclerosis, spinal muscular atrophy, critical illness neuropathy, spinal cord injury or peripheral nerve injury), orthopedic injury, casting, and other post-surgical forms of limb immobilization, chronic disease (including cancer, congestive heart failure, chronic pulmonary disease, chronic renal failure, chronic liver disease, diabetes mellitus, Cushing syndrome and chronic infections such as HIV/AIDS or tuberculosis), burns, sepsis, other illnesses requiring mechanical ventiliation, drug-induced muscle disease (such as glucorticoid-induced myopathy and statin-induced myopathy), genetic diseases that primarily affect skeletal muscle (such as muscular dystrophy and myotonic dystrophy), autoimmune diseases that affect skeletal muscle (such as polymyositis and dermatomyositis), spaceflight, or age-related sarcopenia.

Thus, provided is a method for treating or preventing muscle atrophy, comprising: administering to a subject at least one disclosed compound; at least one disclosed pharmaceutical composition; and/or at least one disclosed product in a dosage and amount effective to treat the disorder in the subject.

Also provided is a method for increasing muscle mass, comprising: administering to a subject at least one disclosed compound; at least one disclosed pharmaceutical composition; and/or at least one disclosed product in a dosage and amount effective to treat the disorder in the subject.

a. Preventing or Treating Muscle Atrophy

In one aspect, the invention relates to a method for preventing or treating muscle atrophy in an animal, the method comprising administering to the animal a compound of the formula:

wherein each is an optional covalent bond, and R⁰ is optionally present; wherein n is 0 or 1; wherein R⁰, when present, is hydrogen; wherein R^1a is selected from C1-C6 alkyl and —C(O)ZR¹⁰; wherein R^1b is selected from C1-C6 alkyl, or wherein R^1a and R^1b are optionally covalently bonded and, together with the intermediate carbon, comprise an optionally substituted C3-C5 cycloalkyl or C2-C5 heterocycloalkyl; wherein one of R^2a and R^2b is —OR¹¹, and the other is hydrogen, or R^2a and R^2b together comprise ═O; wherein each of R^3a and R^3b is independently selected from hydrogen, hydroxyl, C1-C6 alkyl, and C1-C6 alkoxyl, provided that R^3a and R^3b are not simultaneously hydroxyl, wherein R³ and R^3b are optionally covalently bonded and, together with the intermediate carbon, comprise an optionally substituted C3-C5 cycloalkyl or C2-C5 heterocycloalkyl; wherein each of R⁴, R⁵, and R⁶ is independently selected from C1-C6 alkyl; wherein R⁷ is selected from C1-C6 alkyl, —CH₂OR¹² and —C(O)ZR¹²; wherein R⁸ is selected from hydrogen and C1-C6 alkyl; wherein each of R^9a and R^9b is independently selected from hydrogen and C1-C6 alkyl, provided that R^9a and R^9b are not simultaneously hydrogen; or wherein R^9a and R^9b are covalently bonded and, along with the intermediate carbon, together comprise C3-C5 cycloalkyl or C2-C5 heterocycloalkyl; wherein R¹⁰ is selected from hydrogen and C1-C6 alkyl; wherein R¹¹ is selected from hydrogen, C1-C6 alkyl, C1-C5 heteroalkyl, C3-C6 cycloalkyl, C4-C6 heterocycloalkyl, phenyl, heteroaryl, and —C(O)R¹⁴; wherein R¹¹, where permitted, is substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; wherein R¹² is selected from hydrogen and optionally substituted organic residue having from 1 to 20 carbons; wherein Z is selected from —O— and —NR¹³—; wherein R¹³ is selected from hydrogen and C1-C4 alkyl; or, wherein Z is N, R¹² and R¹³ are covalently bonded and —NR¹²R¹³ comprises a moiety of the formula:

wherein Y is selected from —O—, —S—, —SO—, —SO₂—, —NH—, —NCH₃—; and wherein R¹⁴ is C1-C6 alkyl and substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof, in an amount effective to prevent or treat muscle atrophy in the animal, wherein the amount is greater than about 1000 mg per day when the compound is ursolic acid, beta-boswellic acid, corosolic acid, betulinic acid, or UA0713.

In a further aspect, the compound administered is a disclosed compound or a product of a disclosed method of making a compound.

In a further aspect, the animal is a mammal. In a yet further aspect, the mammal is a primate. In a still further aspect, the mammal is a human. In an even further aspect, the human is a patient.

In a further aspect. the animal is a domesticated animal. In a still further aspect, the domesticated animal is a domesticated fish, domesticated crustacean, or domesticated mollusk. In a yet further aspect, the domesticated animal is poultry. In an even further aspect, the poultry is selected from chicken, turkey, duck, and goose. In a still further aspect, the domesticated animal is livestock. In a yet further aspect, the livestock animal is selected from pig, cow, horse, goat, bison, and sheep.

In a further aspect, the effective amount is a therapeutically effective amount. In a still further aspect, the effective amount is a prophylactically effective amount. In a yet further aspect, muscle atrophy is prevented by administration of the compound. In an even further aspect, muscle atrophy is treated by administration of the compound. In a still further aspect, the method further comprises the step of identifying the mammal in need of treatment of muscle atrophy. In a yet further aspect, the method further comprises the step of identifying the mammal in a need of prevention of muscle atrophy. In an even further aspect, the mammal has been diagnosed with a need for treatment of muscle atrophy prior to the administering step.

In a further aspect, the compound is not ursolic acid, beta-boswellic acid, corosolic acid, betulinic acid, or UA0713. In a still further aspect, the compound is ursolic acid, beta-boswellic acid, corosolic acid, betulinic acid, or UA0713. In yet further aspect, the compound is not administered as a foodstuff.

b. Increasing Muscle Mass and/or Strength

In one aspect, the invention relates to a method for increasing muscle mass and/or muscular strength in an animal, the method comprising administering to the animal a compound of the formula:

wherein each is an optional covalent bond, and R⁰ is optionally present; wherein n is 0 or 1; wherein R⁰, when present, is hydrogen; wherein R^1a is selected from C1-C6 alkyl and —C(O)ZR¹⁰; wherein R^1b is selected from C1-C6 alkyl, or wherein R^1a and R^1b are optionally covalently bonded and, together with the intermediate carbon, comprise an optionally substituted C3-C5 cycloalkyl or C2-C5 heterocycloalkyl; wherein one of R^2a and R^2b is —OR¹¹, and the other is hydrogen, or R^2a and R^2b together comprise ═O; wherein each of R^3a and R^3b is independently selected from hydrogen, hydroxyl, C1-C6 alkyl, and C1-C6 alkoxyl, provided that R^3a and R^3b are not simultaneously hydroxyl, wherein R^3a and R^3b are optionally covalently bonded and, together with the intermediate carbon, comprise an optionally substituted C3-C5 cycloalkyl or C2-C5 heterocycloalkyl; wherein each of R⁴, R⁵, and R⁶ is independently selected from C1-C6 alkyl; wherein R⁷ is selected from C1-C6 alkyl, —CH₂OR¹² and —C(O)ZR¹²; wherein R⁸ is selected from hydrogen and C1-C6 alkyl; wherein each of R^9a and R^9b is independently selected from hydrogen and C1-C6 alkyl, provided that R^9a and R^9b are not simultaneously hydrogen; or wherein R^9a and R^9b are covalently bonded and, along with the intermediate carbon, together comprise C3-C5 cycloalkyl or C2-C5 heterocycloalkyl; wherein R¹⁰ is selected from hydrogen and C1-C6 alkyl; wherein R¹¹ is selected from hydrogen, C1-C6 alkyl, C1-C5 heteroalkyl, C3-C6 cycloalkyl, C4-C6 heterocycloalkyl, phenyl, heteroaryl, and —C(O)R¹⁴; wherein R¹¹, where permitted, is substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; wherein R¹² is selected from hydrogen and optionally substituted organic residue having from 1 to 20 carbons; wherein Z is selected from —O— and —NR¹³—; wherein R¹³ is selected from hydrogen and C1-C4 alkyl; or, wherein Z is N, R¹² and R¹³ are covalently bonded and —NR¹²R¹³ comprises a moiety of the formula:

wherein Y is selected from —O—, —S—, —SO—, —SO₂—, —NH—, —NCH₃—; and wherein R¹⁴ is C1-C6 alkyl and substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof, in an amount effective to prevent or treat muscle atrophy in the animal, wherein the amount is greater than about 1000 mg per day when the compound is ursolic acid, beta-boswellic acid, corosolic acid, betulinic acid, or UA0713. In a further aspect, the compound administered is a disclosed compound or a product of a disclosed method of making a compound.

In a further aspect, the compound administered is a disclosed compound or a product of a disclosed method of making a compound.

In a further aspect, the animal is a mammal. In a yet further aspect, the mammal is a primate. In a still further aspect, the mammal is a human. In an even further aspect, the human is a patient.

In a further aspect. the animal is a domesticated animal. In a still further aspect, the domesticated animal is a domesticated fish, domesticated crustacean, or domesticated mollusk. In a yet further aspect, the domesticated animal is poultry. In an even further aspect, the poultry is selected from chicken, turkey, duck, and goose. In a still further aspect, the domesticated animal is livestock. In a yet further aspect, the livestock animal is selected from pig, cow, horse, goat, bison, and sheep.

In a further aspect, the effective amount is a therapeutically effective amount. In a still further aspect, the effective amount is a prophylactically effective amount. In a yet further aspect, muscle atrophy is prevented by administration of the compound. In an even further aspect, muscle atrophy is treated by administration of the compound. In a still further aspect, the method further comprises the step of identifying the mammal in need of treatment of muscle atrophy. In a yet further aspect, the method further comprises the step of identifying the mammal in need of prevention of muscle atrophy. In an even further aspect, the mammal has been diagnosed with a need for treatment of muscle atrophy prior to the administering step.

In a further aspect, the compound is not ursolic acid, beta-boswellic acid, corosolic acid, betulinic acid, or UA0713. In a still further aspect, the compound is ursolic acid, beta-boswellic acid, corosolic acid, betulinic acid, or UA0713. In yet further aspect, the compound is not administered as a foodstuff.

c. Enhancing Muscle Formation

In one aspect, the invention relates to a method of enhancing muscle formation in a mammal, the method comprising administering to the mammal a compound of the formula:

wherein R¹¹ is selected from hydrogen, C1-C6 alkyl, C1-C5 heteroalkyl, C3-C6 cycloalkyl, C4-C6 heterocycloalkyl, phenyl, heteroaryl, and —C(O)R¹⁴; wherein R¹¹, where permitted, is substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; wherein R¹² is selected from hydrogen and optionally substituted organic residue having from 1 to 20 carbons; wherein Z is selected from —O— and —NR¹³—; wherein R¹³ is selected from hydrogen and C1-C4 alkyl; or, R¹² and R¹³, when present, are covalently bonded and —NR¹²R¹³ comprises a moiety represented by the formula:

wherein X is selected from O, S, SO, SO₂, NH and NCH₃; and wherein R¹⁴ is C1-C6 alkyl and substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof, in an amount of at least about 200 mg/kg and effective to enhance muscle formation in the mammal.

In a further aspect, the compound administered is a disclosed compound or a product of a disclosed method of making a compound.

In a further aspect, the mammal is a human. In a still further aspect, the human is a patient. In a yet further aspect, administration of the compound prevents muscle atrophy in the mammal. In an even further aspect, administration of the compound treats muscle atrophy in the mammal. In a still further aspect, administration of of the compound increases muscle mass in the mammal. In a yet further aspect, administration of the compound increases muscular strength in the mammal.

In a further aspect, the compound is administered in an effective amount. In a yet further aspect, the effective amount is a therapeutically effective amount. In a still further aspect, the effective amount is a prophylactically effective amount. In a still further aspect, the method further comprises the step of identifying the mammal in need of treatment of muscle atrophy. In a yet further aspect, the method further comprises the step of identifying the mammal in need of prevention of muscle atrophy. In an even further aspect, the mammal has been diagnosed with a need for treatment of muscle atrophy prior to the administering step.

In a further aspect. the mammal is a domesticated animal. In a yet further aspect, domesticated animal is livestock. In a yet further aspect, the livestock animal is selected from pig, cow, horse, goat, bison, and sheep.

In a further aspect, the compound is not ursolic acid. In a still further aspect, the compound is ursolic acid. In a further aspect, the compound is not ursolic acid, beta-boswellic acid, corosolic acid, betulinic acid, or UA0713. In a still further aspect, the compound is ursolic acid, beta-boswellic acid, corosolic acid, betulinic acid, or UA0713. In yet further aspect, the compound is not administered as a foodstuff.

3. Enhancing Tissue Growth In Vitro

In one aspect, the invention relates to a method of enhancing tissue growth in vitro, the method comprising administering to the tissue a compound of the formula:

wherein each is an optional covalent bond, and R⁰ is optionally present; wherein n is 0 or 1; wherein R⁰, when present, is hydrogen; wherein R^1a is selected from C1-C6 alkyl and —C(O)ZR¹⁰; wherein R^1b is selected from C1-C6 alkyl, or wherein R^1a and R^1b are optionally covalently bonded and, together with the intermediate carbon, comprise an optionally substituted C3-C5 cycloalkyl or C2-C5 heterocycloalkyl; wherein one of R^2a and R^2b is —OR¹¹, and the other is hydrogen, or R^2a and R^2b together comprise ═O; wherein each of R^3a and R^3b is independently selected from hydrogen, hydroxyl, C1-C6 alkyl, and C1-C6 alkoxyl, provided that R^3a and R^3b are not simultaneously hydroxyl, wherein R^3a and R^3b are optionally covalently bonded and, together with the intermediate carbon, comprise an optionally substituted C3-C5 cycloalkyl or C2-C5 heterocycloalkyl; wherein each of R⁴, R⁵, and R⁶ is independently selected from C1-C6 alkyl; wherein R⁷ is selected from C1-C6 alkyl, —CH₂OR¹² and —C(O)ZR¹²; wherein R⁸ is selected from hydrogen and C1-C6 alkyl; wherein each of R^9a and R^9b is independently selected from hydrogen and C1-C6 alkyl, provided that R^9a and R^9b are not simultaneously hydrogen; or wherein R^9a and R^9b are covalently bonded and, along with the intermediate carbon, together comprise C3-C5 cycloalkyl or C2-C5 heterocycloalkyl; wherein R¹⁰ is selected from hydrogen and C1-C6 alkyl; wherein R¹¹ is selected from hydrogen, C1-C6 alkyl, C1-C5 heteroalkyl, C3-C6 cycloalkyl, C4-C6 heterocycloalkyl, phenyl, heteroaryl, and —C(O)R¹⁴; wherein R¹¹, where permitted, is substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; wherein R¹² is selected from hydrogen and optionally substituted organic residue having from 1 to 20 carbons; wherein Z is selected from —O— and —NR¹³—; wherein R¹³ is selected from hydrogen and C1-C4 alkyl; or, wherein Z is N, R¹² and R¹³ are covalently bonded and —NR¹²R¹³ comprises a moiety of the formula:

wherein Y is selected from —O—, —S—, —SO—, —SO₂—, —NH—, —NCH₃—; and wherein R¹⁴ is C1-C6 alkyl and substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof, in an amount effective to enhance growth of the tissue.

In a further aspect, the compound administered is a disclosed compound or a product of a disclosed method of making a compound.

In a further aspect, the tissue comprises animal cells. In a still further aspect, the animal cells are muscle cells. In a yet further aspect, the muscle cells are myosatellite cells. In an even further aspect, the myosatellite cells are grown on a scaffold.

4. Manufacture of a Medicament

In one aspect, the invention relates to a method for the manufacture of a medicament for inhibiting muscle atrophy and for increasing muscle mass in a mammal comprising combining a therapeutically effective amount of a disclosed compound or product of a disclosed method with a pharmaceutically acceptable carrier or diluent.

In a further aspect, the medicament is modulates muscle growth. In a still further aspect, the medicament inhibits muscle atrophy. In a yet further aspect, the medicament increases muscle mass. In an even further aspect, the medicament induces skeletal muscle hypertrophy.

5. Methods of Testing for Performance Enhancing Use

In one aspect, the invention relates to a method of testing for performance enhancing use of a ursolic acid analog in an animal, the method comprising: (a) obtaining a biological test sample from the animal; and (b) measuring the amount of a compound of formula:

wherein each is an optional covalent bond, and R⁰ is optionally present; wherein n is 0 or 1; wherein R⁰, when present, is hydrogen; wherein R^1a is selected from C1-C6 alkyl and —C(O)ZR¹⁰; wherein R^1b is selected from C1-C6 alkyl, or wherein R^1a and R^1b are optionally covalently bonded and, together with the intermediate carbon, comprise an optionally substituted C3-C5 cycloalkyl or C2-C5 heterocycloalkyl; wherein one of R^1a and R^2b is —OR¹¹, and the other is hydrogen, or R^2a and R^2b together comprise ═O; wherein each of R^3a and R^3b is independently selected from hydrogen, hydroxyl, C1-C6 alkyl, and C1-C6 alkoxyl, provided that R^3a and R^3b are not simultaneously hydroxyl, wherein R^3a and R^3b are optionally covalently bonded and, together with the intermediate carbon, comprise an optionally substituted C3-C5 cycloalkyl or C2-C5 heterocycloalkyl; wherein each of R⁴, R⁵, and R⁶ is independently selected from C1-C6 alkyl; wherein R⁷ is selected from C1-C6 alkyl, —CH₂OR¹² and —C(O)ZR¹²; wherein R⁸ is selected from hydrogen and C1-C6 alkyl; wherein each of R^9a and R^9b is independently selected from hydrogen and C1-C6 alkyl, provided that R^9a and R^9b are not simultaneously hydrogen; or wherein R^9a and R^9b are covalently bonded and, along with the intermediate carbon, together comprise C3-C5 cycloalkyl or C2-C5 heterocycloalkyl; wherein R¹⁰ is selected from hydrogen and C1-C6 alkyl; wherein R¹¹ is selected from hydrogen, C1-C6 alkyl, C1-C5 heteroalkyl, C3-C6 cycloalkyl, C4-C6 heterocycloalkyl, phenyl, heteroaryl, and —C(O)R¹⁴; wherein R¹¹, where permitted, is substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; wherein R¹² is selected from hydrogen and optionally substituted organic residue having from 1 to 20 carbons; wherein Z is selected from —O— and —NR¹³—; wherein R¹³ is selected from hydrogen and C1-C4 alkyl; or, wherein Z is N, R¹² and R¹³ are covalently bonded and —NR¹²R¹³ comprises a moiety of the formula:

wherein Y is selected from —O—, —S—, —SO—, —SO₂—, —NH—, —NCH₃—; and wherein R¹⁴ is C1-C6 alkyl and substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof, in the test sample to determine whether a superphysiological amount of the compound is present in the biological test sample; wherein the superphysiological amount of the compound in the biological test sample is indicative of performance enhancing use of the compound.

In a further aspect, the superphysiological amount is greater than the peak concentration from administration at a level of about 1000 mg per day. In a still further aspect, the superphysiological amount is the amount that results from administration of the compound at a level greater than 200 mg per day. In a still further aspect, the superphysiological amount is the amount resulting from administration of the compound at a level greater than 200 mg per day. In an even further aspect, the biological test sample is obtained about 12 hours to about 96 hours following administration of the compound.

In a further aspect, the animal is a mammal. In a yet further aspect, the animal is a domesticated animal. In a still further aspect, the mammal is a human.

In a further aspect, the biological sample is blood, urine, saliva, hair, muscle, skin, fat, or breath.

6. Use of Compounds

In one aspect, the invention relates to the use of a compound for increasing muscle mass in a mammal, the compound having a structure represented by a formula:

wherein each is an optional covalent bond, and R⁰ is optionally present; wherein n is 0 or 1; wherein R⁰, when present, is hydrogen; wherein R^1a is selected from C1-C6 alkyl and —C(O)ZR¹⁰; wherein R^1b is selected from C1-C6 alkyl, or wherein R^1a and R^1b are optionally covalently bonded and, together with the intermediate carbon, comprise an optionally substituted C3-C5 cycloalkyl or C2-C5 heterocycloalkyl; wherein one of R^2a and R^2b is —OR¹¹, and the other is hydrogen, or R^2a and R^2b together comprise ═O; wherein each of R^3a and R^3b is independently selected from hydrogen, hydroxyl, C1-C6 alkyl, and C1-C6 alkoxyl, provided that R^3a and R^3b are not simultaneously hydroxyl, wherein R^3a and R^3b are optionally covalently bonded and, together with the intermediate carbon, comprise an optionally substituted C3-C5 cycloalkyl or C2-C5 heterocycloalkyl; wherein each of R⁴, R⁵, and R⁶ is independently selected from C1-C6 alkyl; wherein R⁷ is selected from C1-C6 alkyl, —CH₂OR¹² and —C(O)ZR¹²; wherein R⁸ is selected from hydrogen and C1-C6 alkyl; wherein each of R^9a and R^9b is independently selected from hydrogen and C1-C6 alkyl, provided that R^9a and R^9b are not simultaneously hydrogen; or wherein R^9a and R^9b are covalently bonded and, along with the intermediate carbon, together comprise C3-C5 cycloalkyl or C2-C5 heterocycloalkyl; wherein R¹⁰ is selected from hydrogen and C1-C6 alkyl; wherein R¹¹ is selected from hydrogen, C1-C6 alkyl, C1-C5 heteroalkyl, C3-C6 cycloalkyl, C4-C6 heterocycloalkyl, phenyl, heteroaryl, and —C(O)R¹⁴; wherein R¹¹, where permitted, is substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; wherein R¹² is selected from hydrogen and optionally substituted organic residue having from 1 to 20 carbons; wherein Z is selected from —O— and —NR¹³—; wherein R¹³ is selected from hydrogen and C1-C4 alkyl; or, R¹² and R¹³, when present, are covalently bonded and —NR¹²R¹³ comprises a moiety represented by the formula:

wherein X is selected from O, S, SO, SO₂, NH and NCH₃; and wherein R¹⁴ is C1-C6 alkyl and substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof.

In a further aspect, a use is the treatment of a mammal. In a yet further aspect, the mammal is a human. In a still further aspect, the human is a patient. In a yet further aspect, a use is administration of the compound to a mammal to prevent muscle atrophy. In a yet further aspect, a use is administration of the compound to increase muscular strength in the mammal. In a further aspect. the mammal is a domesticated animal. In a yet further aspect, domesticated animal is livestock. In a yet further aspect, the livestock animal is selected from pig, cow, horse, goat, bison, and sheep.

In a further aspect, a use is administration of the compound in an effective amount. In a yet further aspect, the effective amount is a therapeutically effective amount. In a still further aspect, the effective amount is a prophylactically effective amount. In a still further aspect, prior to use the mammal in need of treatment of muscle atrophy is identified. In a yet further aspect, prior to use the mammal in need of prevention of muscle atrophy is identified. In an even further aspect, the mammal has been diagnosed with a need for treatment of muscle atrophy prior to the administering step.

In a further aspect, the compound is not ursolic acid. In a still further aspect, the compound is ursolic acid. In a further aspect, the compound is not ursolic acid, beta-boswellic acid, corosolic acid, betulinic acid, or UA0713. In a still further aspect, the compound is ursolic acid, beta-boswellic acid, corosolic acid, betulinic acid, or UA0713. In yet further aspect, the compound is not used as a foodstuff. In an even further aspect, the compound is used in an amount is greater than about 1000 mg per day when the compound is ursolic acid, beta-boswellic acid, corosolic acid, betulinic acid, or UA0713.

7. Kits

In one aspect, the invention relates to a kit comprising at least one compound having a structure represented by a formula:

wherein each is an optional covalent bond, and R⁰ is optionally present; wherein n is 0 or 1; wherein R⁰, when present, is hydrogen; wherein R^1a is selected from C1-C6 alkyl and —C(O)ZR¹⁰; wherein R^1b is selected from C1-C6 alkyl; or wherein R^1a and R^1b are covalently bonded and, along with the intermediate carbon, together comprise optionally substituted C3-C5 cycloalkyl or optionally substituted C2-C5 heterocycloalkyl; wherein R^2a and R^2b are independently selected from hydrogen and —OR¹¹, provided that at least one of R^2a and R^2b is —OR¹¹; or wherein R^2a and R^2b together comprise ═O; wherein each of R^3a and R^3b is independently selected from hydrogen, hydroxyl, C1-C6 alkyl, and C1-C6 alkoxyl, provided that R^3a and R^3b are not simultaneously hydroxyl; or wherein R^3a and R^3b are covalently bonded and, along with the intermediate carbon, together comprise optionally substituted C3-C5 cycloalkyl or optionally substituted C2-C5 heterocycloalkyl; wherein each of R⁴, R⁵, and R⁶ is independently selected from C1-C6 alkyl; wherein R⁷ is selected from C1-C6 alkyl, —CH₂OR¹², and —C(O)ZR¹²; wherein R⁸ is selected from hydrogen and C1-C6 alkyl; wherein each of R^9a and R^9b is independently selected from hydrogen and C1-C6 alkyl, provided that R^9a and R^9b are not simultaneously hydrogen; or wherein R^9a and R^9b are covalently bonded and, along with the intermediate carbon, together comprise optionally substituted C3-C5 cycloalkyl or optionally substituted C2-C5 heterocycloalkyl; wherein R¹⁰ is selected from hydrogen and C1-C6 alkyl; wherein each R¹¹ is independently selected from hydrogen, C1-C6 alkyl, C1-C5 heteroalkyl, C3-C6 cycloalkyl, C4-C6 heterocycloalkyl, phenyl, heteroaryl, and —C(O)R¹⁴; wherein R¹¹, where permitted, is substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; wherein R¹² is selected from hydrogen and optionally substituted organic residue having from 1 to 20 carbons; wherein Z is selected from —O— and —NR¹³—; wherein R¹³ is selected from hydrogen and C1-C4 alkyl; or, wherein Z is N, R¹² and R¹³ are covalently bonded and —NR¹²R¹³ comprises a moiety of the formula:

wherein Y is selected from —O—, —S—, —SO—, —SO₂—, —NH—, —NCH₃—; and wherein R¹⁴ is C1-C6 alkyl and substituted with 0-2 groups selected from cyano, acyl, fluoro, chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, hydroxyl, acetoxyl, methoxyl, ethoxyl, propoxyl, and butoxyl; or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof, and one or more of: (a) a protein supplement; (b) an anabolic agent; (c) a catabolic agent; (d) a dietary supplement; (e) at least one agent known to treat a disorder associated with muscle wasting; (f) instructions for treating a disorder associated with cholinergic activity; or (g) instructions for using the compound to increase muscle mass and/or muscular strength.

In a further aspect, the kit ocomprises a disclosed compound or a product of a disclosed method.

In a further aspect, the at least one compound and the at least one agent are co-formulated. In a still further aspect, the at least one compound and the at least one agent are co-packaged.

The kits can also comprise compounds and/or products co-packaged, co-formulated, and/or co-delivered with other components. For example, a drug manufacturer, a drug reseller, a physician, a compounding shop, or a pharmacist can provide a kit comprising a disclosed compound and/or product and another component for delivery to a patient.

It is contemplated that the disclosed kits can be used in connection with the disclosed methods of making, the disclosed methods of using, and/or the disclosed compositions.

8. Non-Medical Uses

Also provided are the uses of the disclosed compounds and products as pharmacological tools in the development and standardization of in vitro and in vivo test systems for the evaluation of the effects of modulators of muscle hypertrophy or inhibitors of muscle atrophy related activity in laboratory animals such as cats, dogs, rabbits, monkeys, rats and mice, as part of the search for new therapeutic agents of increase muscle mass and/or inhibit muscle hypertrophy.

F. EXPERIMENTAL

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric.

Certain materials, reagents and kits were obtained from specific vendors as indicated below, and as appropriate the vendor catalog, part or other number specifying the item are indicated. Vendors indicated below are as follows: “Ambion” is Ambion, a division of Life Technologies Corporation, Austin, Tex., USA; “Applied Biosystems” is Applied Biosystems, a division of Life Technologies Corporation, Carlsbad, Calif., USA; “Boehringer Mannheim” is Boehringer Mannheim Corporatin, Indiapolis, Ind., USA; “CardinalHealth” is Cardinal Health, Inc., Dublin, Ohio, USA; “Cell Signaling” is Cell Signaling Technology, Inc., Beverly, Mass., USA; “Columbus Inst” is Columbus Instruments International, Columbus, Ohio, USA; “Harlan” is Harlan Laboratories, Indianapolis, Ind., USA; “Instrumedics” is Instrumedics, Inc., Richmond, Ill., USA; “Invitrogen” is Invitrogen Corporation, Carlsbad, Calif., USA; “Microm” is the Microm division (Walldorf, Germany) of Thermo Fisher Scientific Inc., Rockford, Ill., USA; “Millipore” is Millipore Corporation, Billerica, Mass., USA; a division of Merck KGaA, Darmstadt, Germany; “Ortho” is Ortho Clinical Diagnostics, Rochester, N.Y., USA; “Pierce” is Pierce Biotechnology, Inc., Milwaukee, Wis., USA, a division of Thermo Fisher Scientific, Inc.; “R&D Systems” is R&D Systems Inc., Minneapolis, Minn., USA; “Roche Diagnostics” is Roche Diagnostics Corporation, Indianapolis, Ind., USA; “Sakura” is Sakura Finetek USA, Inc., Torrance, Calif., USA; “Santa Cruz” is Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA; and, “Sigma” is Sigma-Aldrich Corporation, Saint Louis, Mo., USA.

1. General Methods

a. Human Subject Protocol.

The study referred to herein was approved by the Institutional Review Board at the University of Iowa, and involved seven healthy adults who gave their informed consent before participating. One week prior to the fasting study, subjects made one visit to the Clinical Research Unit (“CRU”) for anthropometric measurements, a dietary interview that established each subject's routine food intake and food preferences, and baseline determinations of blood hemoglobin (“Hb”) Al c turbidimetric immunoinhibition using the BM/Hitachi 911 analyzer (Boehringer Mannheim); plasma triglycerides and plasma free T4 and TSH by electrochemiluminescence immunoassay using the Elecsys® System (Roche Diagnostics); plasma CRP by immuno-turbidimetric assay using the Roche Cobas Integra® high-sensitivity assay (Roche Diagnostics); and, plasma TNF-α levels using the Quantikine® Kit (R&D Systems). To ensure that subjects were eating their routine diet prior to the fasting study, subjects ate only meals prepared by the CRU dietician (based on the dietary interview) for 48 hours before the fasting study. The fasting study began at t =0 hours, when subjects were admitted to the CRU and began fasting. While fasting, subjects remained in the CRU and were encouraged to maintain their routine physical activities. Water was allowed ad libitum, but caloric intake was not permitted. At about 40 hours, a percutaneous biopsy was taken from the vastus lateralis muscle using a Temno® Biopsy Needle (CardinalHealth; Cat #T1420) under ultrasound guidance. Subjects then ate a CRU-prepared mixed meal, and at t=46 hours, a muscle biopsy was taken from the contralateral vastus lateralis muscle. Plasma glucose and insulin levels were measured at t=36, 40, 42 and 46 hours; the Elecsys® system was used to quantitate plasma insulin. Our study protocol of humans with spinal cord injury was described previously (Adams C M, et al. (2011) Muscle Nerve. 43(1):65-75).

b. Microarray Analysis of Human Skeletal Muscle Mrna Levels.

Following harvest, skeletal muscle samples were immediately placed in RNAlater (Ambion) and stored at −80° C. until further use. Total RNA was extracted using TRIzol solution (Invitrogen), and microarray hybridizations were performed at the University of Iowa DNA Facility, as described previously (Lamb J, et al. (2006) Science (New York, N.Y. 313(5795):1929-1935). The loge hybridization signals as shown herein reflect the mean signal intensity of all exon probes specific for an individual mRNA. To determine which human skeletal muscle mRNAs were significantly altered by fasting (P≤0.02), paired t-tests were used to compare fasted and fed loge signals. To determine which mouse skeletal muscle mRNAs were significantly altered by ursolic acid (P≤0.005), unpaired t-tests were used to compare loge signals in mice fed control diet or diet supplemented with ursolic acid. Highly expressed mRNAs were defined as those significantly altered mRNAs that were repressed from or induced to a loge signal>8. These raw microarray data from humans and mice have been deposited in NCBI's Gene Expression Omnibus (“GEO”) and are accessible through GEO Series accession numbers GSE28016 and GSE28017, respectively. Exon array studies of the effects of fasting on mouse skeletal muscle, and the effects of spinal cord injury on human skeletal muscle were described previously (Adams C M, et al. (2011) Muscle & nerve 43(1):65-75; Ebert S M, et al. (2010) Molecular Endocrinology 24(4):790-799).

c. Quantitative Real-Time RT-PCR (QPCR).

TRIzol-extracted mRNA was treated with DNase I using the Turbo DNA free kit (Ambion). qPCR analysis of human mRNA and mouse IGF-I mRNA was performed using TaqMan Gene Expression Assays (Applied Biosystems). First strand cDNA was synthesized from 2 μg of RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Part No. 4368814). The real time PCR contained, in a final volume of 20 μl, 20 ng of reverse transcribed RNA, 1 μl of 20× TaqMan Gene Expression Assay, and 10 μl of TaqMan Fast Universal PCR Master Mix (Applied Biosystems; Part No. 4352042). qPCR was carried out using a 7500 Fast Real-Time PCR System (Applied Biosystems) in 9600 emulation mode. qPCR analysis of mouse atrogin-1 and MuRF1 mRNA levels was performed as previously described (Ebert S M, et al. (2010) Molecular Endocrinology 24(4):790-799). All qPCR reactions were performed in triplicate and the cycle threshold (Ct) values were averaged to give the final results. To analyze the data, the ΔCt method was used, with the level of 36B4 mRNA serving as the invariant control.

d. Mouse Protocols.

Male C57BL/6 mice, ages 6-8 weeks, were obtained from NCI, housed in colony cages with 12 h light/12 h dark cycles, and used for experiments within 3 weeks of their arrival. Unless otherwise indicated, mice were maintained on standard chow (Harlan; Teklad Diet, Formula 7013, NIH-31 Modified Open Formula Mouse/Rat Sterilizable Diet). Metformin (Sigma) was dissolved in 0.9% NaCl at a concentration of 250 mg/ml. Ursolic acid (Enzo Life Sciences) was dissolved in corn oil at a concentration of 200 mg/ml (for i.p. injections); alternatively, the ursolic acid was added directly to standard chow (Harlan; Teklad Diet, Formula 7013) or standard high fat diet (Harlan; Teklad Diet, Formula TD. 93075) as a customized chow. Oleanolic acid (Sigma) was dissolved in corn oil at a concentration of 200 mg/ml. Mice were fasted by removing food, but not water, for 24 hours. Fasting blood glucose levels were obtained from the tail vein with an ACCU-CHEK® Aviva glucose meter (Roche Diagnostics). Unilateral hindlimb muscle denervation was performed by transsecting the sciatic nerve under anesthesia, and was followed by administration of ursolic acid (200 mg/kg) or vehicle alone (corn oil) via i.p injection twice daily for 7 days. Forelimb grip strength was determined using a grip strength meter equipped with a triangular pull bar (Columbus Inst). Each mouse was subjected to 5 consecutive tests to obtain the peak value. Plasma IGF-I and leptin levels were measured by RIA at the Vanderbilt University Hormone Assay Core Facility. Plasma cholesterol, triglyceride, creatinine, bilirubin and ALT were measured using the VITROS® 350 Chemistry System (Ortho). All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Iowa.

e. Histological Analysis.

Following harvest, tissues were immediately placed in isopentane that had been chilled to −160° C. with liquid N₂. Muscles were embedded in tissue freezing medium, and 10 μm sections from the mid-belly were prepared using a Microm HM 505 E cryostat equipped with a CryoJane sectioning system (Instrumedics). Adipose tissue was fixed in 10% neutral buffered formalin, embedded in paraffin, and then 4 μm sections were prepared using a Microm HM355 S motorized microtome (Microm). Hematoxylin and eosin stains were performed using a DRS-601 automatic slide stainer (Sakura), and examined on an Olympus IX-71 microscope equipped with a DP-70 camera. Image analysis was performed using ImageJ software (public domain, available from the National Institutes of Health, USA). Muscle fiber diameter was measured using the lesser diameter method, as described elsewhere (Dubowitz V, et al. (2007) Muscle biopsy: a practical approach (Saunders Elsevier, Philadelphia) 3rd Ed pp XIII, 611 s).

f. Analysis of IGF-I and Insulin-Mediated Protein Phosphorylation.

Mouse quadriceps muscles were snap frozen in liquid N2, and Triton-X 100 soluble protein extracts were prepared as described previously (Ebert S M, et al. (2010) Molecular endocrinology 24(4):790-799). Mouse C2C12 myoblasts were obtained from American Type Culture Collection (“ATCC”), and maintained in Dulbecco's modified Eagle's medium (DMEM; ATCC #30-2002) containing antibiotics (100 units/ml penicillin, 100 μg/ml streptomycin sulfate) and 10% (v/v) fetal bovine serum (FBS). On day 0, myotubes were set-up in 6-well plates at a density of 2.5×10⁵ cells/well. On day 2, differentiation into myotubes was induced by replacing 10% FBS with 2% horse serum. On day 7, myotubes were serum-starved by washing 2 times with phosphate buffered saline, and then adding fresh serum-free media. After 16 hours of serum-starvation, 10 μM ursolic acid (from a 10 mM stock prepared in DMSO), or an equal volume of DMSO, with or without 10 nM mouse IGF-I (Sigma; Cat. No. I8779) or 10 nM bovine insulin (Sigma; Cat. No. I6634) was directly added to the media. For analysis of Akt, S6K, ERK and FoxO phosphorylation, myotubes were incubated in the presence or absence of ursolic acid, IGF-I and/or insulin for 20 min, and then harvested into SDS lysis buffer (10 mM Tris-HCl, pH 7.6, 100 mM NaCl, 1% (w/v) SDS, 1 μg/ml pepstatin A, 2 μg/ml aprotonin, 10 μg/ml leupeptin, 200 μM phenylmethylsulfonyl fluoride and a 1:100 dilution of phosphatase inhibitor cocktail 3 (Sigma). An aliquot of each muscle extract or cell lysate was mixed with 0.25 volume of sample buffer (250 mM Tris-HCl, pH 6.8, 10% SDS, 25% glycerol, 0.2% (w/v) bromophenol blue, and 5% (w/v) 2-mercaptoethanol) and heated for 5 min at 95° C., whereas a separate aliquot was used to determine protein concentration by the BCA kit (Pierce). Samples (25 μg) were subjected to 8% SDS-PAGE, then transferred to Hybond-C extra nitrocellulose filters (Millipore). Immunoblots were performed at 4° C. for 16 h using a 1:2000 dilution of antibodies detecting total Akt, phospho-Akt(Ser473), total S6K, phospho-S6K(T421/S424), total ERK1/2, phospho-ERK(T202/Y204), FoxO3a, or phospho-FoxO1(T24)/FoxO3a(T32) (Cell Signaling). For analysis of IGF-1 receptor or insulin receptor phosphorylation, myotubes were incubated in the presence or absence of ursolic acid, IGF-I and/or insulin for 2 min, and then harvested into RIPA buffer (10 mM Tris-HCL, pH 7.4, 150 mM NaCl, 0.1% (w/v) SDS, 1% (w/v) Triton X-100, 1% Na deoxycholate, 5 mM EDTA, 1 mM NaF, 1 mM Na orthovanadate, 1 μg/ml pepstatin A, 2 μg/ml aprotonin, 10 μug/ml leupeptin, 200 μM phenylmethylsulfonyl fluoride, 1:100 dilution of phosphatase inhibitor cocktail 2 (Sigma) and a 1:100 dilution of phosphatase inhibitor cocktail 3 (Sigma). The protein concentration was measured using the BCA kit, after which the extract was diluted to a concentration of 1 mg/ml in RIPA buffer (final volume 500 μl). Then 2 μg anti-IGF-1 receptor β antibody (Cell Signaling) or 2 μg anti-insulin receptor β antibody (Santa Cruz) was added with 50 μl protein G plus Sepharose beads (Santa Cruz), and then the samples were rotated at 4° C. for 16 h. Immunoprecipitates were washed three times for 20 min with 1 ml RIPA buffer and then mixed with 100 μl sample buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 5% glycerol, 0.04% (w/v) bromophenol blue and 5% (w/v) 2-mercaptoethanol), then boiled for 5 min. Immunoprecipitates were subjected to 8% SDS-PAGE. For analysis of total IGF-1 receptor, phospho-insulin receptor and total insulin receptor, proteins were transferred to Hybond-C extra nitrocellulose filters (Millipore). For analysis of phospho-IGF-1 receptor, proteins were transferred to PVDF membranes (Bio-Rad). Immunoblots were performed at room temperature using a 1:2000 dilution of anti-IGF-1 receptor β antibody, 1:5000 dilution of mouse anti-phospho-tyrosine 4G10 monoclonal antibody (Millipore), a 1:2000 dilution of anti-insulin receptor β, or 1:2000 dilution of anti-phospho-insulin receptor β (Y1162/1163) (Santa Cruz).

g. PTP1B Inhibition Via RNA Interference.

The plasmidspCMV-miR-PTP1B #1 and pCMV-miR-PTP1B #2 were generated by ligating PTPN1-specific oligonucleotide duplexes (Invitrogen) into the pcDNA6.2GW/EmGFP miR plasmid (Invitrogen), which contains a CMV promoter driving co-cistronic expression of engineered pre-miRNAs and EmGFP. pCMV-miR-control encodes a non-targeting pre-miRNA hairpin sequence (miR-neg control; Invitrogen) in pcDNA6.2GW/EmGFP miR plasmid. Male C57BL/6 mice were obtained from NCI at ages 6-8 weeks, and used for experiments within 3 weeks of their arrival. Electroporation of mouse tibialis anterior muscles and isolation of skeletal muscle RNA was performed as described previously (Ebert S M, et al. (2010) Molecular endocrinology 24(4):790-799). First strand cDNA was synthesized in a 20 μl reaction that contained 2 μg of RNA, random hexamer primers and components of the High Capacity cDNA reverse transcription kit (Applied Biosystems). qPCR analysis of PTPN1 mRNA levels was performed using a Taqman expression assay as described previously (Ebert S M, et al. (2010) Molecular endocrinology 24(4):790-799). qPCR was carried out using a 7500 Fast Real-Time PCR System (Applied Biosystems). All qPCR reactions were performed in triplicate and the cycle threshold (Ct) values were averaged to give the final results. Fold changes were determined by the ΔCt method, with level of 36B4 mRNA serving as the invariant control. Skeletal muscle sections were prepared and transfected (EmGFP-positive) muscle fibers were identified and measured as described previously (Ebert S M, et al. (2010) Molecular endocrinology 24(4):790-799).

h. Measurement of Serum Ursolic Acid Levels.

Ursolic acid is extracted from serum using a 10:1 mixture of hexane:propanol (recovery>90%), and then conjugated via its carboxylic acid group to 2-(2,3-naphthalimino)ethyl trifluoromethanesulfonate (Invitrogen; Ne-OTf), a moiety that enhances TUV and fluorescence detection. Derivatized samples are then analyzed on a Waters Acquity UPLC equipped with a 100×2.1 mm C18 HSS column with 1.8 μm beads (Waters Part No. 186003533) and a TUV detector.

2. Identification of Therapeutics to Treat Muscle Atrophy

Skeletal muscle atrophy is common and debilitating condition that lacks a pharmacologic therapy. To identify and develop new therapeutic approaches to this pathophysiological condition (FIG. 1), an approach using gene expression signatures to connect small molecules, genes, and disease was used. Briefly, 63 mRNAs were identified that were regulated by fasting in both human and mouse muscle, and 29 mRNAs that were regulated by both fasting and spinal cord injury in human muscle. These two unbiased mRNA expression signatures of muscle atrophy were used to query the Connectivity Map, an algorithm that allows gene signature datasets to be used to find relationships between small molecules, genes, and disease.

Three complimentary studies to characterize global atrophy-associated changes in skeletal muscle mRNA levels in humans and mice were carried. These three studies determined the effects of: A) fasting on human skeletal muscle mRNA levels, B) spinal cord injury (“SCI”) on human skeletal mRNA levels (Adams C M, et al. (2011) Muscle & nerve 43(1):65-75) and C) fasting on mouse skeletal muscle mRNA levels (Ebert S M, et al. (2010) Molecular endocrinology 24(4):790-799). In each study, exon expression arrays were used that quantitated levels of >16,000 mRNAs. Although there were many significant changes in each study, analysis focused on mRNAs whose levels were similarly altered in at least two atrophy models. Thus, by comparing the effects of fasting on human and mouse skeletal muscle, there were two sets of mRNAs identified: a) 31 mRNAs that were increased by fasting in both species, and b) 32 mRNAs that were decreased by fasting in both species. These evolutionarily conserved, fasting-regulated skeletal muscle mRNAs were termed “atrophy signature-1.” Next, the effects of fasting and SCI on human skeletal muscle were determined and two sets of mRNAs were identitied: a) 18 mRNAs that were increased by fasting and SCI, and b) 17 mRNAs that were decreased by fasting and SCI. This second group of mRNAs was termed “atrophy signature-2.” Almost all of the mRNAs in atrophy signatures-1 and -2 have previously uncharacterized roles in normal or atrophied skeletal muscle. It was next hypothesized that pharmacologic compounds whose effects on cellular mRNA levels were opposite to atrophy signatures-1 and -2 might inhibit skeletal muscle atrophy. To identify candidate compounds, the Connectivity Map (Lamb J, et al. (2006) Science (New York, N.Y. 313(5795):1929-1935) was used to compare atrophy signatures-1 and -2 to mRNA expression signatures of >1300 bioactive small molecules. These results identified several predicted inhibitors of human skeletal muscle atrophy, including ursolic acid. As a proof-of-concept of the utility of atrophy signatures-1 and -2 described herein, the effects of ursolic acid were assessed in mice, and surprisingly it was discovered ursolic acid inhibited muscle atrophy and promoted muscle hypertrophy.

3. Effects of Fasting on Skeletal Muscle mRNA Expression in Humans.

Prolonged fasting induces muscle atrophy, but its effects on global mRNA expression in human skeletal muscle were not known heretofore. In order to determine the relationship between global mRNA expression and human skeletal muscle status, seven healthy adult human volunteers (3 male and 4 female) with ages ranging from 25 to 69 years (mean=46 years) were studied. The overall study design is shown in FIG. 2A. The mean body mass index of these subjects (±SEM) was 25±1. Their mean weight was 69.4±4.8 kg. Baseline circulating levels of hemoglobin A1c (HbA1c), triglycerides (TG), thyroid-stimulating hormone (TSH), free thyroxine (free T4), C-reactive protein (CRP) and tumor necrosis factor-α (TNF-α) were within normal limits (FIG. 2A). The table (FIG. 2A, insert) shows baseline circulating metabolic and inflammatory markers. The graph shows plasma glucose and insulin levels (FIG. 2A). Data are means±SEM from the seven study subjects. In some cases, the error bars are too small to see. While staying in the University of Iowa Clinical Research Unit, the subjects fasted for 40 h by forgoing food but not water. The mean weight loss during the fast was 1.7±0.1 kg (3±0% of the initial body weight).

After the 40 h fast, a muscle biopsy was obtained from the subjects' vastus lateralis (VL) muscle. Immediately after the muscle biopsy, the subjects ate a mixed meal. Five hours later (six hours after the first biopsy), a second muscle biopsy from their contralateral VL muscle. Thus, each subject had a muscle biopsy under fasting and nonfasting conditions. As expected, plasma glucose and insulin levels were low at the end of the 40 h fast, rose after the meal, and returned to baseline by the time of the second biopsy (FIG. 2A). These data indicate comparable levels of plasma glucose and insulin at the times of the first (fasting) and second (nonfasting) muscle biopsies.

To determine the effect of fasting on skeletal muscle mRNA expression, RNA was isolated from the paired muscle biopsies and then analyzed it with exon expression arrays. Using P≤0.02 (by paired t-test) as criteria for statistical significance, it was found that 281 mRNAs were higher in the fasting state and 277 were lower (out of >17,000 mRNAs measured; see FIG. 2B). A complete list of these fasting-responsive mRNAs is shown below in Table 1 (“Change” is the mean loge change or difference between fasting and fed states). The data in Table 1 is for all mRNAs in this study whose levels were increased or decreased by fasting (P≤0.02 by paired t-test).

Representative fasting-responsive human skeletal muscle mRNAs, and the effect of fasting on their log2 hybridization signals, as assessed by Affymetrix Human Exon 1.0 ST arrays are shown in FIG. 2B. In each subject, the fasting signal was normalized to the nonfasting signal from the same subject. Data are means±SEM from 7 subjects. P≤0.02 by paired t-test for all mRNAs shown. The complete set of 458 fasting-responsive mRNAs is shown in Table 1. Most of the differentially expressed mRNAs identified as altered by fasting surprisingly did not have previously known roles in muscle atrophy. However, fasting increased several mRNAs that encode proteins with known roles in catabolic processes such as fat oxidation, reverse cholesterol transport, thermogenesis, inhibition of protein synthesis, autophagy, ubiquitin-mediated proteolysis, glutamine transport and heme catabolism (FIG. 2B). Of these, atrogin-1, MuRF1 and ZFAND5 mRNAs encode proteins known to be required for skeletal muscle atrophy in mice (Bodine S C, et al. (2001) Science (New York, N.Y. 294(5547):1704-1708; Hishiya A, et al. (2006) The EMBO journal 25(3):554-564). Conversely, fasting significantly decreased several mRNAs encoding proteins with known roles in anabolic processes such as glycogen synthesis, lipid synthesis and uptake, polyamine synthesis, iron uptake, angiogenesis, and mitochondrial biogenesis (FIG. 2B). Of these, PGC-1α mRNA encodes a protein that inhibits atrophy-associated gene expression and skeletal muscle atrophy in mice (Sandri M, et al. (2006) Proceedings of the National Academy of Sciences of the United States of America 103(44):16260-16265).

The results were further validated using qPCR to to analyze RNA from paired fed and fasted skeletal muscle biopsy samples obtained from seven healthy human subjects (see FIG. 3; data are means±SEM; *P≤0.01 by paired t-test.). In each subject, the fasting mRNA level was normalized to the nonfasting level, which was set at 1. The mRNA encoding myostatin (MSTN) is a control transcript whose level was not altered by fasting, as assessed by exon expression arrays. Taken together, these data established an mRNA expression signature of fasting in human skeletal muscle.

TABLE 1
FASTING-RESPONSIVE HUMAN MRNAS.
	Change
Affymetrix		Gene	Accession	(Fasting −
ID	mRNA	Assignment	No.	Fed)	SEM	P
3062082	PDK4	NM_002612 //	NM_002612	2.15	0.34	0.000
		PDK4 // pyruvate
		dehydrogenase
		kinase, isozyme 4 //
		7q21.3 // 5166
2319340	SLC25A33	NM_032315 //	NM_032315	1.42	0.41	0.007
		SLC25A33 // solute
		carrier family 25,
		member 33 //
		1p36.22 // 84275
3165957	IFNK	NM_020124 //	NM_020124	0.96	0.28	0.007
		IFNK // interferon,
		kappa // — //
		56832 ///
		ENST00000276943 // IF
3424158	MYF6	NM_002469 //	NM_002469	0.95	0.12	0.000
		MYF6 // myogenic
		factor 6 (herculin) //
		12q21 // 4618 ///
		ENST00000
3422144	LGR5	NM_003667 //	NM_003667	0.88	0.12	0.000
		LGR5 // leucine-
		rich repeat-
		containing G
		protein-coupled
		receptor 5
2356115	TXNIP	NM_006472 //	NM_006472	0.85	0.22	0.004
		TXNIP //
		thioredoxin
		interacting
		protein // 1q21.1 //
		10628 /// ENS
3233605	PFKFB3	NM_004566 //	NM_004566	0.84	0.18	0.002
		PFKFB3 // 6-
		phosphofructo-2-
		kinase/fructose-2,6-
		biphosphatase 3 //
3151607	FBXO32	NM_058229 //	NM_058229	0.82	0.19	0.002
		FBXO32 // F-box
		protein 32 //
		8q24.13 // 114907 ///
		NM_148177 // FB
2745547	GAB1	NM_207123 //	NM_207123	0.71	0.08	0.000
		GAB1 // GRB2-
		associated binding
		protein 1 // 4q31.21 //
		2549 /// NM
3173479	FOXD4L3	NM_199135 //	NM_199135	0.68	0.25	0.017
		FOXD4L3 //
		forkhead box D4-
		like 3 // 9q13 //
		286380 ///
		NM_012184/
3199500	CER1	NM_005454 //	NM_005454	0.64	0.24	0.019
		CER1 // cerberus 1,
		cysteine knot
		superfamily,
		homolog
		(Xenopus lae
3444309	TAS2R9	NM_023917 //	NM_023917	0.63	0.22	0.015
		TAS2R9 // taste
		receptor, type 2,
		member 9 // 12p13 //
		50835 /// EN
3452323	SLC38A2	NM_018976 //	NM_018976	0.62	0.13	0.001
		SLC38A2 // solute
		carrier family 38,
		member 2 // 12q //
		54407 /// E
3381843	UCP3	NM_003356 //	NM_003356	0.59	0.04	0.000
		UCP3 // uncoupling
		protein 3
		(mitochondrial,
		proton carrier) //
		11q
3147508	KLF10	NM_005655 //	NM_005655	0.58	0.11	0.001
		KLF10 // Kruppel-
		like factor 10 //
		8q22.2 // 7071 ///
		NM_001032282
3982534	LPAR4	NM_005296 //	NM_005296	0.57	0.17	0.008
		LPAR4 //
		lysophosphatidic
		acid receptor 4 //
		Xq13-q21.1 // 2846 ///
3384321	RAB30	NM_014488 //	NM_014488	0.56	0.21	0.019
		RAB30 // RAB30,
		member RAS
		oncogene family //
		11q12-q14 // 27314 //
3256192	C10orf116	NM_006829 //	NM_006829	0.55	0.19	0.013
		C10orf116 //
		chromosome 10
		open reading frame
		116 // 10q23.2 //
		109
2705690	GHSR	NM_198407 //	NM_198407	0.54	0.20	0.016
		GHSR // growth
		hormone
		secretagogue
		receptor // 3q26.31 //
		2693 ///
3326938	LOC100130104	AF274942 //	AF274942	0.53	0.16	0.009
		LOC100130104 //
		PNAS-17 // 11p13 //
		100130104
2318656	PER3	NM_016831 //	NM_016831	0.52	0.16	0.009
		PER3 // period
		homolog 3
		(Drosophila) //
		1p36.23 // 8863 ///
		ENST00
3209623	ZFAND5	NM_001102420 //	NM_001102420	0.51	0.13	0.005
		ZFAND5 // zinc
		finger, AN1-type
		domain 5 // 9q13-
		q21 // 7763 ///
3741300	OR1D4	NM_003552 //	NM_003552	0.50	0.19	0.019
		OR1D4 // olfactory
		receptor, family 1,
		subfamily D,
		member 4 // 17p
2899176	HIST1H2BD	NM_138720 //	NM_138720	0.49	0.16	0.010
		HIST1H2BD //
		histone cluster 1,
		H2bd // 6p21.3 //
		3017 /// NM_02106
3439256	RPS11	ENST00000270625 //	ENST00000270625	0.49	0.11	0.002
		RPS11 //
		ribosomal protein
		S11 // 19q13.3 //
		6205 /// BC10002
2973232	KIAA0408	NM_014702 //	NM_014702	0.49	0.14	0.006
		KIAA0408 //
		KIAA0408 //
		6q22.33 // 9729 ///
		NM_001012279 //
		C6orf17
3291151	RHOBTB1	NM_014836 //	NM_014836	0.48	0.09	0.001
		RHOBTB1 // Rho-
		related BTB
		domain containing
		1 // 10q21.2 // 9886/
2358136	C1orf51	BC027999 //	BC027999	0.48	0.17	0.016
		C1orf51 //
		chromosome 1
		open reading frame
		51 // 1q21.2 //
		148523 //
3948936		—	—	0.47	0.18	0.020
3944129	HMOX1	NM_002133 //	NM_002133	0.46	0.13	0.006
		HMOX1 // heme
		oxygenase
		(decycling) 1 //
		22q12|22q13.1 //
		3162 ///
2968652	SESN1	NM_014454 //	NM_014454	0.46	0.12	0.004
		SESN1 // sestrin 1 //
		6q21 // 27244 ///
		ENST00000302071 //
		SESN1 //
2951881	PXT1	NM_152990 //	NM_152990	0.45	0.14	0.008
		PXT1 //
		peroxisomal, testis
		specific 1 //
		6p21.31 // 222659 ///
		ENS
2819747	POLR3G	NM_006467 //	NM_006467	0.45	0.13	0.007
		POLR3G //
		polymerase (RNA)
		III (DNA directed)
		polypeptide G
		(32 kD)
2957384	GSTA2	NM_000846 //	NM_000846	0.44	0.10	0.002
		GSTA2 //
		glutathione S-
		transferase A2 //
		6p12.1 // 2939 ///
		NM_1536
4014387	RPSA	NM_002295 //	NM_002295	0.44	0.16	0.018
		RPSA // ribosomal
		protein SA //
		3p22.2 // 3921 ///
		NM_001012321 //
3021158	C7orf58	NM_024913 //	NM_024913	0.44	0.07	0.000
		C7orf58 //
		chromosome 7
		open reading frame
		58 // 7q31.31 //
		79974/
2976155	OLIG3	NM_175747 //	NM_175747	0.44	0.12	0.006
		OLIG3 //
		oligodendrocyte
		transcription factor
		3 // 6q23.3 //
		167826
3261886	C10orf26	NM_017787 //	NM_017787	0.44	0.17	0.019
		C10orf26 //
		chromosome 10
		open reading frame
		26 // 10q24.32 //
		5483
2489169		—	—	0.42	0.12	0.006
2790062	TMEM154	NM_152680 //	NM_152680	0.42	0.14	0.012
		TMEM154 //
		transmembrane
		protein 154 //
		4q31.3 // 201799 ///
		ENST00
3792656	CCDC102B	NM_024781 //	NM_024781	0.42	0.12	0.007
		CCDC102B //
		coiled-coil domain
		containing 102B //
		18q22.1 // 79839
3554282	INF2	NM_022489 //	NM_022489	0.41	0.14	0.012
		INF2 // inverted
		formin, FH2 and
		WH2 domain
		containing //
		14q32.33
2614142	NR1D2	NM_005126 //	NM_005126	0.39	0.15	0.019
		NR1D2 // nuclear
		receptor subfamily
		1, group D, member
		2 // 3p24.2
3404636	GABARAPL1	NM_031412 //	NM_031412	0.39	0.10	0.004
		GABARAPL1 //
		GABA(A) receptor-
		associated protein
		like 1 // 12p13.2
3063856	tcag7.1177	ENST00000292369 //	ENST00000292369	0.39	0.09	0.003
		tcag7.1177 //
		opposite strand
		transcription unit to
		STAG3 //
3461981	TSPAN8	NM_004616 //	NM_004616	0.39	0.14	0.015
		TSPAN8 //
		tetraspanin 8 //
		12q14.1-q21.1 //
		7103 ///
		ENST0000039333
2908154	C6orf206	BC029519 //	BC029519	0.39	0.09	0.003
		C6orf206 //
		chromosome 6
		open reading frame
		206 // 6p21.1 //
		221421
3415046	FLJ33996	AK091315 //	AK091315	0.39	0.15	0.019
		FLJ33996 //
		hypothetical protein
		FLJ33996 //
		12q13.13 // 283401 ///
3326400	CAT	NM_001752 //	NM_001752	0.39	0.09	0.003
		CAT //catalase //
		11p13 // 847 ///
		ENST00000241052 //
		CAT // catal
2390322	OR2M5	NM_001004690 //	NM_001004690	0.38	0.12	0.011
		OR2M5 // olfactory
		receptor, family 2,
		subfamily M,
		member 5 //
2402536	TRIM63	NM_032588 //	NM_032588	0.38	0.12	0.009
		TRIM63 // tripartite
		motif-containing
		63 // 1p34-p33 //
		84676 /// E
2976768	CITED2	NM_006079 //	NM_006079	0.37	0.10	0.005
		CITED2 //
		Cbp/p300-
		interacting
		transactivator, with
		Glu/Asp-rich ca
3218528	ABCA1	NM_005502 //	NM_005502	0.37	0.14	0.016
		ABCA1 // ATP-
		binding cassette,
		sub-family A
		(ABC1), member 1 //
		9q3
3377861	DKFZp761E198	NM_138368 //	NM_138368	0.37	0.06	0.000
		DKFZp761E198 //
		DKFZp761E198
		protein // 11q13.1 //
		91056 /// BC1091
2961347	FILIP1	NM_015687 //	NM_015687	0.37	0.10	0.005
		FILIP1 // filamin A
		interacting protein
		1 // 6q14.1 //
		27145 /// EN
3097580	C8orf22	NM_001007176 //	NM_001007176	0.37	0.08	0.002
		C8orf22 //
		chromosome 8
		open reading frame
		22 // 8q11 //
		492307
3755655	FBXL20	NM_032875 //	NM_032875	0.35	0.08	0.002
		FBXL20 // F-box
		and leucine-rich
		repeat protein 20 //
		17q12 // 8496
3057505	CCL26	NM_006072 //	NM_006072	0.35	0.12	0.012
		CCL26 //
		chemokine (C-C
		motif) ligand 26 //
		7q11.23 // 10344 ///
		EN
3307795	C10orf118	NM_018017 //	NM_018017	0.35	0.13	0.020
		C10orf118 //
		chromosome 10
		open reading frame
		118 // 10q25.3 //
		550
3654699	NUPR1	NM_001042483 //	NM_001042483	0.35	0.10	0.007
		NUPR1 // nuclear
		protein 1 //
		16p11.2 // 26471 ///
		NM_012385 //
3778252	ANKRD12	NM_015208 //	NM_015208	0.34	0.08	0.002
		ANKRD12 //
		ankyrin repeat
		domain 12 //
		18p11.22 // 23253 ///
		NM_001
2662560	C3orf24	NM_173472 //	NM_173472	0.34	0.08	0.002
		C3orf24 //
		chromosome 3
		open reading frame
		24 // 3p25.3 //
		115795/
3896370	RP5-1022P6.2	NM_019593 //	NM_019593	0.34	0.10	0.007
		RP5-1022P6.2 //
		hypothetical protein
		KIAA1434 //
		20p12.3 // 56261/
3389566	KBTBD3	NM_198439 //	NM_198439	0.34	0.08	0.003
		KBTBD3 // kelch
		repeat and BTB
		(POZ) domain
		containing 3 //
		11q22.3
3247818	FAM133B	NM_152789 //	NM_152789	0.34	0.11	0.010
		FAM133B // family
		with sequence
		similarity 133,
		member B // 7q21.2
2457988	ZNF706	AF275802 //	AF275802	0.34	0.12	0.016
		ZNF706 // zinc
		finger protein 706 //
		8q22.3 // 51123 ///
		BC015925 //
3525234	IRS2	NM_003749 //	NM_003749	0.34	0.09	0.004
		IRS2 // insulin
		receptor substrate
		2 // 13q34 // 8660 ///
		ENST00000
2730281	ODAM	NM_017855 //	NM_017855	0.34	0.12	0.016
		ODAM //
		odontogenic,
		ameloblast
		asssociated //
		4q13.3 // 54959 ///
3768969	ABCA5	NM_018672 //	NM_018672	0.33	0.10	0.008
		ABCA5 // ATP-
		binding cassette,
		sub-family A
		(ABC1), member 5 //
		17q
3687494	MAPK3	NM_001040056 //	NM_001040056	0.33	0.09	0.004
		MAPK3 // mitogen-
		activated protein
		kinase 3 // 16p11.2 //
		5595/
3405396	CREBL2	NM_001310 //	NM_001310	0.33	0.07	0.002
		CREBL2 // cAMP
		responsive element
		binding protein-like
		2 // 12p13/
3647504	PMM2	NM_000303 //	NM_000303	0.33	0.10	0.008
		PMM2 //
		phosphomannomutase
		2 // 16p13.3-
		p13.2 // 5373 ///
		ENST00000
3392840	BUD13	NM_032725 //	NM_032725	0.33	0.07	0.002
		BUD13 // BUD13
		homolog
		(S. cerevisiae) //
		11q23.3 // 84811 ///
		ENST
3453837	TUBA1A	NM_006009 //	NM_006009	0.33	0.07	0.002
		TUBA1A // tubulin,
		alpha 1a // 12q12-
		q14.3 // 7846 ///
		ENST00000301
2409310	ELOVL1	NM_022821 //	NM_022821	0.32	0.09	0.005
		ELOVL1 //
		elongation of very
		long chain fatty
		acids (FEN1/Elo2,
		SUR
3837707	ZNF114	NM_153608 //	NM_153608	0.31	0.09	0.007
		ZNF114 // zinc
		finger protein 114 //
		19q13.32 // 163071 ///
		ENST000
3504434	XPO4	NM_022459 //	NM_022459	0.31	0.10	0.009
		XPO4 // exportin 4 //
		13q11 // 64328 ///
		ENST00000255305 //
		XPO4 //
2431877		—	—	0.31	0.11	0.017
3837836	PSCD2	NM_017457 //	NM_017457	0.31	0.05	0.000
		PSCD2 // pleckstrin
		homology, Sec7
		and coiled-coil
		domains 2 (cytoh
3869396	ZNF432	NM_014650 //	NM_014650	0.31	0.09	0.006
		ZNF432 // zinc
		finger protein 432 //
		19q13.33 // 9668 ///
		ENST00000
3981120	OGT	NM_181672 //	NM_181672	0.31	0.10	0.013
		OGT // O-linked N-
		acetylglucosamine
		(GlcNAc)
		transferase (UDP-
		N-ace
2622607	SLC38A3	NM_006841 //	NM_006841	0.30	0.11	0.016
		SLC38A3 // solute
		carrier family 38,
		member 3 // 3p21.3 //
		10991 //
3978812	FOXR2	NM_198451 //	NM_198451	0.30	0.09	0.008
		FOXR2 // forkhead
		box R2 // Xp11.21 //
		139628 ///
		ENST00000339140/
3571904	NPC2	NM_006432 //	NM_006432	0.30	0.10	0.011
		NPC2 // Niemann-
		Pick disease, type
		C2 // 14q24.3 //
		10577 /// NM_00
2417945	PTGER3	NM_198715 //	NM_198715	0.30	0.11	0.017
		PTGER3 //
		prostaglandin E
		receptor 3 (subtype
		EP3) // 1p31.2 //
		573
3059393	SEMA3E	NM_012431 //	NM_012431	0.30	0.09	0.009
		SEMA3E // sema
		domain,
		immunoglobulin
		domain (Ig), short
		basic doma
2336456	MGC52498	NM_001042693 //	NM_001042693	0.30	0.10	0.011
		MGC52498 //
		hypothetical protein
		MGC52498 //
		1p32.3 // 348378 //
3726772	CROP	NM_016424 //	NM_016424	0.30	0.11	0.016
		CROP // cisplatin
		resistance-
		associated
		overexpressed
		protein // 17
2784265	IL2	NM_000586 // IL2 //	NM_000586	0.29	0.11	0.019
		interleukin 2 //
		4q26-q27 // 3558 ///
		ENST00000226730 // IL2
2495782	LIPT1	NM_145197 //	NM_145197	0.29	0.10	0.012
		LIPT1 //
		lipoyltransferase
		1 // 2q11.2 // 51601 ///
		NM_145198 //
		LI
2377094	PFKFB2	NM_006212 //	NM_006212	0.29	0.10	0.012
		PFKFB2 // 6-
		phosphofructo-2-
		kinase/fructose-2,6-
		biphosphatase 2 //
2469213	KLF11	NM_003597 //	NM_003597	0.29	0.10	0.011
		KLF11 // Kruppel-
		like factor 11 //
		2p25 // 8462 ///
		ENST00000305883
3662387	HERPUD1	NM_014685 //	NM_014685	0.29	0.07	0.003
		HERPUD1 //
		homocysteine-
		inducible,
		endoplasmic
		reticulum
		stress-ind
3771215	ACOX1	NM_004035 //	NM_004035	0.29	0.10	0.013
		ACOX1 // acyl-
		Coenzyme A
		oxidase 1,
		palmitoyl // 17q24-
		q25|17q25.1
3203135	TOPORS	NM_005802 //	NM_005802	0.28	0.11	0.018
		TOPORS //
		topoisomerase I
		binding,
		arginine/serine-
		rich // 9p21 //
2805482		—	—	0.28	0.09	0.008
3247757	UBE2D1	NM_003338 //	NM_003338	0.28	0.08	0.007
		UBE2D1 //
		ubiquitin-
		conjugating enzyme
		E2D 1 (UBC4/5
		homolog, yeast
3444147	KLRC1	NM_002259 //	NM_002259	0.28	0.10	0.015
		KLRC1 // killer cell
		lectin-like receptor
		subfamily C,
		member 1 //
3348891	C11orf57	NM_018195 //	NM_018195	0.28	0.09	0.011
		C11orf57 //
		chromosome 11
		open reading frame
		57 // 11q23.1 //
		55216
3906942	SERINC3	NM_006811 //	NM_006811	0.28	0.07	0.003
		SERINC3 // serine
		incorporator 3 //
		20q13.1-q13.3 //
		10955 /// NM_1
2930418	UST	NM_005715 // UST //	NM_005715	0.28	0.06	0.002
		uronyl-2-
		sulfotransferase //
		6q25.1 // 10090 ///
		ENST0000036
3188200	OR1L1	NM_001005236 //	NM_001005236	0.28	0.09	0.011
		OR1L1 // olfactory
		receptor, family 1,
		subfamily L,
		member 1 //
3856075	ZNF682	NM_033196 //	NM_033196	0.28	0.10	0.017
		ZNF682 // zinc
		finger protein 682 //
		19p12 // 91120 ///
		NM_00107734
3385951	NOX4	NM_016931 //	NM_016931	0.28	0.06	0.002
		NOX4 // NADPH
		oxidase 4 //
		11q14.2-q21 //
		50507 ///
		ENST00000263317
3523881	KDELC1	NM_024089 //	NM_024089	0.28	0.06	0.002
		KDELC1 // KDEL
		(Lys-Asp-Glu-Leu)
		containing 1 //
		13q33 // 79070 ///
2632778	EPHA6	NM_001080448 //	NM_001080448	0.28	0.09	0.010
		EPHA6 // EPH
		receptor A6 //
		3q11.2 // 285220 ///
		ENST00000389672
3373272	OR5W2	NM_001001960 //	NM_001001960	0.28	0.10	0.015
		OR5W2 // olfactory
		receptor, family 5,
		subfamily W,
		member 2 //
4017694	IRS4	NM_003604 //	NM_003604	0.28	0.10	0.016
		IRS4 // insulin
		receptor substrate
		4 // Xq22.3 // 8471 ///
		ENST0000
3545311	KIAA1737	NM_033426 //	NM_033426	0.28	0.07	0.003
		KIAA1737 //
		KIAA1737 //
		14q24.3 // 85457 ///
		ENST00000361786 //
		KIA
3753860	CCL5	NM_002985 //	NM_002985	0.28	0.05	0.001
		CCL5 // chemokine
		(C-C motif) ligand
		5 // 17q11.2-q12 //
		6352 /// E
3617312	SLC12A6	NM_001042496 //	NM_001042496	0.27	0.07	0.005
		SLC12A6 // solute
		carrier family 12
		(potassium/chloride
		transpor
3351315	UBE4A	NM_004788 //	NM_004788	0.27	0.07	0.004
		UBE4A //
		ubiquitination
		factor E4A (UFD2
		homolog, yeast) //
		11q23.3
3755396	CCDC49	NM_017748 //	NM_017748	0.27	0.09	0.013
		CCDC49 // coiled-
		coil domain
		containing 49 //
		17q12 // 54883 ///
		EN
2870889	C5orf13	NM_004772 //	NM_004772	0.27	0.09	0.010
		C5orf13 //
		chromosome 5
		open reading frame
		13 // 5q22.1 //
		9315 ///
2775259	RASGEF1B	NM_152545 //	NM_152545	0.27	0.10	0.015
		RASGEF1B //
		RasGEF domain
		family, member
		1B // 4q21.21-
		q21.22 // 15
3165624		—	—	0.27	0.06	0.003
2771654	CENPC1	NM_001812 //	NM_001812	0.27	0.09	0.013
		CENPC1 //
		centromere protein
		C 1 // 4q12-q13.3 //
		1060 /// ENST0000
3784670	C18orf21	NM_031446 //	NM_031446	0.27	0.08	0.008
		C18orf21 //
		chromosome 18
		open reading frame
		21 // 18q12.2 //
		83608
2364231	DDR2	NM_001014796 //	NM_001014796	0.26	0.10	0.018
		DDR2 // discoidin
		domain receptor
		tyrosine kinase 2 //
		1q23.3 //
3921442	SH3BGR	NM_007341 //	NM_007341	0.26	0.08	0.007
		SH3BGR // SH3
		domain binding
		glutamic acid-rich
		protein // 21q22.3
2627368	C3orf49	BC015210 //	BC015210	0.26	0.06	0.003
		C3orf49 //
		chromosome 3
		open reading frame
		49 // 3p14.1 //
		132200
3250699	EIF4EBP2	NM_004096 //	NM_004096	0.26	0.10	0.018
		EIF4EBP2 //
		eukaryotic
		translation initiation
		factor 4E binding
		pro
3237788	PLXDC2	NM_032812 //	NM_032812	0.26	0.09	0.013
		PLXDC2 // plexin
		domain containing
		2 // 10p12.32-
		p12.31 // 84898 //
3285926	ZNF33B	NM_006955 //	NM_006955	0.26	0.10	0.018
		ZNF33B // zinc
		finger protein 33B //
		10q11.2 // 7582 ///
		ENST000003
3304475	ARL3	NM_004311 //	NM_004311	0.26	0.08	0.008
		ARL3 // ADP-
		ribosylation factor-
		like 3 // 10q23.3 //
		403 /// ENST00
3364306	SOX6	NM_017508 //	NM_017508	0.26	0.08	0.010
		SOX6 // SRY (sex
		determining region
		Y)-box6 // 11p15.3 //
		55553 //
3185498	SLC31A2	NM_001860 //	NM_001860	0.25	0.09	0.015
		SLC31A2 // solute
		carrier family 31
		(copper
		transporters),
		member 2
3998766	KAL1	NM_000216 //	NM_000216	0.25	0.07	0.006
		KAL1 // Kallmann
		syndrome 1
		sequence //
		Xp22.32 // 3730 ///
		ENST000
3143266	PSKH2	NM_033126 //	NM_033126	0.25	0.07	0.006
		PSKH2 // protein
		serine kinase H2 //
		8q21.2 // 85481 ///
		ENST000002
3458911	CTDSP2	NM_005730 //	NM_005730	0.25	0.06	0.003
		CTDSP2 // CTD
		(carboxy-terminal
		domain, RNA
		polymerase II,
		polypept
3195034	PTGDS	NM_000954 //	NM_000954	0.25	0.08	0.010
		PTGDS //
		prostaglandin D2
		synthase 21 kDa
		(brain) // 9q34.2-
		q34.3 //
3854066	C19orf42	NM_024104 //	NM_024104	0.25	0.08	0.010
		C19orf42 //
		chromosome 19
		open reading frame
		42 // 19p13.11 //
		7908
3819474	ANGPTL4	NM_139314 //	NM_139314	0.25	0.06	0.004
		ANGPTL4 //
		angiopoietin-like
		4 // 19p13.3 //
		51129 ///
		NM_001039667
3944084	TOM1	NM_005488 //	NM_005488	0.25	0.07	0.006
		TOM1 // target of
		myb1 (chicken) //
		22q13.1 // 10043 ///
		ENST000003
3848243	INSR	NM_000208 //	NM_000208	0.24	0.09	0.014
		INSR // insulin
		receptor // 19p13.3-
		p13.2 // 3643 ///
		NM_001079817
3168415	CLTA	NM_007096 //	NM_007096	0.24	0.08	0.009
		CLTA // clathrin,
		light chain (Lca) //
		9p13 // 1211 ///
		NM_00107667
2609462	CAV3	NM_033337 //	NM_033337	0.24	0.07	0.007
		CAV3 // caveolin 3 //
		3p25 // 859 ///
		NM_001234 //
		CAV3 // caveolin
3393834	C11orf60	BC022856 //	BC022856	0.24	0.06	0.003
		C11orf60 //
		chromosome 11
		open reading frame
		60 // 11q23.3 //
		56912
3755614	STAC2	NM_198993 //	NM_198993	0.24	0.07	0.009
		STAC2 // SH3 and
		cysteine rich
		domain 2 // 17q12 //
		342667 /// ENST
3627363	NARG2	NM_024611 ///	NM_024611	0.24	0.06	0.003
		NARG2 // NMDA
		receptor regulated
		2 // 15q22.2 //
		79664 ///
		NM_00101
3212976	ZCCHC6	NM_024617 //	NM_024617	0.24	0.08	0.014
		ZCCHC6 // zinc
		finger, CCHC
		domain containing
		6 // 9q21 // 79670 //
3275922	PRKCQ	NM_006257 //	NM_006257	0.24	0.05	0.002
		PRKCQ // protein
		kinase C, theta //
		10p15 // 5588 ///
		ENST000002631
3023825	C7orf45	BC017587 //	BC017587	0.23	0.09	0.020
		C7orf45 //
		chromosome 7
		open reading frame
		45 // 7q32.2 //
		136263 //
3832906	IL29	NM_172140 //	NM_172140	0.23	0.08	0.015
		IL29 // interleukin
		29 (interferon,
		lambda 1) //
		19q13.13 // 282618
3529156	NGDN	NM_015514 //	NM_015514	0.23	0.08	0.012
		NGDN //
		neuroguidin, EIF4E
		binding protein //
		14q11.2 // 25983 ///
2620448	CLEC3B	NM_003278 //	NM_003278	0.23	0.08	0.014
		CLEC3B // C-type
		lectin domain
		family 3, member B //
		3p22-p21.3 //
3481296	SGCG	NM_000231 //	NM_000231	0.23	0.09	0.019
		SGCG //
		sarcoglycan,
		gamma (35 kDa
		dystrophin-
		associated
		glycoprotei
3135184	RB1CC1	NM_014781 //	NM_014781	0.23	0.07	0.008
		RB1CC1 // RB1-
		inducible coiled-
		coil 1 // 8q11 //
		9821 ///
		NM_001083
2421843	GBP3	NM_018284 //	NM_018284	0.23	0.06	0.004
		GBP3 // guanylate
		binding protein 3 //
		1p22.2 // 2635 ///
		ENST00000
3385003	CREBZF	NM_001039618 //	NM_001039618	0.23	0.09	0.020
		CREBZF //
		CREB/ATF bZIP
		transcription
		factor //
		11q14 // 58487/
3610804	IGF1R	NM_000875 //	NM_000875	0.23	0.08	0.013
		IGF1R // insulin-
		like growth factor 1
		receptor // 15q26.3 //
		3480/
3606304	AKAP13	NM_006738 //	NM_006738	0.23	0.04	0.000
		AKAP13 // A
		kinase (PRKA)
		anchor protein 13 //
		15q24-q25 // 11214/
2565579	ANKRD39	NM_016466 //	NM_016466	0.23	0.05	0.003
		ANKRD39 //
		ankyrin repeat
		domain 39 // 2q11.2 //
		51239 ///
		ENST0000
2722151	RBPJ	NM_005349 //	NM_005349	0.22	0.07	0.008
		RBPJ //
		recombination
		signal binding
		protein for
		immunoglobulin
		kap
3031533	GIMAP4	NM_018326 //	NM_018326	0.22	0.08	0.017
		GIMAP4 //
		GTPase, IMAP
		family member 4 //
		7q36.1 // 55303 ///
		ENST0
3725481	UBE2Z	NM_023079 //	NM_023079	0.22	0.06	0.004
		UBE2Z //
		ubiquitin-
		conjugating enzyme
		E2Z // 17q21.32 //
		65264 ///
3549575	IFI27	NM_005532 //	NM_005532	0.22	0.08	0.016
		IFI27 // interferon,
		alpha-inducible
		protein 27 // 14q32 //
		3429 //
3725035	NFE2L1	NM_003204 //	NM_003204	0.22	0.07	0.011
		NFE2L1 // nuclear
		factor (erythroid-
		derived 2)-like 1 //
		17q21.3 //
3348748	C11orf1	NM_022761 //	NM_022761	0.22	0.07	0.008
		C11orf1 //
		chromosome 11
		open reading frame
		1 // 11q13-q22 //
		64776
3722039	RAMP2	NM_005854 //	NM_005854	0.22	0.05	0.003
		RAMP2 // receptor
		(G protein-coupled)
		activity modifying
		protein 2
3886704	STK4	NM_006282 //	NM_006282	0.22	0.07	0.012
		STK4 //
		serine/threonine
		kinase 4 // 20q11.2-
		q13.2 // 6789 ///
		ENST
3645901	FLJ14154	NM_024845/	NM_024845	0.22	0.06	0.005
		FLJ14154 //
		hypothetical protein
		FLJ14154 //
		16p13.3 // 79903 ///
		N
3367673	MPPED2	NM_001584 //	NM_001584	0.22	0.08	0.017
		MPPED2 //
		metallophosphoesterase
		domain
		containing 2 //
		11p13 // 74
3219885	PTPN3	NM_002829 //	NM_002829	0.22	0.05	0.003
		PTPN3 // protein
		tyrosine
		phosphatase, non-
		receptor type 3 //
		9q31
3791466		—	—	0.22	0.06	0.007
3717635	ZNF207	NM_001098507 //	NM_001098507	0.22	0.08	0.015
		ZNF207 // zinc
		finger protein 207 //
		17q11.2 // 7756 ///
		NM_0034
2648141	MBNL1	NM_021038 //	NM_021038	0.22	0.07	0.009
		MBNL1 //
		muscleblind-like
		(Drosophila) //
		3q25 // 4154 ///
		NM_20729
2436938	PBXIP1	NM_020524 //	NM_020524	0.21	0.05	0.002
		PBXIP1 // pre-B-
		cell leukemia
		homeobox
		interacting protein
		1 // 1q2
3299705	PANK1	NM_148977 //	NM_148977	0.21	0.06	0.007
		PANK1 //
		pantothenate kinase
		1 // 10q23.31 //
		53354 ///
		NM_148978/
3628923	FAM96A	NM_032231 //	NM_032231	0.21	0.05	0.003
		FAM96A // family
		with sequence
		similarity 96,
		member A //
		15q22.31
2353669	CD2	NM_001767 // CD2 //	NM_001767	0.21	0.06	0.006
		CD2 molecule //
		1p13 // 914 ///
		ENST00000369478 //
		CD2 // CD
3474450	PLA2G1B	NM_000928 //	NM_000928	0.21	0.08	0.016
		PLA2G1B //
		phospholipase A2,
		group IB
		(pancreas) //
		12q23-q24.1 //
3722417	NBR1	NM_031858 //	NM_031858	0.21	0.08	0.017
		NBR1 // neighbor
		of BRCA1 gene 1 //
		17q21.31 // 4077 ///
		NM_005899
3234760	CUGBP2	NM_001025077 //	NM_001025077	0.21	0.06	0.004
		CUGBP2 // CUG
		triplet repeat, RNA
		binding protein 2 //
		10p13 //
3627422	RORA	NM_134260 //	NM_134260	0.21	0.06	0.006
		RORA // RAR-
		related orphan
		receptor A //
		15q21-q22 // 6095 ///
		NM_0
3382061	XRRA1	NM_182969 //	NM_182969	0.21	0.08	0.017
		XRRA1 // X-ray
		radiation resistance
		associated 1 //
		11q13.4 // 1435
3015338	STAG3	NM_012447 //	NM_012447	0.21	0.06	0.007
		STAG3 // stromal
		antigen 3 // 7q22.1 //
		10734 ///
		ENST00000317296/
2665720	ZNF385D	NM_024697 //	NM_024697	0.21	0.07	0.013
		ZNF385D // zinc
		finger protein
		385D // 3p24.3 //
		79750 /// ENST0000
3154185	TMEM71	NM_144649 //	NM_144649	0.21	0.06	0.009
		TMEM71 //
		transmembrane
		protein 71 //
		8q24.22 // 137835 ///
		ENST000
3789947	NEDD4L	NM_015277 //	NM_015277	0.21	0.08	0.016
		NEDD4L // neural
		precursor cell
		expressed,
		developmentally
		down-reg
2688933	CD200R2	ENST00000383679 //	ENST00000383679	0.21	0.08	0.016
		CD200R2 //
		CD200 cell surface
		glycoprotein
		receptor isoform 2
3379644	CPT1A	NM_001876 //	NM_001876	0.21	0.04	0.001
		CPT1A // carnitine
		palmitoyltransferase
		1A (liver) //
		11q13.1-q13.2
3677795	CREBBP	NM_004380 //	NM_004380	0.21	0.05	0.004
		CREBBP // CREB
		binding protein
		(Rubinstein-Taybi
		syndrome) // 16p13
2358320	TARS2	NM_025150 //	NM_025150	0.21	0.06	0.007
		TARS2 // threonyl-
		tRNA synthetase 2,
		mitochondrial
		(putative) // 1q
3228373	TSC1	NM_000368 //	NM_000368	0.20	0.06	0.006
		TSC1 // tuberous
		sclerosis 1 // 9q34 //
		7248 ///
		NM_001008567 //
		TS
3362795	RNF141	NM_016422 //	NM_016422	0.20	0.08	0.019
		RNF141 // ring
		finger protein 141 //
		11p15.4 // 50862 ///
		ENST00000
3673684	CDT1	NM_030928 //	NM_030928	0.20	0.07	0.015
		CDT1 // chromatin
		licensing and DNA
		replication factor
		1 // 16q24.3
3042881	HOXA7	NM_006896 //	NM_006896	0.20	0.02	0.000
		HOXA7 //
		homeobox A7 //
		7p15-p14 // 3204 ///
		ENST00000396347 //
		HOX
3381817	UCP2	NM_003355 //	NM_003355	0.20	0.05	0.005
		UCP2 // uncoupling
		protein 2
		(mitochondrial,
		proton carrier) //
		11q
3415068	ANKRD33	NM_182608 //	NM_182608	0.20	0.06	0.006
		ANKRD33 //
		ankyrin repeat
		domain 33 //
		12q13.13 // 341405 ///
		ENST0
3633403	SIN3A	NM_015477 //	NM_015477	0.20	0.07	0.014
		SIN3A // SIN3
		homolog A,
		transcription
		regulator (yeast) //
		15q24.2
3380901	NUMA1	NM_006185 //	NM_006185	0.19	0.04	0.002
		NUMA1 // nuclear
		mitotic apparatus
		protein 1 // 11q13 //
		4926 /// E
2598099	BARD1	NM_000465 //	NM_000465	0.19	0.07	0.015
		BARD1 // BRCA1
		associated RING
		domain 1 // 2q34-
		q35 // 580 /// ENST
3139722	NCOA2	NM_006540 //	NM_006540	0.19	0.06	0.010
		NCOA2 // nuclear
		receptor coactivator
		2 // 8q13.3 // 10499 ///
		ENST
3641871	LINS1	NM_018148 //	NM_018148	0.19	0.06	0.013
		LINS1 // lines
		homolog 1
		(Drosophila) //
		15q26.3 // 55180 ///
		NM_00
3401217	TULP3	NM_003324 //	NM_003324	0.19	0.06	0.008
		TULP3 // tubby like
		protein 3 // 12p13.3 //
		7289 ///
		ENST0000022824
3741997	ANKFY1	NM_016376 //	NM_016376	0.19	0.06	0.008
		ANKFY1 // ankyrin
		repeat and FYVE
		domain containing
		1 // 17p13.3 //
2622742	C3orf45	BC028000 //	BC028000	0.19	0.06	0.013
		C3orf45 //
		chromosome 3
		open reading frame
		45 // 3p21.31 //
		132228/
3845352	UQCR	NM_006830 //	NM_006830	0.19	0.06	0.014
		UQCR // ubiquinol-
		cytochrome c
		reductase, 6.4 kDa
		subunit // 19p13.3
3960356	BAIAP2L2	NM_025045 //	NM_025045	0.19	0.07	0.018
		BAIAP2L2 //
		BAI1-associated
		protein 2-like 2 //
		22q13.1 // 80115 //
3645947	CLUAP1	NM_015041 //	NM_015041	0.19	0.06	0.012
		CLUAP1 //
		clusterin associated
		protein 1 // 16p13.3 //
		23059 /// NM
3835544	ZNF227	NM_182490 //	NM_182490	0.18	0.06	0.011
		ZNF227 // zinc
		finger protein 227 //
		— // 7770 ///
		ENST0000031304
3368748	FBXO3	NM_033406 //	NM_033406	0.18	0.07	0.020
		FBXO3 // F-box
		protein 3 // 11p13 //
		26273 ///
		NM_012175 //
		FBXO3/
3621623	ELL3	NM_025165 //	NM_025165	0.18	0.05	0.005
		ELL3 // elongation
		factor RNA
		polymerase II-like
		3 // 15q15.3 // 80
3430552	PWP1	NM_007062 //	NM_007062	0.18	0.07	0.016
		PWP1 // PWP1
		homolog
		(S. cerevisiae) //
		12q23.3 // 11137 ///
		ENST00
2844908	BTNL9	NM_152547 //	NM_152547	0.18	0.05	0.005
		BTNL9 //
		butyrophilin-like
		9 // 5q35.3 //
		153579 ///
		ENST0000032770
4021508	ZNF280C	NM_017666 //	NM_017666	0.18	0.07	0.018
		ZNF280C // zinc
		finger protein
		280C // Xq25 //
		55609 ///
		ENST000003
2489071	TET3	NM_144993 //	NM_144993	0.18	0.04	0.003
		TET3 // tet
		oncogene family
		member 3 // 2p13.1 //
		200424 ///
		ENST00
2516879	HOXD8	NM_019558 //	NM_019558	0.18	0.06	0.015
		HOXD8 //
		homeobox D8 //
		2q31.1 // 3234 ///
		ENST00000313173 //
		HOXD8
3740704	SMYD4	NM_052928 //	NM_052928	0.18	0.06	0.012
		SMYD4 // SET and
		MYND domain
		containing 4 //
		17p13.3 // 114826 ///
3975467	UTX	NM_021140 //	NM_021140	0.18	0.06	0.013
		UTX //
		ubiquitously
		transcribed
		tetratricopeptide
		repeat, X chromos
3699044	RFWD3	NM_018124 //	NM_018124	0.18	0.06	0.011
		RFWD3 // ring
		finger and WD
		repeat domain 3 //
		16q22.3 // 55159 ///
3473083	MED13L	NM_015335 //	NM_015335	0.18	0.02	0.000
		MED13L //
		mediator complex
		subunit 13-like //
		12q24.21 // 23389 ///
2332711	PPIH	NM_006347 //	NM_006347	0.17	0.06	0.017
		PPIH //
		peptidylprolyl
		isomerase H
		(cyclophilin H) //
		1p34.1 // 104
3556990	JUB	NM_032876 // JUB //	NM_032876	0.17	0.04	0.004
		jub, ajuba
		homolog
		(Xenopus laevis) //
		14q11.2 //
		84962 ///
2780143	BDH2	NM_020139 //	NM_020139	0.17	0.05	0.006
		BDH2 // 3-
		hydroxybutyrate
		dehydrogenase,
		type 2 // 4q24 //
		56898 //
3899495	C20orf12	NM_001099407 //	NM_001099407	0.17	0.05	0.008
		C20orf12 //
		chromosome 20
		open reading frame
		12 // 20p11.23 // 5
3290875	ANK3	NM_020987 //	NM_020987	0.17	0.03	0.001
		ANK3 // ankyrin 3,
		node of Ranvier
		(ankyrin G) //
		10q21 // 288 ///
3576014	C14orf102	NM_017970 //	NM_017970	0.17	0.04	0.002
		C14orf102 //
		chromosome 14
		open reading frame
		102 // 14q32.11 // 55
3644887	ATP6V0C	NM_001694 //	NM_001694	0.17	0.06	0.017
		ATP6V0C //
		ATPase, H+
		transporting,
		lysosomal 16 kDa,
		V0 subunit c/
2648378	RAP2B	NM_002886 //	NM_002886	0.17	0.06	0.017
		RAP2B // RAP2B,
		member of RAS
		oncogene family //
		3q25.2 // 5912 ///
2362892	ATP1A2	NM_000702 //	NM_000702	0.16	0.06	0.015
		ATP1A2 // ATPase,
		Na+/K+
		transporting, alpha
		2 (+) polypeptide // 1
2361488	RHBG	NM_020407 //	NM_020407	0.16	0.06	0.014
		RHBG // Rh
		family, B
		glycoprotein //
		1q21.3 // 57127 ///
		ENST000003
3415915	PFDN5	NM_002624 //	NM_002624	0.16	0.05	0.011
		PFDN5 // prefoldin
		subunit 5 // 12q12 //
		5204 ///
		NM_145897 //
		PFDN
3433796	PEBP1	NM_002567 //	NM_002567	0.16	0.04	0.004
		PEBP1 //
		phosphatidyl-
		ethanolamine binding
		protein 1 //
		12q24.23 //
3788302	SMAD4	NM_005359 //	NM_005359	0.16	0.05	0.012
		SMAD4 // SMAD
		family member 4 //
		18q21.1 // 4089 ///
		ENST0000039841
3436236	ZNF664	NM_152437 //	NM_152437	0.16	0.06	0.016
		ZNF664 // zinc
		finger protein 664 //
		12q24.31 //144348 ///
		ENST000
3441542	TMEM16B	NM_020373 //	NM_020373	0.16	0.06	0.018
		TMEM16B //
		transmembrane
		protein 16B //
		12p13.3 // 57101 ///
		ENST00
3456353	CALCOCO1	NM_020898 //	NM_020898	0.16	0.05	0.010
		CALCOCO1 //
		calcium binding
		and coiled-coil
		domain 1 //
		12q13.13 //
3888721	PTPN1	NM_002827 //	NM_002827	0.16	0.06	0.020
		PTPN1 // protein
		tyrosine
		phosphatase, non-
		receptor type 1 //
		20q13
3138204	CYP7B1	NM_004820 //	NM_004820	0.15	0.05	0.014
		CYP7B1 //
		cytochrome P450,
		family 7, subfamily
		B, polypeptide 1 //
3278401	FRMD4A	NM_018027 //	NM_018027	0.15	0.05	0.009
		FRMD4A // FERM
		domain containing
		4A // 10p13 //
		55691 ///
		ENST00000
3904226	RBM39	NM_184234 //	NM_184234	0.15	0.05	0.015
		RBM39 // RNA
		binding motif
		protein 39 //
		20q11.22 // 9584 ///
		NM_00
3791850	SERPINB13	NM_012397 //	NM_012397	0.15	0.04	0.005
		SERPINB13 //
		serpin peptidase
		inhibitor, clade B
		(ovalbumin),
		membe
3665603	CTCF	NM_006565 //	NM_006565	0.15	0.04	0.004
		CTCF // CCCTC-
		binding factor (zinc
		finger protein) //
		16q21-q22.3/
3969802	BMX	NM_203281 //	NM_203281	0.15	0.05	0.016
		BMX // BMX non-
		receptor tyrosine
		kinase // Xp22.2 //
		660 /// NM_001
3621276	HISPPD2A	NM_014659 //	NM_014659	0.14	0.04	0.005
		HISPPD2A //
		histidine acid
		phosphatase
		domain containing
		2A // 15q1
2325113	C1orf213	NM_138479 //	NM_138479	0.14	0.05	0.012
		C1orf213 //
		chromosome 1
		open reading frame
		213 // 1p36.12 //
		14889
3681956	KIAA0430	NM_014647 //	NM_014647	0.14	0.05	0.018
		KIAA0430 //
		KIAA0430 //
		16p13.11 // 9665 ///
		ENST00000396368 //
		KIA
3415193	GRASP	NM_181711 //	NM_181711	0.14	0.05	0.019
		GRASP // GRP1
		(general receptor
		for
		phosphoinositides
		1)-associated
3249369	LRRTM3	NM_178011 //	NM_178011	0.14	0.05	0.011
		LRRTM3 // leucine
		rich repeat
		transmembrane
		neuronal 3 //
		10q21.3/
3874023	PTPRA	NM_002836 //	NM_002836	0.14	0.04	0.004
		PTPRA // protein
		tyrosine
		phosphatase,
		receptor type, A //
		20p13 //
3809621	FECH	NM_001012515 //	NM_001012515	0.14	0.04	0.009
		FECH //
		ferrochelatase
		(protoporphyria) //
		18q21.3 // 2235 /// N
3351385	MLL	NM_005933 //	NM_005933	0.14	0.05	0.016
		MLL //
		myeloid/lymphoid
		or mixed-lineage
		leukemia (trithorax
		homolo
3288707	ERCC6	NM_000124 //	NM_000124	0.14	0.05	0.016
		ERCC6 // excision
		repair cross-
		complementing
		rodent repair
		deficien
3624607	MYO5A	NM_000259 //	NM_000259	0.14	0.04	0.006
		MYO5A // myosin
		VA (heavy chain
		12, myoxin) //
		15q21 // 4644 ///
		EN
3353859	OR4D5	NM_001001965 //	NM_001001965	0.14	0.05	0.017
		OR4D5 // olfactory
		receptor, family 4,
		subfamily D,
		member 5 //
2823797	TSLP	NM_033035 //	NM_033035	0.14	0.05	0.013
		TSLP // thymic
		stromal
		lymphopoietin //
		5q22.1 // 85480 ///
		NM_1385
2414366	PPAP2B	NM_003713 //	NM_003713	0.13	0.04	0.007
		PPAP2B //
		phosphatidic acid
		phosphatase type
		2B // 1pter-p22.1 // 8
3878308	CSRP2BP	NM_020536 //	NM_020536	0.13	0.05	0.019
		CSRP2BP //
		CSRP2 binding
		protein // 20p11.23 //
		57325 ///
		NM_177926
4025771	CD99L2	NM_031462 //	NM_031462	0.13	0.04	0.007
		CD99L2 // CD99
		molecule-like 2 //
		Xq28 // 83692 ///
		NM_134446 // CD
3414776	LETMD1	NM_015416 //	NM_015416	0.13	0.05	0.014
		LETMD1 //
		LETM1 domain
		containing 1 //
		12q13.13 // 25875 ///
		NM_001
3645253	SRRM2	NM_016333 //	NM_016333	0.13	0.04	0.007
		SRRM2 //
		serine/arginine
		repetitive matrix
		2 // 16p13.3 //
		23524 //
2440700	ADAMTS4	NM_005099 //	NM_005099	0.13	0.03	0.005
		ADAMTS4 //
		ADAM
		metallopeptidase
		with
		thrombospondin
		type 1 motif,
2609870	BRPF1	NM_001003694 //	NM_001003694	0.13	0.04	0.012
		BRPF1 //
		bromodomain and
		PHD finger
		containing, 1 //
		3p26-p25 //
3632298	ADPGK	NM_031284 //	NM_031284	0.13	0.04	0.007
		ADPGK // ADP-
		dependent
		glucokinase //
		15q24.1 // 83440 ///
		ENST0000
3184940	GNG10	NM_001017998 //	NM_001017998	0.13	0.04	0.011
		GNG10 // guanine
		nucleotide binding
		protein (G protein),
		gamma 1
3223776	C5	NM_001735 // C5 //	NM_001735	0.13	0.04	0.008
		complement
		component 5 //
		9q33-q34 // 727 ///
		ENST00000223642
3922100	MX1	NM_002462 //	NM_002462	0.12	0.04	0.015
		MX1 // myxovirus
		(influenza virus)
		resistance 1,
		interferon-inducib
3960478	CSNK1E	NM_001894 //	NM_001894	0.12	0.04	0.018
		CSNK1E // casein
		kinase 1, epsilon //
		22q13.1 // 1454 ///
		NM_152221
3715703	SUPT6H	NM_003170 //	NM_003170	0.11	0.03	0.005
		SUPT6H //
		suppressor of Ty 6
		homolog
		(S. cerevisiae) //
		17q11.2 //
2322818	PADI3	NM_016233 //	NM_016233	0.11	0.03	0.006
		PADI3 // peptidyl
		arginine deiminase,
		type III // 1p36.13 //
		51702
2393740	KIAA0562	NM_014704 //	NM_014704	0.11	0.03	0.009
		KIAA0562 //
		KIAA0562 //
		1p36.32 // 9731 ///
		ENST00000378230 //
		KIAA
3784509	ZNF271	NM_001112663 //	NM_001112663	0.11	0.04	0.020
		ZNF271 // zinc
		finger protein 271 //
		18q12 // 10778 ///
		NM_00662
3372253	CUGBP1	NM_006560 //	NM_006560	0.11	0.04	0.011
		CUGBP1 // CUG
		triplet repeat, RNA
		binding protein 1 //
		11p11 // 106
2948259	TRIM26	NM_003449 //	NM_003449	0.11	0.03	0.006
		TRIM26 // tripartite
		motif-containing
		26 // 6p21.3 //
		7726 /// ENST
3191900	NUP214	NM_005085 //	NM_005085	0.11	0.03	0.003
		NUP214 //
		nucleoporin
		214 kDa // 9q34.1 //
		8021 ///
		ENST00000359428
3105581	CA3	NM_005181 // CA3 //	NM_005181	0.11	0.03	0.003
		carbonic
		anhydrase III,
		muscle specific //
		8q13-q22 // 761/
3832457	RYR1	NM_000540 //	NM_000540	0.11	0.03	0.006
		RYR1 // ryanodine
		receptor 1 (skeletal) //
		19q13.1 // 6261 ///
		NM_0
3936256	BCL2L13	NM_015367 //	NM_015367	0.10	0.02	0.002
		BCL2L13 // BCL2-
		like 13 (apoptosis
		facilitator) // 22q11 //
		23786/
3599280	PIAS1	NM_016166 //	NM_016166	0.10	0.04	0.017
		PIAS1 // protein
		inhibitor of
		activated STAT, 1 //
		15q // 8554 ///
3755976	MED24	NM_014815 //	NM_014815	0.10	0.04	0.019
		MED24 // mediator
		complex subunit 24 //
		17q21.1 // 9862 ///
		NM_0010
3656418	SRCAP	NM_006662 //	NM_006662	0.10	0.04	0.017
		SRCAP // Snf2-
		related CREBBP
		activator protein //
		16p11.2 // 10847
3943101	DEPDC5	NM_014662 //	NM_014662	0.09	0.01	0.000
		DEPDC5 // DEP
		domain containing
		5 // 22q12.3 // 9681 ///
		NM_0010071
3960685	DMC1	NM_007068 //	NM_007068	0.09	0.03	0.013
		DMC1 // DMC1
		dosage suppressor
		of mck1 homolog,
		meiosis-specific ho
2434776	CDC42SE1	NM_001038707 //	NM_001038707	0.08	0.03	0.014
		CDC42SE1 //
		CDC42 small
		effector 1 // 1q21.2 //
		56882 ///
		NM_020
3438417	SFRS8	NM_004592 //	NM_004592	0.08	0.03	0.016
		SFRS8 // splicing
		factor,
		arginine/serine-rich
		8 (suppressor-of-
		whi
3457696	PAN2	NM_014871 //	NM_014871	0.08	0.02	0.008
		PAN2 // PAN2
		polyA specific
		ribonuclease
		subunit homolog
		(S. cerevi
2534615	SCLY	NM_016510 //	NM_016510	0.08	0.02	0.004
		SCLY //
		selenocysteine
		lyase // 2q37.3 //
		51540 ///
		ENST00000254663
2765865	RELL1	NM_001085400 //	NM_001085400	0.07	0.02	0.002
		RELL1 // RELT-
		like 1 // 4p14 //
		768211 ///
		NM_001085399 //
		RELL1
3765642	INTS2	NM_020748 //	NM_020748	0.05	0.01	0.005
		INTS2 // integrator
		complex subunit 2 //
		17q23.2 // 57508 ///
		ENST0
2906607	NFYA	NM_002505 //	NM_002505	−0.07	0.02	0.011
		NFYA // nuclear
		transcription factor
		Y, alpha // 6p21.3 //
		4800 ///
3168102	CREB3	NM_006368 //	NM_006368	−0.07	0.02	0.010
		CREB3 // cAMP
		responsive element
		binding protein 3 //
		9pter-p22.1/
3939365	SMARCB1	NM_003073 //	NM_003073	−0.07	0.02	0.013
		SMARCB1 //
		SWI/SNF related,
		matrix associated,
		actin dependent
		regu
3415229	NR4A1	NM_002135 //	NM_002135	−0.07	0.03	0.015
		NR4A1 // nuclear
		receptor subfamily
		4, group A, member
		1 // 12q13/
2437801	ARHGEF2	NM_004723 //	NM_004723	−0.09	0.02	0.002
		ARHGEF2 //
		rho/rac guanine
		nucleotide
		exchange factor
		(GEF) 2 // 1q
3645565	THOC6	NM_024339 //	NM_024339	−0.10	0.04	0.018
		THOC6 // THO
		complex 6 homolog
		(Drosophila) //
		16p13.3 // 79228 ///
2406766	MRPS15	NM_031280 //	NM_031280	−0.11	0.03	0.003
		MRPS15 //
		mitochondrial
		ribosomal protein
		S15 // 1p35-p34.1 //
		6496
3553141	KIAA0329	NM_014844 //	NM_014844	−0.11	0.04	0.018
		KIAA0329 //
		KIAA0329 //
		14q32.31 // 9895 ///
		ENST00000359520 //
		KIA
3297666	DYDC1	NM_138812 //	NM_138812	−0.11	0.02	0.000
		DYDC1 // DPY30
		domain containing
		1 // 10q23.1 //
		143241 ///
		ENST000
3625674	RFXDC2	NM_022841 //	NM_022841	−0.12	0.04	0.012
		RFXDC2 //
		regulatory factor X
		domain containing
		2 // 15q21.3 // 648
2926969	PDE7B	NM_018945 //	NM_018945	−0.12	0.04	0.013
		PDE7B //
		phosphodiesterase
		7B // 6q23-q24 //
		27115 ///
		ENST00000308
3525313	COL4A1	NM_001845 //	NM_001845	−0.12	0.04	0.014
		COL4A1 //
		collagen, type IV,
		alpha 1 // 13q34 //
		1282 ///
		ENST00000
2438892	FCRL5	NM_031281 //	NM_031281	−0.12	0.04	0.009
		FCRL5 // Fc
		receptor-like 5 //
		1q21 // 83416 ///
		ENST00000361835 //
3220846	SUSD1	NM_022486 //	NM_022486	−0.12	0.03	0.006
		SUSD1 // sushi
		domain containing
		1 // 9q31.3-q33.1 //
		64420 /// ENS
3598430	SLC24A1	NM_004727 //	NM_004727	−0.12	0.05	0.019
		SLC24A1 // solute
		carrier family 24
		(sodium/potassium/
		calcium excha
3506431	RNF6	NM_005977 //	NM_005977	−0.12	0.04	0.011
		RNF6 // ring finger
		protein (C3H2C3
		type) 6 // 13q12.2 //
		6049 ///
3696057	SLC12A4	NM_005072 //	NM_005072	−0.12	0.02	0.001
		SLC12A4 // solute
		carrier family 12
		(potassium/chloride
		transporter
2519577	COL3A1	NM_000090 //	NM_000090	−0.12	0.04	0.012
		COL3A1 //
		collagen, type III,
		alpha 1 (Ehlers-
		Danlos syndrome
		type
3734479	TMEM104	NM_017728 //	NM_017728	−0.13	0.04	0.015
		TMEM104 //
		transmembrane
		protein 104 //
		17q25.1 // 54868 ///
		ENST00
3345157	PIWIL4	NM_152431 //	NM_152431	−0.13	0.05	0.015
		PIWIL4 // piwi-like
		4 (Drosophila) //
		11q21 // 143689 ///
		ENST00000
2949471	NEU1	NM_000434 //	NM_000434	−0.13	0.04	0.013
		NEU1 // sialidase 1
		(lysosomal
		sialidase) // 6p21.3 //
		4758 /// ENS
2599670	CRYBA2	NM_057093 //	NM_057093	−0.13	0.04	0.014
		CRYBA2 //
		crystallin, beta A2 //
		2q34-q36 // 1412 ///
		NM_005209 //
3922444	ABCG1	NM_207628 //	NM_207628	−0.13	0.03	0.003
		ABCG1 // ATP-
		binding cassette,
		sub-family G
		(WHITE), member 1 // 21
2760371	WDR1	NM_017491 //	NM_017491	−0.14	0.05	0.019
		WDR1 // WD
		repeat domain 1 //
		4p16.1 // 9948 ///
		NM_005112 //
		WDR1
2835440	TCOF1	NM_001008656 //	NM_001008656	−0.14	0.04	0.007
		TCOF1 // Treacher
		Collins-
		Franceschetti
		syndrome 1 // 5q32-
		q33.1
2451544	MYOG	NM_002479 //	NM_002479	−0.14	0.05	0.018
		MYOG //
		myogenin
		(myogenic factor 4) //
		1q31-q41 // 4656 ///
		ENST00
3745504	SCO1	NM_004589 //	NM_004589	−0.14	0.03	0.003
		SCO1 // SCO
		cytochrome oxidase
		deficient homolog 1
		(yeast) // 17p12
2835213	PPARGC1B	NM_133263 //	NM_133263	−0.14	0.04	0.006
		PPARGC1B //
		peroxisome
		proliferator-
		activated receptor
		gamma, coact
3704567	CBFA2T3	NM_005187 //	NM_005187	−0.14	0.05	0.020
		CBFA2T3 // core-
		binding factor, runt
		domain, alpha
		subunit 2; trans
2893562	RREB1	NM_002955 //	NM_002955	−0.14	0.04	0.006
		RREB1 // ras
		responsive element
		binding protein 1 //
		6p25 // 6239/
2672712	SCAP	NM_012235 //	NM_012235	−0.14	0.04	0.009
		SCAP // SREBF
		chaperone //
		3p21.31 // 22937 ///
		ENST00000265565 //
2768197	CORIN	NM_006587 //	NM_006587	−0.14	0.05	0.011
		CORIN // corin,
		serine peptidase //
		4p13-p12 // 10699 ///
		ENST00000
2495279	VWA3B	NM_144992 //	NM_144992	−0.14	0.04	0.006
		VWA3B // von
		Willebrand factor A
		domain containing
		3B // 2q11.2 //
2903588	PFDN6	NM_014260 //	NM_014260	−0.14	0.05	0.014
		PFDN6 // prefoldin
		subunit 6 // 6p21.3 //
		10471 ///
		ENST00000399112
3031383	REPIN1	NM_013400 //	NM_013400	−0.15	0.05	0.018
		REPIN1 //
		replication initiator
		1 // 7q36.1 // 29803 ///
		NM_014374
3754469	ACACA	NM_198839 //	NM_198839	−0.15	0.05	0.010
		ACACA // acetyl-
		Coenzyme A
		carboxylase alpha //
		17q21 // 31 /// NM—
3767480	AXIN2	NM_004655 //	NM_004655	−0.15	0.05	0.013
		AXIN2 // axin 2
		(conductin, axil) //
		17q23-q24 // 8313 ///
		ENST0000
2954506	CRIP3	NM_206922 //	NM_206922	−0.15	0.06	0.018
		CRIP3 // cysteine-
		rich protein 3 //
		6p21.1 // 401262 ///
		ENST000003
3845263	ADAMTSL5	NM_213604 //	NM_213604	−0.15	0.06	0.016
		ADAMTSL5 //
		ADAMTS-like 5 //
		19p13.3 // 339366 ///
		ENST00000330475
2565143	STARD7	NM_020151 //	NM_020151	−0.15	0.06	0.016
		STARD7 // StAR-
		related lipid
		transfer (START)
		domain containing
		7/
2321960	PLEKHM2	NM_015164 //	NM_015164	−0.16	0.05	0.009
		PLEKHM2 //
		pleckstrin
		homology domain
		containing, family
		M (with RU
3829174	GPATCH1	NM_018025 //	NM_018025	−0.16	0.03	0.001
		GPATCH1 // G
		patch domain
		containing 1 //
		19q13.11 // 55094 ///
		ENS
2798586	AHRR	NM_020731 //	NM_020731	−0.16	0.05	0.011
		AHRR // aryl-
		hydrocarbon
		receptor repressor //
		5p15.3 // 57491 ///
2362991	CASQ1	NM_001231 //	NM_001231	−0.16	0.06	0.015
		CASQ1 //
		calsequestrin 1
		(fast-twitch,
		skeletal muscle) //
		1q21 //
3954525	ZNF280B	NM_080764 //	NM_080764	−0.16	0.04	0.005
		ZNF280B // zinc
		finger protein
		280B // 22q11.22 //
		140883 /// ENST0
4020991	ACTRT1	NM_138289 //	NM_138289	−0.16	0.05	0.007
		ACTRT1 // actin-
		related protein T1 //
		Xq25 // 139741 ///
		ENST000003
3982975	POU3F4	NM_000307 //	NM_000307	−0.16	0.05	0.013
		POU3F4 // POU
		class 3 homeobox
		4 // Xq21.1 // 5456 ///
		ENST00000373
3963990	PKDREJ	NM_006071 //	NM_006071	−0.16	0.03	0.001
		PKDREJ //
		polycystic kidney
		disease (polycystin)
		and REJ homolog (s
2436401	JTB	NM_006694 // JTB //	NM_006694	−0.16	0.06	0.014
		jumping
		translocation
		breakpoint //
		1q21 // 10899 ///
		NM_002
2759654	ABLIM2	NM_032432 //	NM_032432	−0.16	0.05	0.007
		ABLIM2 // actin
		binding LIM
		protein family,
		member 2 // 4p16-
		p15 //
2437329	CLK2	NM_003993 //	NM_003993	−0.16	0.06	0.016
		CLK2 // CDC-like
		kinase 2 // 1q21 //
		1196 ///
		NR_002711 //
		CLK2P //
3401119	ITFG2	NM_018463 //	NM_018463	−0.16	0.04	0.004
		ITFG2 // integrin
		alpha FG-GAP
		repeat containing
		2 // 12p13.33 // 5
3599709	GLCE	NM_015554 //	NM_015554	−0.16	0.06	0.014
		GLCE // glucuronic
		acid epimerase //
		15q23 // 26035 ///
		ENST0000026
3882413	C20orf114	NM_033197 //	NM_033197	−0.16	0.06	0.020
		C20orf114 //
		chromosome 20
		open reading frame
		114 // 20q11.21 //
		92
3712922	C17orf39	NM_024052 //	NM_024052	−0.16	0.06	0.017
		C17orf39 //
		chromosome 17
		open reading frame
		39 // 17p11.2 //
		79018
2473376	EFR3B	BC049384 //	BC049384	−0.17	0.05	0.009
		EFR3B // EFR3
		homolog B
		(S. cerevisiae) //
		2p23.3 // 22979 ///
		ENST0
2607262	STK25	NM_006374 //	NM_006374	−0.17	0.06	0.015
		STK25 //
		serine/threonine
		kinase 25 (STE20
		homolog, yeast) //
		2q37.
3755580	CACNB1	NM_199247 //	NM_199247	−0.17	0.06	0.013
		CACNB1 //
		calcium channel,
		voltage-dependent,
		beta 1 subunit //
		17q
3402150	NTF3	NM_001102654 //	NM_001102654	−0.17	0.06	0.020
		NTF3 //
		neurotrophin 3 //
		12p13 // 4908 ///
		NM_002527 //
		NTF3 //
3014714	ARPC1B	NM_005720 //	NM_005720	−0.17	0.06	0.020
		ARPC1B // actin
		related protein 2/3
		complex, subunit
		1B, 41 kDa // 7
3723071	DBF4B	NM_145663 //	NM_145663	−0.17	0.04	0.002
		DBF4B // DBF4
		homolog B
		(S. cerevisiae) //
		17q21.31|17q21 //
		80174
2371255	SMG7	NM_173156 //	NM_173156	−0.17	0.06	0.014
		SMG7 // Smg-7
		homolog, nonsense
		mediated mRNA
		decay factor
		(C. eleg
3217487	ALG2	NM_033087 //	NM_033087	−0.17	0.06	0.011
		ALG2 //
		asparagine-linked
		glycosylation 2
		homolog
		(S. cerevisiae, a
3352159	LOC100130353	AK130019 //	AK130019	−0.17	0.06	0.018
		LOC100130353 //
		hypothetical protein
		LOC100130353 //
		11q23.3 // 1001
3401259	TEAD4	NM_003213 //	NM_003213	−0.17	0.07	0.020
		TEAD4 // TEA
		domain family
		member 4 //
		12p13.3-p13.2 //
		7004 /// NM
3114618	RNF139	NM_007218 //	NM_007218	−0.17	0.06	0.015
		RNF139 // ring
		finger protein 139 //
		8q24 // 11236 ///
		ENST00000303
2991150	TSPAN13	NM_014399 //	NM_014399	−0.18	0.05	0.006
		TSPAN13 //
		tetraspanin 13 //
		7p21.1 // 27075 ///
		ENST00000262067 //
2875193	P4HA2	NM_004199 //	NM_004199	−0.18	0.05	0.007
		P4HA2 //
		procollagen-
		proline, 2-
		oxoglutarate 4-
		dioxygenase
		(proline
4011743	SLC7A3	NM_032803 //	NM_032803	−0.18	0.06	0.009
		SLC7A3 // solute
		carrier family 7
		(cationic amino
		acid transporter,
3194015	LCN9	NM_001001676 //	NM_001001676	−0.18	0.06	0.011
		LCN9 // lipocalin 9 //
		9q34.3 // 392399 ///
		ENST00000277526 // L
3741040	MNT	NM_020310 //	NM_020310	−0.18	0.04	0.003
		MNT // MAX
		binding protein //
		17p13.3 // 4335 ///
		ENST00000174618/
3901851	ABHD12	NM_001042472 //	NM_001042472	−0.18	0.05	0.004
		ABHD12 //
		abhydrolase domain
		containing 12 //
		20p11.21 // 26090
2324919	EPHB2	NM_017449 //	NM_017449	−0.18	0.06	0.010
		EPHB2 // EPH
		receptor B2 //
		1p36.1-p35 // 2048 ///
		NM_004442 //
		EPH
3185976	COL27A1	NM_032888 //	NM_032888	−0.18	0.06	0.009
		COL27A1 //
		collagen, type
		XXVII, alpha 1 //
		9q32 // 85301 ///
		ENST0
2855434	C5orf39	NM_001014279 //	NM_001014279	−0.18	0.05	0.007
		C5orf39 //
		chromosome 5
		open reading frame
		39 // 5p12 //
		389289
2334476	MAST2	NM_015112 //	NM_015112	−0.18	0.02	0.000
		MAST2 //
		microtubule
		associated
		serine/threonine
		kinase 2 // 1p34.1
3962734	TTLL1	NM_001008572 //	NM_001008572	−0.18	0.03	0.001
		TTLL1 // tubulin
		tyrosine ligase-like
		family, member 1 //
		22q13.
4017538	COL4A6	NM_033641 //	NM_033641	−0.18	0.03	0.000
		COL4A6 //
		collagen, type IV,
		alpha 6 // Xq22 //
		1288 ///
		NM_001847
3141589	IL7	NM_000880 // IL7 //	NM_000880	−0.19	0.05	0.006
		interleukin 7 //
		8q12-q13 // 3574 ///
		ENST00000263851 //
		IL7
2436826	KCNN3	NM_002249 //	NM_002249	−0.19	0.06	0.008
		KCNN3 //
		potassium
		intermediate/small
		conductance
		calcium-activated
3521174	ABCC4	NM_005845 //	NM_005845	−0.19	0.07	0.017
		ABCC4 // ATP-
		binding cassette,
		sub-family C
		(CFTR/MRP),
		member 4 //
3768280	C17orf58	NM_181656 //	NM_181656	−0.19	0.07	0.017
		C17orf58 //
		chromosome 17
		open reading frame
		58 // 17q24.2 //
		28401
2363784	HSPA6	NM_002155 //	NM_002155	−0.19	0.06	0.011
		HSPA6 // heat
		shock 70 kDa
		protein 6
		(HSP70B′) // 1q23 //
		3310 /// E
3928211	GRIK1	NM_175611 //	NM_175611	−0.19	0.06	0.011
		GRIK1 // glutamate
		receptor,
		ionotropic, kainate
		1 // 21q22.11 // 2
2758978	EVC2	NM_147127 //	NM_147127	−0.19	0.06	0.012
		EVC2 // Ellis van
		Creveld syndrome
		2 (limbin) //
		4p16.2-p16.1 // 13
3740664	C17orf91	NM_032895 //	NM_032895	−0.19	0.07	0.015
		C17orf91 //
		chromosome 17
		open reading frame
		91 // 17p13.3 //
		84981
2782267	NEUROG2	NM_024019 //	NM_024019	−0.20	0.06	0.010
		NEUROG2 //
		neurogenin 2 //
		4q25 // 63973 ///
		ENST00000313341 //
		NEU
3826542	ZNF738	BC034499 //	BC034499	−0.20	0.05	0.003
		ZNF738 // zinc
		finger protein 738 //
		19p12 // 148203 ///
		AK291002 //
3966000	TYMP	NM_001113756 //	NM_001113756	−0.20	0.05	0.003
		TYMP // thymidine
		phosphorylase //
		22q13|22q13.33 //
		1890 /// NM
3607447	ABHD2	NM_007011 //	NM_007011	−0.20	0.05	0.005
		ABHD2 //
		abhydrolase domain
		containing 2 //
		15q26.1 // 11057 ///
		NM
3236448	SUV39H2	NM_024670 //	NM_024670	−0.20	0.07	0.011
		SUV39H2 //
		suppressor of
		variegation 3-9
		homolog 2
		(Drosophila) //
2528504	SPEG	NM_005876 //	NM_005876	−0.20	0.06	0.009
		SPEG // SPEG
		complex locus //
		2q35 // 10290 ///
		ENST00000312358 //
2730746	SLC4A4	NM_001098484 //	NM_001098484	−0.20	0.06	0.007
		SLC4A4 // solute
		carrier family 4,
		sodium bicarbonate
		cotranspor
2544662	DNMT3A	NM_175629 //	NM_175629	−0.20	0.06	0.007
		DNMT3A // DNA
		(cytosine-5-)-
		methyltransferase 3
		alpha // 2p23 // 17
2937625	C6orf208	BC101251 //	BC101251	−0.20	0.06	0.007
		C6orf208 //
		chromosome 6
		open reading frame
		208 // 6q27 //
		80069 ///
3233157	UCN3	NM_053049 //	NM_053049	−0.20	0.08	0.017
		UCN3 // urocortin 3
		(stresscopin) //
		10p15.1 // 114131 ///
		ENST0000
2548172	FEZ2	NM_001042548 //	NM_001042548	−0.21	0.03	0.000
		FEZ2 //
		fasciculation and
		elongation protein
		zeta 2 (zygin II)/
3877809	OTOR	NM_020157 //	NM_020157	−0.21	0.08	0.019
		OTOR // otoraplin //
		20p12.1-p11.23 //
		56914 ///
		ENST00000246081 //
3839400	C19orf63	NM_175063 //	NM_175063	−0.21	0.04	0.002
		C19orf63 //
		chromosome 19
		open reading frame
		63 // 19q13.33 //
		2843
3875108	C20orf196	AK292708 //	AK292708	−0.21	0.06	0.006
		C20orf196 //
		chromosome 20
		open reading frame
		196 // 20p12.3 //
		1498
2970985	TSPYL4	NM_021648 //	NM_021648	−0.21	0.07	0.011
		TSPYL4 // TSPY-
		like 4 // 6q22.1 //
		23270 ///
		ENST00000368611 //
		TSP
3189580	ZBTB43	NM_014007 //	NM_014007	−0.21	0.08	0.017
		ZBTB43 // zinc
		finger and BTB
		domain containing
		43 // 9q33-q34 // 2
3407926	CMAS	NM_018686 //	NM_018686	−0.21	0.03	0.000
		CMAS // cytidine
		monophosphate N-
		acetylneuraminic
		acid synthetase/
3249886	TET1	NM_030625 //	NM_030625	−0.21	0.06	0.007
		TET1 // tet
		oncogene 1 //
		10q21 // 80312 ///
		ENST00000373644 //
		TET
3151970	MTSS1	NM_014751 //	NM_014751	−0.21	0.07	0.009
		MTSS1 //
		metastasis
		suppressor 1 //
		8p22 // 9788 ///
		ENST0000032506
3937183	DGCR8	NM_022720 //	NM_022720	−0.21	0.06	0.008
		DGCR8 //
		DiGeorge
		syndrome critical
		region gene 8 //
		22q11.2 // 544
3958253	C22orf28	BC016707 //	BC016707	−0.22	0.08	0.019
		C22orf28 //
		chromosome 22
		open reading frame
		28 // 22q12 //
		51493 //
3607503	ABHD2	NM_007011 //	NM_007011	−0.22	0.07	0.010
		ABHD2 //
		abhydrolase domain
		containing 2 //
		15q26.1 // 11057 ///
		NM
2799030	SLC6A19	NM_001003841 //	NM_001003841	−0.22	0.06	0.007
		SLC6A19 // solute
		carrier family 6
		(neutral amino acid
		transport
3870611	LILRB3	NM_001081450 //	NM_001081450	−0.22	0.08	0.016
		LILRB3 //
		leukocyte
		immunoglobulin-
		like receptor,
		subfamily B (w
3857811	C19orf12	NM_031448 //	NM_031448	−0.22	0.08	0.019
		C19orf12 //
		chromosome 19
		open reading frame
		12 // 19q12 //
		83636/
2500667	FBLN7	NM_153214 //	NM_153214	−0.22	0.08	0.019
		FBLN7 // fibulin 7 //
		2q13 // 129804 ///
		ENST00000331203 //
		FBLN7/
3523156	TMTC4	NM_032813 //	NM_032813	−0.22	0.07	0.010
		TMTC4 //
		transmembrane and
		tetratricopeptide
		repeat containing
		4 //
2612371	EAF1	NM_033083 //	NM_033083	−0.22	0.07	0.008
		EAF1 // ELL
		associated factor 1 //
		3p24.3 // 85403 ///
		ENST00000396
3988638	LONRF3	NM_001031855 //	NM_001031855	−0.23	0.08	0.012
		LONRF3 // LON
		peptidase N-
		terminal domain
		and ring finger 3 // X
3114240	C8orf32	BC008781 //	BC008781	−0.23	0.08	0.016
		C8orf32 //
		chromosome 8
		open reading frame
		32 // 8q24.13 //
		55093 //
2460368	TTC13	NM_024525 //	NM_024525	−0.23	0.08	0.014
		TTC13 //
		tetratricopeptide
		repeat domain 13 //
		1q42.2 // 79573 ///
2428425	PPM1J	NM_005167 //	NM_005167	−0.23	0.06	0.003
		PPM1J // protein
		phosphatase 1J
		(PP2C domain
		containing) //
		1p13.2
3194986	LCN12	NM_178536 //	NM_178536	−0.23	0.06	0.004
		LCN12 // lipocalin
		12 // 9q34.3 //
		286256 ///
		ENST00000371633 //
		LC
3642875	RAB11FIP3	NM_014700 //	NM_014700	−0.23	0.07	0.010
		RAB11FIP3 //
		RAB11 family
		interacting protein
		3 (class II) // 16p13
2532378	CHRND	NM_000751 //	NM_000751	−0.23	0.08	0.018
		CHRND //
		cholinergic
		receptor, nicotinic,
		delta // 2q33-q34 //
		1144
2995667	ADCYAP1R1	NM_001118 //	NM_001118	−0.23	0.05	0.002
		ADCYAP1R1 //
		adenylate cyclase
		activating
		polypeptide 1
		(pituitary)
3390641	ARHGAP20	NM_020809 //	NM_020809	−0.23	0.05	0.003
		ARHGAP20 // Rho
		GTPase activating
		protein 20 //
		11q22.3-q23.1 // 57
2830465	MYOT	NM_006790 //	NM_006790	−0.23	0.07	0.007
		MYOT // myotilin //
		5q31 // 9499 ///
		ENST00000239926 //
		MYOT // myo
2452069	PIK3C2B	NM_002646 //	NM_002646	−0.23	0.02	0.000
		PIK3C2B //
		phosphoinositide-3-
		kinase, class 2,
		beta polypeptide //
3744127	HES7	NM_032580 //	NM_032580	−0.23	0.09	0.019
		HES7 // hairy and
		enhancer of split 7
		(Drosophila) //
		17p13.1 // 84
3327057	FLJ14213	NM_024841 //	NM_024841	−0.23	0.07	0.007
		FLJ14213 // protor-
		2 // 11p13-p12 //
		79899 ///
		ENST00000378867 // F
2664332	COLQ	NM_005677 //	NM_005677	−0.23	0.07	0.006
		COLQ // collagen-
		like tail subunit
		(single strand of
		homotrimer) of
3829160	C19orf40	NM_152266 //	NM_152266	−0.23	0.08	0.012
		C19orf40 //
		chromosome 19
		open reading frame
		40 // 19q13.11 //
		9144
3708798	SENP3	NM_015670 //	NM_015670	−0.23	0.06	0.005
		SENP3 //
		SUMO1/sentrin/SM
		T3 specific
		peptidase 3 //
		17p13 // 26168
2358700	MGC29891	NM_144618 //	NM_144618	−0.23	0.09	0.019
		MGC29891 //
		hypothetical
		protein
		MGC29891 //
		1q21.2 //
		126626 /// E
2755111	KLKB1	NM_000892 //	NM_000892	−0.24	0.08	0.012
		KLKB1 //
		kallikrein B,
		plasma
		(Fletcher factor)
		1 // 4q34-q35 // 38
2568968	UXS1	NM_025076 //	NM_025076	−0.24	0.08	0.011
		UXS1 // UDP-
		glucuronate
		decarboxylase 1 //
		2q12.2 // 80146 ///
		BC00
2748923	GUCY1B3	NM_000857 //	NM_000857	−0.24	0.07	0.007
		GUCY1B3 //
		guanylate
		cyclase 1,
		soluble, beta 3 //
		4q31.3-q33 // 29
3816509	GADD45B	NM_015675 //	NM_015675	−0.24	0.09	0.016
		GADD45B //
		growth arrest and
		DNA-damage-
		inducible, beta //
		19p13.3
3376410	SLC22A24	BC034394 //	BC034394	−0.24	0.07	0.007
		SLC22A24 // solute
		carrier family 22,
		member 24 //
		11q12.3 // 283238
3286393	ZNF32	NM_006973 //	NM_006973	−0.24	0.08	0.010
		ZNF32 // zinc
		finger protein 32 //
		10q22-q25 // 7580 ///
		NM_0010053
2540157	ODC1	NM_002539 //	NM_002539	−0.24	0.09	0.020
		ODC1 // ornithine
		decarboxylase 1 //
		2p25 // 4953 ///
		ENST000002341
2994835	CHN2	NM_004067 //	NM_004067	−0.24	0.09	0.017
		CHN2 // chimerin
		(chimaerin) 2 //
		7p15.3 // 1124 ///
		NM_001039936/
3603199	IDH3A	NM_005530 //	NM_005530	−0.24	0.05	0.001
		IDH3A // isocitrate
		dehydrogenase 3
		(NAD+) alpha //
		15q25.1-q25.2/
3040454	TWISTNB	NM_001002926 //	NM_001002926	−0.24	0.09	0.017
		TWISTNB //
		TWIST neighbor //
		7p15.3 // 221830 ///
		ENST0000022256
2497301	TMEM182	NM_144632 //	NM_144632	−0.24	0.07	0.007
		TMEM182 //
		transmembrane
		protein 182 //
		2q12.1 // 130827 ///
		ENST00
3766716	TEX2	NM_018469 //	NM_018469	−0.25	0.07	0.007
		TEX2 // testis
		expressed 2 //
		17q23.3 // 55852 ///
		ENST00000258991
3458819	CYP27B1	NM_000785 //	NM_000785	−0.25	0.08	0.009
		CYP27B1 //
		cytochrome P450,
		family 27,
		subfamily B,
		polypeptide 1/
3368940	ABTB2	NM_145804 //	NM_145804	−0.25	0.08	0.010
		ABTB2 // ankyrin
		repeat and BTB
		(POZ) domain
		containing 2 //
		11p13
3298924	MMRN2	NM_024756 //	NM_024756	−0.25	0.07	0.006
		MMRN2 //
		multimerin 2 //
		10q23.2 // 79812 ///
		ENST00000372027 //
		MM
3529951	KIAA1305	NM_025081 //	NM_025081	−0.25	0.08	0.011
		KIAA1305 //
		KIAA1305 //
		14q12 // 57523 ///
		BC008219 //
		KIAA1305 //
3006572	AUTS2	NM_015570 //	NM_015570	−0.25	0.09	0.017
		AUTS2 // autism
		susceptibility
		candidate 2 //
		7q11.22 // 26053 ///
3025500	BPGM	NM_001724 //	NM_001724	−0.25	0.10	0.018
		BPGM // 2,3-
		bisphosphoglycerate
		mutase // 7q31-
		q34 // 669 ///
		NM_19
2494709	CNNM4	NM_020184 //	NM_020184	−0.26	0.09	0.016
		CNNM4 // cyclin
		M4 // 2p12-p11.2 //
		26504 ///
		ENST00000377075 //
		CN
3329983	PTPRJ	NM_002843 //	NM_002843	−0.26	0.08	0.010
		PTPRJ // protein
		tyrosine
		phosphatase,
		receptor type, J //
		11p11.2
2769346	LNX1	NM_032622 //	NM_032622	−0.26	0.09	0.015
		LNX1 // ligand of
		numb-protein X 1 //
		4q12 // 84708 ///
		ENST0000030
3867195	FAM83E	NM_017708 //	NM_017708	−0.26	0.09	0.013
		FAM83E // family
		with sequence
		similarity 83,
		member E //
		19q13.32-
3790529	GRP	NM_002091 //	NM_002091	−0.26	0.05	0.001
		GRP // gastrin-
		releasing peptide //
		18q21.1-q21.32 //
		2922 /// NM_0
3987029	TMEM164	NM_032227 //	NM_032227	−0.26	0.10	0.018
		TMEM164 //
		transmembrane
		protein 164 //
		Xq22.3 // 84187 ///
		ENST000
3526454	GRTP1	NM_024719 //	NM_024719	−0.26	0.09	0.015
		GRTP1 // growth
		hormone regulated
		TBC protein 1 //
		13q34 // 79774/
2438344	GPATCH4	NM_182679 //	NM_182679	−0.26	0.07	0.006
		GPATCH4 // G
		patch domain
		containing 4 //
		1q22 // 54865 ///
		NM_0155
3132927	NKX6-3	NM_152568 //	NM_152568	−0.27	0.09	0.014
		NKX6-3 // NK6
		homeobox 3 //
		8p11.21 // 157848 ///
		ENST00000343444/
2672376	TESSP2	NM_182702 //	NM_182702	−0.27	0.09	0.013
		TESSP2 // testis
		serine protease 2 //
		3p21.31 // 339906 ///
		ENST000
2730347	C4orf35	NM_033122 //	NM_033122	−0.27	0.10	0.019
		C4orf35 //
		chromosome 4
		open reading frame
		35 // 4q13.3 //
		85438 //
3921068	ETS2	NM_005239 //	NM_005239	−0.27	0.03	0.000
		ETS2 // v-ets
		erythroblastosis
		virus E26 oncogene
		homolog 2 (avian)
2532894	DGKD	NM_152879 //	NM_152879	−0.27	0.07	0.003
		DGKD //
		diacylglycerol
		kinase, delta
		130 kDa // 2q37.1 //
		8527 /// N
4018454	AMOT	NM_133265 //	NM_133265	−0.27	0.09	0.012
		AMOT //
		angiomotin // Xq23 //
		154796 ///
		NM_001113490 //
		AMOT // an
3070507	RNF148	NM_198085 //	NM_198085	−0.27	0.10	0.017
		RNF148 // ring
		finger protein 148 //
		7q31.33 // 378925 ///
		BC029264
3832256	SPINT2	NM_021102 //	NM_021102	−0.27	0.10	0.017
		SPINT2 // serine
		peptidase inhibitor,
		Kunitz type, 2 //
		19q13.1 //
3371225	CHST1	NM_003654 //	NM_003654	−0.27	0.07	0.005
		CHST1 //
		carbohydrate
		(keratan sulfate
		Gal-6)
		sulfotransferase 1 //
3870494	TFPT	NM_013342 //	NM_013342	−0.27	0.09	0.010
		TFPT // TCF3
		(E2A) fusion
		partner (in
		childhood
		Leukemia) // 19q13
3863811	PSG9	NM_002784 //	NM_002784	−0.28	0.09	0.011
		PSG9 // pregnancy
		specific beta-1-
		glycoprotein 9 //
		19q13.2 // 5678
3160175	VLDLR	NM_003383 //	NM_003383	−0.28	0.08	0.007
		VLDLR // very low
		density lipoprotein
		receptor // 9p24 //
		7436 ///
2794704	ASB5	NM_080874 //	NM_080874	−0.28	0.11	0.019
		ASB5 // ankyrin
		repeat and SOCS
		box-containing 5 //
		4q34.2 // 14045
3908901	KCNB1	NM_004975 //	NM_004975	−0.28	0.09	0.009
		KCNB1 //
		potassium voltage-
		gated channel,
		Shab-related
		subfamily, m
3390852	FLJ45803	NM_207429 //	NM_207429	−0.28	0.10	0.015
		FLJ45803 //
		FLJ45803 protein //
		11q23.1 // 399948 ///
		ENST000003554
2600689	EPHA4	NM_004438 //	NM_004438	−0.29	0.07	0.003
		EPHA4 // EPH
		receptor A4 //
		2q36.1 // 2043 ///
		ENST00000281821 // E
3469597	NUAK1	NM_014840 //	NM_014840	−0.29	0.09	0.009
		NUAK1 // NUAK
		family, SNF1-like
		kinase, 1 //
		12q23.3 // 9891 ///
		EN
3607232	ISG20L1	NM_022767 //	NM_022767	−0.29	0.10	0.015
		ISG20L1 //
		interferon
		stimulated
		exonuclease gene
		20 kDa-like 1 // 1
2358426	ADAMTSL4	AK023606 //	AK023606	−0.29	0.11	0.016
		ADAMTSL4 //
		ADAMTS-like 4 //
		1q21.2 // 54507
3853609	CYP4F2	NM_001082 //	NM_001082	−0.29	0.11	0.016
		CYP4F2 //
		cytochrome P450,
		family 4, subfamily
		F, polypeptide 2 //
2936971	KIF25	NM_030615 //	NM_030615	−0.30	0.09	0.008
		KIF25 // kinesin
		family member 25 //
		6q27 // 3834 ///
		NM_005355 //
2997272	EEPD1	NM_030636 //	NM_030636	−0.30	0.09	0.010
		EEPD1 //
		endonuclease/
		exonuclease/
		phosphatase
		family domain
		contain
3961253	RPS19BP1	NM_194326 //	NM_194326	−0.30	0.10	0.013
		RPS19BP1 //
		ribosomal protein
		S19 binding protein
		1 // 22q13.1 // 9
3082373	VIPR2	NM_003382 //	NM_003382	−0.30	0.10	0.011
		VIPR2 // vasoactive
		intestinal peptide
		receptor 2 // 7q36.3 //
		7434
2340961	IL12RB2	NM_001559 //	NM_001559	−0.30	0.08	0.005
		IL12RB2 //
		interleukin 12
		receptor, beta 2 //
		1p31.3-p31.2 //
		3595
2736462	BMPR1B	NM_001203 //	NM_001203	−0.30	0.08	0.004
		BMPR1B // bone
		morphogenetic
		protein receptor,
		type IB // 4q22-q24
3774504		—	—	−0.30	0.11	0.016
3395958	OR8B4	NM_001005196 //	NM_001005196	−0.30	0.11	0.018
		OR8B4 // olfactory
		receptor, family 8,
		subfamily B,
		member 4 //
2806231	BXDC2	NM_018321 //	NM_018321	−0.31	0.10	0.013
		BXDC2 // brix
		domain containing
		2 // 5p13.2 // 55299 ///
		ENST000003
2396858	NPPB	NM_002521 //	NM_002521	−0.31	0.11	0.016
		NPPB // natriuretic
		peptide precursor
		B // 1p36.2 // 4879 ///
		ENST0
3233322	C10orf18	NM_017782 //	NM_017782	−0.31	0.06	0.001
		C10orf18 //
		chromosome 10
		open reading frame
		18 // 10p15.1 //
		54906
2439101	FCRL1	NM_052938 //	NM_052938	−0.31	0.06	0.001
		FCRL1 // Fc
		receptor-like 1 //
		1q21-q22 // 115350 ///
		ENST000003681
2413907	DHCR24	NM_014762 //	NM_014762	−0.31	0.11	0.014
		DHCR24 // 24-
		dehydrocholesterol
		reductase // 1p33-
		p31.1 // 1718 ///
3231186	C9orf37	NM_032937 //	NM_032937	−0.31	0.09	0.008
		C9orf37 //
		chromosome 9
		open reading frame
		37 // 9q34.3 //
		85026 //
2669955	XIRP1	NM_194293 //	NM_194293	−0.32	0.11	0.013
		XIRP1 // xin actin-
		binding repeat
		containing 1 //
		3p22.2 // 165904
3345222	AMOTL1	NM_130847 //	NM_130847	−0.32	0.11	0.012
		AMOTL1 //
		angiomotin like 1 //
		11q14.3 // 154810 ///
		ENST0000031782
2573326	FLJ14816	BC112205 //	BC112205	−0.32	0.11	0.016
		FLJ14816 //
		hypothetical protein
		FLJ14816 // 2q14.2 //
		84931 /// BC1
3349437	UNQ2550	AY358815 //	AY358815	−0.32	0.09	0.005
		UNQ2550 //
		SFVP2550 //
		11q23.1 //
		100130653
3951117	ACR	NM_001097 //	NM_001097	−0.32	0.12	0.017
		ACR // acrosin //
		22q13-
		qter|22q13.33 // 49 ///
		ENST00000216139 //
2489140		—	—	−0.32	0.07	0.002
2562115	LSM3	CR457185 // LSM3 //	CR457185	−0.32	0.11	0.011
		LSM3 homolog,
		U6 small nuclear
		RNA associated
		(S. cerevisiae
3572975	NGB	NM_021257 //	NM_021257	−0.33	0.09	0.004
		NGB // neuroglobin //
		14q24.3 // 58157 ///
		ENST00000298352 //
		NGB/
2439350	OR6N1	NM_001005185 //	NM_001005185	−0.33	0.10	0.009
		OR6N1 // olfactory
		receptor, family 6,
		subfamily N,
		member 1 //
3590275	CHAC1	NM_024111 //	NM_024111	−0.33	0.12	0.014
		CHAC1 // ChaC,
		cation transport
		regulator homolog
		1 (E. coli) // 15
2397898	HSPB7	NM_014424 //	NM_014424	−0.33	0.12	0.015
		HSPB7 // heat
		shock 27 kDa
		protein family,
		member 7
		(cardiovascular)
2364677	PBX1	NM_002585 //	NM_002585	−0.34	0.07	0.001
		PBX1 // pre-B-cell
		leukemia
		homeobox 1 // 1q23 //
		5087 ///
		ENST0000
2474409	DNAJC5G	NM_173650 //	NM_173650	−0.34	0.09	0.004
		DNAJC5G // DnaJ
		(Hsp40) homolog,
		subfamily C,
		member 5 gamma //
		2p2
3581373		—	—	−0.34	0.12	0.014
3508330	HSPH1	NM_006644 //	NM_006644	−0.34	0.13	0.019
		HSPH1 // heat
		shock
		105 kDa/110 kDa
		protein 1 // 13q12.3 //
		10808 ///
3751164	DHRS13	NM_144683 //	NM_144683	−0.35	0.10	0.006
		DHRS13 //
		dehydrogenase/
		reductase (SDR
		family)
		member 13 //
		17q11.2
2908179	VEGFA	NM_001025366 //	NM_001025366	−0.35	0.13	0.016
		VEGFA // vascular
		endothelial growth
		factor A // 6p12 //
		7422 //
3962448	dJ222E13.2	NR_002184 //	NR_002184	−0.35	0.12	0.014
		dJ222E13.2 //
		similar to CGI-96 //
		22q13.2 // 91695 ///
		BC073834 //
3747638	LOC201164	BC031263 //	BC031263	−0.35	0.09	0.004
		LOC201164 //
		similar to CG12314
		gene product //
		17p11.2 // 201164 //
2821981	TMEM157	NM_198507 //	NM_198507	−0.35	0.12	0.015
		TMEM157 //
		transmembrane
		protein 157 //
		5q21.1 // 345757 ///
		ENST00
3123675	PPP1R3B	NM_024607 //	NM_024607	−0.35	0.12	0.014
		PPP1R3B // protein
		phosphatase 1,
		regulatory
		(inhibitor) subunit
		3B
2656837	ST6GAL1	NM_173216 //	NM_173216	−0.35	0.13	0.016
		ST6GAL1 // ST6
		beta-galactosamide
		alpha-2,6-
		sialyltranferase
		1 // 3
3746574	PMP22	NM_000304 //	NM_000304	−0.36	0.09	0.004
		PMP22 //
		peripheral myelin
		protein 22 // 17p12-
		p11.2 // 5376 ///
		NM
2771342	EPHA5	NM_004439 //	NM_004439	−0.36	0.09	0.003
		EPHA5 // EPH
		receptor A5 //
		4q13.1 // 2044 ///
		NM_182472 //
		EPHA5/
2888674	MXD3	NM_031300 //	NM_031300	−0.36	0.12	0.012
		MXD3 // MAX
		dimerization
		protein 3 // 5q35.3 //
		83463 ///
		ENST00000
2353477	ATP1A1	NM_000701 //	NM_000701	−0.36	0.11	0.007
		ATP1A1 // ATPase,
		Na+/K+
		transporting, alpha
		1 polypeptide //
		1p21
3956984	ZMAT5	NM_019103 //	NM_019103	−0.36	0.11	0.009
		ZMAT5 // zinc
		finger, matrin type
		5 // 22cen-q12.3 //
		55954 /// NM—
2551651	ATP6V1E2	NM_080653 //	NM_080653	−0.37	0.13	0.017
		ATP6V1E2 //
		ATPase, H+
		transporting,
		lysosomal 31 kDa,
		V1 subunit E2
3578069	C14orf139	BC008299 //	BC008299	−0.37	0.13	0.016
		C14orf139 //
		chromosome 14
		open reading frame
		139 // 14q32.13 //
		796
2428501	SLC16A1	NM_003051 //	NM_003051	−0.37	0.14	0.018
		SLC16A1 // solute
		carrier family 16,
		member 1
		(monocarboxylic
		acid
3061621	TFPI2	NM_006528 //	NM_006528	−0.37	0.09	0.002
		TFPI2 // tissue
		factor pathway
		inhibitor 2 // 7q22 //
		7980 /// ENST
3705516	LOC100131454	AF229804 //	AF229804	−0.38	0.11	0.008
		LOC100131454 //
		similar to
		hCG1646635 //
		17p13.3 //
		100131454 /// EN
3306299	XPNPEP1	NM_020383 //	NM_020383	−0.38	0.14	0.018
		XPNPEP1 // X-
		prolyl
		aminopeptidase
		(aminopeptidase P)
		1, soluble //
2763550	PPARGC1A	NM_013261 //	NM_013261	−0.38	0.13	0.012
		PPARGC1A //
		peroxisome
		proliferator-
		activated receptor
		gamma, coact
2769063	USP46	NM_022832 //	NM_022832	−0.38	0.13	0.013
		USP46 // ubiquitin
		specific peptidase
		46 // 4q12 // 64854 ///
		ENST0
3806459	ST8SIA5	NM_013305 //	NM_013305	−0.38	0.10	0.004
		ST8SIA5 // ST8
		alpha-N-acetyl-
		neuraminide alpha-
		2,8-sialyltransfera
3190151	SLC25A25	NM_001006641 //	NM_001006641	−0.39	0.09	0.003
		SLC25A25 // solute
		carrier family 25
		(mitochondrial
		carrier; pho
2489172	MTHFD2	NM_001040409 //	NM_001040409	−0.39	0.05	0.000
		MTHFD2 //
		methylenetetra-
		hydrofolate
		dehydrogenase
		(NADP+ depende
2952065	PPIL1	NM_016059 //	NM_016059	−0.39	0.10	0.005
		PPIL1 //
		peptidylprolyl
		isomerase
		(cyclophilin)-like 1 //
		6p21.1 //
3382015	CHRDL2	NM_015424 //	NM_015424	−0.39	0.10	0.003
		CHRDL2 //
		chordin-like 2 //
		11q14 // 25884 ///
		ENST00000263671 // C
2711139	ATP13A5	NM_198505 //	NM_198505	−0.40	0.11	0.005
		ATP13A5 //
		ATPase type 13A5 //
		3q29 // 344905 ///
		ENST00000342358/
2633917	RG9MTD1	NM_017819 //	NM_017819	−0.41	0.14	0.013
		RG9MTD1 // RNA
		(guanine-9-)
		methyltransferase
		domain containing 1/
2974671	C6orf192	NM_052831 //	NM_052831	−0.41	0.15	0.018
		C6orf192 //
		chromosome 6
		open reading frame
		192 // 6q22.3-q23.3 //
2982270	FLJ27255	ENST00000355047 //	ENST00000355047	−0.41	0.12	0.007
		FLJ27255 //
		hypothetical
		LOC401281 //
		6q25.3 // 401281 ///
		AK
2778273	PGDS	NM_014485 //	NM_014485	−0.41	0.08	0.001
		PGDS //
		prostaglandin D2
		synthase,
		hematopoietic //
		4q22.3 // 27306
3005332	RCP9	NM_014478 //	NM_014478	−0.41	0.14	0.013
		RCP9 // calcitonin
		gene-related
		peptide-receptor
		component protein
2650393	PPM1L	NM_139245 //	NM_139245	−0.42	0.12	0.006
		PPM1L // protein
		phosphatase 1
		(formerly 2C)-like //
		3q26.1 // 1517
3463056	CSRP2	NM_001321 //	NM_001321	−0.42	0.11	0.005
		CSRP2 // cysteine
		and glycine-rich
		protein 2 // 12q21.1 //
		1466 ///
2459405		—	—	−0.43	0.10	0.003
2570238	NPHP1	NM_000272 //	NM_000272	−0.43	0.06	0.000
		NPHP1 //
		nephronophthisis 1
		(juvenile) // 2q13 //
		4867 /// NM_20718
2840616	NPM1	NM_002520 //	NM_002520	−0.43	0.14	0.010
		NPM1 //
		nucleophosmin
		(nucleolar
		phosphoprotein
		B23, numatrin) // 5
3601051	NEO1	NM_002499 //	NM_002499	−0.43	0.09	0.002
		NEO1 // neogenin
		homolog 1
		(chicken) //
		15q22.3-q23 //
		4756 /// ENS
3936515	TUBA8	NM_018943 //	NM_018943	−0.43	0.10	0.002
		TUBA8 // tubulin,
		alpha 8 // 22q11.1 //
		51807 ///
		ENST00000330423/
2725013	UCHL1	NM_004181 //	NM_004181	−0.44	0.11	0.004
		UCHL1 // ubiquitin
		carboxyl-terminal
		esterase L1
		(ubiquitin thioles
2380590	TGFB2	NM_003238 //	NM_003238	−0.44	0.16	0.017
		TGFB2 //
		transforming
		growth factor, beta
		2 // 1q41 // 7042 ///
		ENS
2496382	NPAS2	NM_002518 //	NM_002518	−0.46	0.10	0.002
		NPAS2 // neuronal
		PAS domain
		protein 2 // 2q11.2 //
		4862 /// ENST00
3841574	LILRB1	NM_006669 //	NM_006669	−0.46	0.16	0.015
		LILRB1 //
		leukocyte
		immunoglobulin-
		like receptor,
		subfamily B (with
3726960	NME2	NM_001018137 //	NM_001018137	−0.47	0.16	0.013
		NME2 // non-
		metastatic cells 2,
		protein (NM23B)
		expressed in //
2649367	PTX3	NM_002852 //	NM_002852	−0.47	0.11	0.002
		PTX3 // pentraxin-
		related gene,
		rapidly induced by
		IL-1 beta // 3q2
2909483	GPR111	NM_153839 //	NM_153839	−0.47	0.13	0.006
		GPR111 // G
		protein-coupled
		receptor 111 //
		6p12.3 // 222611 ///
		EN
2881950	SLC36A2	NM_181776 //	NM_181776	−0.48	0.12	0.004
		SLC36A2 // solute
		carrier family 36
		(proton/amino acid
		symporter),
3441190	FGF6	NM_020996 //	NM_020996	−0.48	0.12	0.004
		FGF6 // fibroblast
		growth factor 6 //
		12p13 // 2251 ///
		ENST0000022
3028911	C7orf34	NM_178829 //	NM_178829	−0.49	0.18	0.019
		C7orf34 //
		chromosome 7
		open reading frame
		34 // 7q34 //
		135927 ///
2830861	EGR1	NM_001964 //	NM_001964	−0.49	0.19	0.020
		EGR1 // early
		growth response 1 //
		5q31.1 // 1958 ///
		ENST000002399
3323891	GAS2	NM_177553 //	NM_177553	−0.49	0.16	0.011
		GAS2 // growth
		arrest-specific 2 //
		11p14.3-p15.2 //
		2620 /// NM_00
2497252	SLC9A2	NM_003048 //	NM_003048	−0.50	0.11	0.002
		SLC9A2 // solute
		carrier family 9
		(sodium/hydrogen
		exchanger), memb
3018484	GPR22	NM_005295 //	NM_005295	−0.51	0.15	0.008
		GPR22 // G
		protein-coupled
		receptor 22 // 7q22-
		q31.1 // 2845 /// EN
2712632	TFRC	NM_003234 //	NM_003234	−0.51	0.12	0.003
		TFRC // transferrin
		receptor (p90,
		CD71) // 3q29 //
		7037 /// ENST00
3214451	NFIL3	NM_005384 //	NM_005384	−0.53	0.14	0.004
		NFIL3 // nuclear
		factor, interleukin 3
		regulated // 9q22 //
		4783 //
2435981	S100A12	NM_005621 //	NM_005621	−0.54	0.19	0.014
		S100A12 // S100
		calcium binding
		protein A12 // 1q21 //
		6283 /// ENS
3320675	RIG	U32331 // RIG //	U32331	−0.54	0.10	0.001
		regulated in glioma //
		11p15.1 // 10530
3290746	SLC16A9	NM_194298 //	NM_194298	−0.54	0.15	0.006
		SLC16A9 // solute
		carrier family 16,
		member 9
		(monocarboxylic
		acid
3055703	NSUN5C	NM_032158 //	NM_032158	−0.57	0.17	0.008
		NSUN5C //
		NOL1/NOP2/Sun
		domain family,
		member 5C //
		7q11.23 // 2602
3265494	TRUB1	NM_139169 //	NM_139169	−0.57	0.17	0.008
		TRUB1 // TruB
		pseudouridine (psi)
		synthase homolog 1
		(E. coli) // 1
3374213	OR1S2	NM_001004459 //	NM_001004459	−0.58	0.20	0.013
		OR1S2 // olfactory
		receptor, family 1,
		subfamily S,
		member 2 //
3318253	OR51L1	NM_001004755 //	NM_001004755	−0.59	0.18	0.009
		OR51L1 //
		olfactory receptor,
		family 51,
		subfamily L,
		member 1/
3294280	DNAJC9	NM_015190 //	NM_015190	−0.59	0.22	0.018
		DNAJC9 // DnaJ
		(Hsp40) homolog,
		subfamily C,
		member 9 //
		10q22.2 //
2899095	HIST1H4A	NM_003538 //	NM_003538	−0.60	0.16	0.005
		HIST1H4A //
		histone cluster 1,
		H4a // 6p21.3 //
		8359 ///
		ENST000003
2378068	G0S2	NM_015714 //	NM_015714	−0.63	0.22	0.016
		G0S2 //
		G0/G1switch 2 //
		1q32.2-q41 //
		50486 ///
		ENST00000367029 //
3737677	LOC100129503	AF218021 //	AF218021	−0.64	0.19	0.007
		LOC100129503 //
		hypothetical protein
		LOC100129503 //
		17q25.3 // 1001
3300115	PPP1R3C	NM_005398 //	NM_005398	−0.69	0.26	0.020
		PPP1R3C // protein
		phosphatase 1,
		regulatory
		(inhibitor) subunit
		3C
3279058	ACBD7	NM_001039844 //	NM_001039844	−0.69	0.13	0.001
		ACBD7 // acyl-
		Coenzyme A
		binding domain
		containing 7 //
		10p13 //
4031156	RPS4Y2	NM_001039567 //	NM_001039567	−0.71	0.17	0.003
		RPS4Y2 //
		ribosomal protein
		S4, Y-linked 2 //
		Yq11.223 // 140032
2979246	RAET1L	NM_130900 //	NM_130900	−0.75	0.26	0.013
		RAET1L // retinoic
		acid early transcript
		1L // 6q25.1 //
		154064 ///
3321150	ARNTL	NM_001178 //	NM_001178	−0.80	0.20	0.004
		ARNTL // aryl
		hydrocarbon
		receptor nuclear
		translocator-like //
		11p
3862873	CYP2A6	NM_000762 //	NM_000762	−1.12	0.34	0.009
		CYP2A6 //
		cytochrome P450,
		family 2, subfamily
		A, polypeptide 6 //

4. Identification of Ursolic Acid as an Inhibitor of Fasting-Induced Muscle Atrophy.

The Connectivity Map describes the effects of >1300 bioactive small molecules on global mRNA expression in several cultured cell lines, and contains search algorithms that permit comparisons between compound-specific mRNA expression signatures and mRNA expression signatures of interest (Lamb J, et al. (2006) Science (New York, N.Y. 313(5795):1929-1935). It was hypothesized herein that querying the Connectivity Map with the mRNA expression signature of fasting (atrophy signature-1) would identify inhibitors of atrophy-associated gene expression and thus, potential inhibitors of muscle atrophy. It was also reasoned herein that increasing the specificity of the query would enhance the output. To this end, as described herein, an evolutionarily conserved mRNA expression signature of fasting was discovered by comparing the effect of fasting on human skeletal muscle to the effect of a 24 h fast on mouse skeletal muscle. The mouse studies were described previously (Ebert S M, et al. (2010) Molecular endocrinology 24(4):790-799). Altogether, 35 mRNAs that were increased by fasting and 40 mRNAs that were decreased by fasting were identified in both human and mouse skeletal muscle (Table 2; the data in column labeled “Change” show mean changes in loge hybridization signals between fasting and fed states for the species indicated, [Mean log₂ mRNA levels for fasted] minus [Mean loge mRNA levels in unfasted]; P-values were determined with paired t-tests). The data shown in Table 2 includes all mRNAs whose levels were increased by fasting in human muscle (P≤0.02) and in mouse muscle (P≤0.05), and all mRNAs whose levels were decreased by fasting in human muscle (P≤0.02) and in mouse muscle (P≤0.05). Of the mRNAs shown in Table 2, 63 mRNAs were represented on the HG-U133A arrays used in the Connectivity Map (FIG. 4A). These mRNAs (31 increased by fasting and 32 decreased by fasting) were used to query the Connectivity Map for candidate small molecule inhibitors of muscle atrophy.

TABLE 2
FASTING-REGULATED MRNAs COMMON TO HUMAN
AND MOUSE SKELETAL MUSCLE.
	Human	Mouse
	Mean Log2	Mean Log2
	Change	Change
		(Fasting-		(Fasting-
mRNA	Protein	Fed)	P	Fed)	P
PDK4	pyruvate dehydrogenase	2.15	0.000	1.91	0.000
	kinase, isozyme 4
TXNIP	thioredoxin interacting	0.85	0.004	0.60	0.038
	protein
FBXO32	F-box protein 32	0.82	0.002	2.13	0.000
SLC38A2	solute carrier family 38,	0.62	0.001	0.33	0.036
	member 2
UCP3	uncoupling protein 3	0.59	0.000	1.02	0.001
	(mitochondrial, proton
	carrier)
ZFAND5	zinc finger, AN1-type	0.51	0.005	0.57	0.001
	domain 5
HMOX1	heme oxygenase	0.46	0.006	0.17	0.035
	(decycling) 1
SESN1	sestrin 1	0.46	0.004	1.51	0.001
GABARAPL1	GABA(A) receptor-	0.39	0.004	1.18	0.000
	associated protein like 1
CAT	catalase	0.39	0.003	0.85	0.001
CITED2	Cbp/p300-interacting	0.37	0.005	0.29	0.010
	transactivator, with Glu/Asp-
	rich carboxy-terminal
	domain
ABCA1	ATP-binding cassette, sub-	0.37	0.016	0.26	0.018
	family A (ABC1), member 1
FBXL20	F-box and leucine-rich	0.35	0.002	0.46	0.001
	repeat protein 20
XPO4	exportin 4	0.31	0.009	0.22	0.022
HERPUD1	homocysteine-inducible,	0.29	0.003	0.27	0.029
	endoplasmic reticulum
	stress-inducible, ubiquitin-
	like domain 1
ACOX1	acyl-Coenzyme A oxidase	0.29	0.013	0.53	0.006
	1, palmitoyl
NOX4	NADPH oxidase 4	0.28	0.002	0.41	0.018
UBE4A	ubiquitination factor E4A	0.27	0.004	1.08	0.010
	(UFD2 homolog, yeast)
INSR	insulin receptor	0.24	0.014	0.58	0.003
IGF1R	insulin-like growth factor 1	0.23	0.013	0.40	0.001
	receptor
PANK1	pantothenate kinase 1	0.21	0.007	0.78	0.000
NBR1	neighbor of BRCA1 gene 1	0.21	0.017	0.39	0.009
RORA	RAR-related orphan	0.21	0.006	0.39	0.006
	receptor A
TMEM71	transmembrane protein 71	0.21	0.009	0.40	0.008
CPT1A	carnitine	0.21	0.001	0.21	0.020
	palmitoyltransferase 1A
	(liver)
UCP2	uncoupling protein 2	0.20	0.005	0.33	0.024
	(mitochondrial, proton
	carrier)
TULP3	tubby like protein 3	0.19	0.008	0.22	0.008
MED13L	mediator complex subunit	0.18	0.000	0.23	0.011
	13-like
CALCOCO1	calcium binding and coiled	0.16	0.010	0.31	0.028
	coil domain 1
MYO5A	myosin VA (heavy chain 12,	0.14	0.006	0.36	0.012
	myoxin)
PPAP2B	phosphatidic acid	0.13	0.007	0.09	0.029
	phosphatase type 2B
SRRM2	serine/arginine repetitive	0.13	0.007	0.24	0.040
	matrix 2
ADPGK	ADP-dependent	0.13	0.007	0.16	0.009
	glucokinase
SUPT6H	suppressor of Ty 6 homolog	0.11	0.005	0.26	0.036
	(S. cerevisiae)
SFRS8	splicing factor,	0.08	0.016	0.13	0.011
	arginine/serine-rich 8
NFYA	nuclear transcription factor	−0.07	0.011	−0.31	0.045
	Y, alpha
MRPS15	mitochondrial ribosomal	−0.11	0.003	−0.25	0.001
	protein S15
PDE7B	phosphodiesterase 7B	−0.12	0.013	−0.51	0.011
WDR1	WD repeat domain 1	−0.14	0.019	−0.21	0.047
ACACA	acetyl-Coenzyme A	−0.15	0.010	−0.22	0.041
	carboxylase alpha
AXIN2	axin 2 (conductin, axil)	−0.15	0.013	−0.12	0.046
CASQ1	calsequestrin 1 (fast-twitch,	−0.16	0.015	−0.26	0.015
	skeletal muscle)
ZNF280B	zinc finger protein 280B	−0.16	0.005	−0.34	0.046
JTB	jumping translocation	−0.16	0.014	−0.42	0.030
	breakpoint
CACNB1	calcium channel, voltage-	−0.17	0.013	−0.43	0.003
	dependent, beta 1 subunit
ALG2	asparagine-linked	−0.17	0.011	−0.39	0.019
	glycosylation 2 homolog
TSPAN13	tetraspanin 13	−0.18	0.006	−0.30	0.028
P4HA2	procollagen-proline, 2-	−0.18	0.007	−0.12	0.012
	oxoglutarate 4-dioxygenase,
	alpha II polypeptide
TTLL1	tubulin tyrosine ligase-like	−0.18	0.001	−0.29	0.043
	family, member 1
SUV39H2	suppressor of variegation 3-	−0.20	0.011	−0.26	0.014
	9 homolog 2 (Drosophila)
SLC4A4	solute carrier family 4,	−0.20	0.007	−0.69	0.003
	sodium bicarbonate
	cotransporter, member 4
DNMT3A	DNA (cytosine-5-)-	−0.20	0.007	−0.48	0.000
	methyltransferase 3 alpha
FEZ2	fasciculation and elongation	−0.21	0.000	−0.50	0.019
	protein zeta 2 (zygin II)
MTSS1	metastasis suppressor 1	−0.21	0.009	−0.22	0.033
TMTC4	transmembrane and	−0.22	0.010	−0.17	0.035
	tetratricopeptide repeat
	containing 4
PPM1J	protein phosphatase 1J	−0.23	0.003	−0.30	0.012
	(PP2C domain containing)
ARHGAP20	Rho GTPase activating	−0.23	0.003	−0.22	0.013
	protein 20
ABTB2	ankyrin repeat and BTB	−0.25	0.010	−0.18	0.005
	(POZ) domain containing 2
CNNM4	cyclin M4	−0.26	0.016	−0.27	0.005
GRTP1	growth hormone regulated	−0.26	0.015	−0.54	0.002
	TBC protein 1
RNF148	ring finger protein 148	−0.27	0.017	−0.35	0.014
SPINT2	serine peptidase inhibitor,	−0.27	0.017	−0.23	0.026
	Kunitz type, 2
PBX1	pre-B-cell leukemia	−0.34	0.001	−0.22	0.000
	homeobox 1
HSPH1	heat shock 105 kDa/110 kDa	−0.34	0.019	−0.20	0.043
	protein 1
VEGFA	vascular endothelial growth	−0.35	0.016	−0.26	0.002
	factor A
PMP22	peripheral myelin protein 22	−0.36	0.004	−0.13	0.012
PPARGC1A	peroxisome proliferative	−0.38	0.012	−0.39	0.030
	activated receptor, gamma,
	coactivator 1 alpha
ST8SIA5	ST8 alpha-N-acetyl-	−0.38	0.004	−0.48	0.011
	neuraminide alpha-2,8-
	sialyltransferase 5
PPIL1	peptidylprolyl isomerase	−0.39	0.005	−0.52	0.016
	(cyclophilin)-like 1
PPM1L	protein phosphatase 1	−0.42	0.006	−0.46	0.000
	(formerly 2C)-like
NEO1	neogenin homolog 1	−0.43	0.002	−0.31	0.037
	(chicken)
TGFB2	transforming growth factor,	−0.44	0.017	−0.30	0.003
	beta 2
PTX3	pentraxin-related gene,	−0.47	0.002	−0.48	0.000
	rapidly induced by IL-1 beta
GAS2	growth arrest-specific 2	−0.49	0.011	−0.23	0.044
TFRC	transferrin receptor (p90,	−0.51	0.003	−1.37	0.011
	CD71)

The left side of FIG. 4B shows the 10 Connectivity Map instances (or data sets) with the most significant positive correlations (P<0.004) to the effect of fasting in skeletal muscle. The connectivity score, represented on the y-axis, is a measure of the strength of the correlation (Lamb J, et al. (2006) Science (New York, N.Y. 313(5795):1929-1935); the compound and cell-line is shown below the bar representing the Connectivity Score. Of these, 6 involved wortmannin or LY-294002 (inhibitors of phosphoinositide 3-kinase (PI3K)) or rapamycin (an inhibitor of the mammalian target of rapamycin complex 1 (mTORC1)). Since PI3K and mTORC1 mediate effects of insulin and IGF-I, and since insulin/IGF-I signaling inhibits muscle atrophy and atrophy-associated changes in skeletal muscle mRNA expression (Bodine S C, et al. (2001) Nat Cell Biol 3(11):1014-1019; Sandri M, et al. (2004) Cell 117(3):399-412), these results lent confidence that the Connectivity Map might be used to identify potential inhibitors of muscle atrophy. The right side of FIG. 4B shows the 10 Connectivity Map instances with the most significant negative correlations (P<0.004) to the effect of fasting in skeletal muscle. These compounds, whose effects on cultured cell lines were opposite to the effect of fasting on muscle, included metformin (an insulin-sensitizing agent widely used to treat type 2 diabetes), as well as ursolic acid. Further experiments focused on metformin and ursolic acid. To test the hypothesis that metformin and ursolic acid might reduce fasting-induced muscle atrophy, each compound was administered, or vehicle alone, via i.p. injection to C57BL/6 mice. The mice were then fasted, and after 12 hours of fasting, the mice received a second dose of the compound or vehicle. After 24 hours of fasting, the blood glucose was measured and muscles were harvested. The data shown in FIGS. 4C-4H are means±SEM from 16 mice. Both metformin (250 mg/kg) and ursolic acid (200 mg/kg) significantly reduced fasting blood glucose (FIGS. 4C and 4D). The effects of metformin and ursolic acid on fasting-induced muscle atrophy were also examined, i.e. the effect of 24 h fast (relative to ad lib feeding) on wet weight of lower hindlimb skeletal muscle (bilateral tibialis anterior (“TA” muscle), gastrocnemius, and soleus; see FIGS. 4E-4G). In the absence of metformin and ursolic acid, fasting reduced muscle weight by 9% (FIG. 4E). Although metformin did not alter muscle weight in fasted mice (FIG. 4F), ursolic acid increased it by 7±2% (FIG. 4G). Moreover, consistent with the predicted inhibitory effect on fasting-induced gene expression described herein, ursolic acid reduced fasting levels of atrogin-1 and MuRF1 mRNA levels in the TA muscles of fasted mice (FIG. 4H; the data shown are normalized to the levels in vehicle-treated mice, which were set at 1). In FIGS. 4E-4H, each data point represents one mouse and the horizontal bars denote the means. In FIGS. 4C-4H, P-values were determined using unpaired t-tests. Thus, ursolic acid, but not metformin, decreased fasting-induced muscle atrophy.

5. Ursolic Acid Reduces Denervation-Induced Muscle Atrophy.

The Connectivity Map was queried with a second mRNA expression signature, atrophy signature-2 (described above), to determine if this muscle atrophy signature would also correlate with ursolic acid, among other compounds. As described above, atrophy signature-2 was an mRNA expression signature identified as described herein for human skeletal muscle mRNAs that were induced or repressed by fasting and also by spinal cord injury (“SCI”). The studies of the effects of SCI on human skeletal muscle gene expression were described previously (Adams C M, et al. (2011) Muscle Nerve. 43(1):65-75). Using this approach with the muscle atrophy expression signatures described herein, there were 18 human mRNAs that were increased by fasting and SCI, and 17 human mRNAs that were decreased by fasting and SCI, and are shown in Table 3 (“Change” represents mean changes in loge hybridization signals for pairs as indicated, e.g. fasting and fed states for column labeled “(Fasting-Fed)” or untrained and trained for the column labeled “(Untrained-Trained)”). The data in Table 3 include all mRNAs whose levels were increased by fasting (P≤0.02) and by SCI (P≤0.05), and all mRNAs whose levels were decreased by fasting (P≤0.02) and by SCI (P≤0.05). P-values in Table 3 were determined with paired t-tests.

TABLE 3
HUMAN SKELETAL MUSCLE MRNAS INDUCED OR
REPRESSED BY FASTING AND SCI.
	EFFECT OF
	FASTING	EFFECT OF SCI
		Change		Change
		(Fasting-		(Untrained-
mRNA	Protein	Fed)	P	Trained)	P
OR1D4	olfactory receptor, family 1,	0.50	0.019	0.65	0.030
	subfamily D, member 4
RHOBTB1	Rho-related BTB domain	0.48	0.001	0.71	0.032
	containing 1
TSPAN8	tetraspanin 8	0.39	0.015	1.79	0.023
FLJ33996	hypothetical protein FLJ33996	0.39	0.019	0.68	0.020
NUPR1	nuclear protein 1	0.35	0.007	0.65	0.030
IRS2	insulin receptor substrate 2	0.34	0.004	0.21	0.035
NPC2	Niemann-Pick disease, type C2	0.30	0.011	0.39	0.042
KLF11	Kruppel-like factor 11	0.29	0.011	0.22	0.034
ZNF682	zinc finger protein 682	0.28	0.017	0.72	0.013
NOX4	NADPH oxidase 4	0.28	0.002	0.56	0.007
PLXDC2	plexin domain containing 2	0.26	0.013	0.38	0.022
CTDSP2	CTD small phosphatase 2	0.25	0.003	0.34	0.021
CAV3	caveolin 3	0.24	0.007	0.56	0.020
IGF1R	insulin-like growth factor 1	0.23	0.013	0.63	0.040
	receptor
FLJ14154	hypothetical protein FLJ14154	0.22	0.005	0.30	0.021
CUGBP2	CUG triplet repeat, RNA	0.21	0.004	0.14	0.034
	binding protein 2
MLL	myeloid/lymphoid or mixed-	0.14	0.016	0.30	0.040
	lineage leukemia
SUPT6H	suppressor of Ty 6 homolog	0.11	0.005	0.19	0.024
MRPS15	mitochondrial ribosomal protein	−0.11	0.003	−0.33	0.001
	S15
RFXDC2	regulatory factor X domain	−0.12	0.012	−0.10	0.037
	containing 2
PDE7B	phosphodiesterase 7B	−0.12	0.013	−0.39	0.011
PFDN6	prefoldin subunit 6	−0.14	0.014	−0.42	0.021
ZNF280B	zinc finger protein 280B	−0.16	0.005	−0.30	0.028
TSPAN13	tetraspanin 13	−0.18	0.006	−0.56	0.023
TTLL1	tubulin tyrosine ligase-like	−0.18	0.001	−0.37	0.020
	family, member 1
CMAS	cytidine monophosphate N-	−0.21	0.000	−0.22	0.025
	acetylneuraminic acid
	synthetase
C8orf32	chromosome 8 open reading	−0.23	0.016	−0.11	0.049
	frame 32
GUCY1B3	guanylate cyclase 1, soluble,	−0.24	0.007	−0.24	0.008
	beta 3
ZNF32	zinc finger protein 32	−0.24	0.010	−0.21	0.030
VLDLR	very low density lipoprotein	−0.28	0.007	−0.16	0.015
	receptor
HSPB7	heat shock 27 kDa protein	−0.33	0.015	−0.77	0.032
	family, member 7
	(cardiovascular)
VEGFA	vascular endothelial growth	−0.35	0.016	−0.43	0.020
	factor A
SLC16A1	solute carrier family 16,	−0.37	0.018	−0.94	0.015
	member 1
PPARGC1A	peroxisome proliferative	−0.38	0.012	−0.74	0.001
	activated receptor, gamma,
	coactivator 1 alpha
C6orf192	chromosome 6 open reading	−0.41	0.018	−0.39	0.042
	frame 192

Of the mRNAs listed in Table 3, 29 were represented on the HG-U133A arrays used in the Connectivity Map (FIG. 5A), but only 10 were common to the 63 mRNAs used in the first Connectivity Map query described above for atrophy signature-1 (IGF-IR, NOX4, SUPT6H, MRPS15, PDE7B, PGC-1a, TSPAN13, TTLL1, VEGFA and ZNF280B). The mRNAs listed in FIG. 5A represent human muscle atrophy signature-2: mRNAs altered by both fasting and SCI in human muscle. These mRNAs, as described above, were used to query the Connectivity Map. Inclusion criteria were: P≤0.02 in fasted human muscle (by t-test), P≤0.05 in untrained, paralyzed muscle (by t-test), and the existence of complimentary probes on HG-U133A arrays. Connectivity Map instances with the most significant positive and negative correlations to the effect of fasting and SCI in human muscle. P<0.005 for all compounds are shown in FIG. 5B. The results partially overlapped with the results of the first search: both search strategies identified LY-294002, wortmannin and rapamycin as predicted mimics of atrophy-inducing stress, and ursolic acid (but not metformin) as a predicted inhibitor (FIG. 5B).

Because atrophy signature-2 utilized data from SCI subjects, it was hypothesized that ursolic acid might reduce denervation-induced muscle atrophy. To test this, the left hindlimb muscles a denervation-induced skeletal muscle atrophy model in mouse was used. Briefly, on day 0, the left hindlimbs of C57BL/6 mice were denervated by transsecting the left sciatic nerve. This approach allowed the right hindlimb to serve as an intra-subject control. Mice were then administered ursolic acid (200 mg/kg) or an equivalent volume of vehicle alone (corn oil) via i.p. injection twice daily for seven days. During this time, mice continued to have ad libitum access to food. On day 7, muscle tissues were harvested for analysis, and the left (denervated) and right (innervated) hindlimb muscles in both groups (ursolic acid vs. vehicle administration) were compared. Ursolic acid significantly decreased denervation-induced muscle loss (FIG. 5C). In FIG. 5C, weights of the left (denervated) lower hindlimb muscles were normalized to weights of the right (innervated) lower hindlimb muscles from the same mouse. Each data point represents one mouse, and horizontal bars denote the means and the P-value was determined using an unpaired t-test. Histologically, this effect of ursolic acid was reflected as an increase in the size of denervated skeletal muscle fiber diameter in denervated gastrocnemius (D) and TA (E) muscles (FIGS. 5D and 5E, respectivel). The data shown in FIGS. 5D and 5E are from >2500 muscle fibers per condition; P<0.0001 by unpaired t-test. Thus, ursolic acid reduced denervation-induced muscle atrophy.

6. Ursolic Acid Induces Skeletal Muscle Hypertrophy.

The results from the denervation-induced muscle atrophy model suggested that ursolic acid reduced muscle atrophy, thus the hypothesis that ursolic acid might promote muscle hypertrophy in the absence of an atrophy-inducing stress was reasonable. Mice were provided ad lib access to either standard chow (control diet) or standard chow supplemented with 0.27% ursolic acid (ursolic acid diet) for 5 weeks before grip strength was measured and tissues were harvested. After five weeks, mice administered ursolic had increased lower hindlimb muscle weight (FIG. 6A), quadriceps weight (FIG. 6B), and upper forelimb muscle (triceps and biceps) weight (FIG. 6C). Each data point in FIGS. 6A-6C represents one mouse, and horizontal bars denote the means. The effect of ursolic acid in this study on skeletal muscle fiber size distribution is shown in FIG. 6D. Each distribution represents measurements of >800 triceps muscle fibers from 7 animals (>100 measurements/animal); P<0.0001. The effect of ursolic acid on peak grip strength (normalized to body weight) is shown in FIG. 6E. Each data point represents one mouse, and horizontal bars denote the means. Non-normalized grip strength data were 157±9 g (control diet) and 181±6 g (ursolic acid diet) (P=0.04).

Moreover, dietary ursolic acid increased the specific force generated by muscles ex vivo (FIG. 7). Briefly, six-week old male C57BL/6 mice were provided either standard diet or diet containing 0.27% ursolic acid for 16 weeks before being euthanized. The lower hindlimb was removed (by transsecting the upper hindlimb mid-way through the femur), and placed in Krebs solution aerated with 95% O2 and 5% CO2. The gastrocnemius, soleus and tibialis anterior muscles, as well as the distal half of the tibia and fibula were then removed and discarded, leaving the extensor digitorum longus and peroneus muscles with their origins and insertions intact. A suture was placed through the proximal tendon and secured to the distal femur fragment. This ex vivo preparation was then mounted vertically in a water jacket bath (Aurora Scientific 1200A Intact Muscle Test System, filled with aerated Krebs solution) by attaching the suture to a servo-controlled lever (superiorly) and clamping the metatarsals (inferiorly). Passive muscle force was adjusted to a baseline of 1 g, and then muscles were stimulated with supramaximal voltage (80 V) at 100 Hz. The mean time from euthanasia to maximal force measurements was 10 min. After force measurements, muscles were removed and weighed in order to calculate specific titanic force. Maximal tetanic force and muscle weight did not differ between the two groups (P=0.20 and 0.26, respectively). Data are means±SEM from 5-6 mice per diet. P-values were determined with a t-test. Together, the data in FIGS. 6 and 7 provide morphological and functional evidence that ursolic acid induced skeletal muscle hypertrophy.

7. Ursolic Acid Induces Trophic Changes in Skeletal Muscle Gene Expression.

The foregoing results suggested that ursolic acid might alter skeletal muscle gene expression. To test this hypothesis, an unbiased approach was used, specifically exon expression arrays were used to analyze gastrocnemius muscle mRNA expression in mice that had been fed diets lacking or containing ursolic acid for 5 weeks. Mice were provided ad lib access to either standard chow (control diet) or standard chow supplemented with 0.27% ursolic acid (ursolic acid diet) for 5 weeks before gastrocnemius muscle RNA was harvested and analyzed by Affymetrix Mouse Exon 1.0 ST arrays (n=4 arrays per diet). Each array assessed pooled gastrocnemius RNA from two mice. Stringent criteria were used for ursolic acid-induced effects on mRNA levels (P<0.005), and mRNAs with low levels of expression were disregarded (i.e. only transcripts that were increased to a mean loge hybridization signal≥8, or repressed from a mean log₂ hybridization signal≥8 were included). The results were that ursolic acid decreased 18 mRNAs and increased 51 mRNAs (out of >16,000 mRNAs analyzed. The results are shown in Table 4 (“Change” is the meang loge change or difference between mice on ursolic acid diet and control diet, i.e. [Mean loge mRNA levels in ursolic acid diet] minus [Mean loge mRNA levels in control diet]).

TABLE 4
MOUSE SKELETAL MUSCLE MRNAS INDUCED
OR REPRESSED BY URSOLIC ACID.
mRNA	Protein	Change	P
Smox	spermine oxidase	0.81	0.001
Lyz2	lysozyme 2	0.71	0.001
C3	complement component 3	0.70	0.000
Tyrobp	TYRO protein tyrosine kinase binding	0.69	0.001
	protein
Lum	lumican	0.61	0.001
Igf1	insulin-like growth factor 1	0.56	0.005
Fmo1	flavin containing monooxygenase 1	0.47	0.000
Ostn	osteocrin	0.43	0.001
Nampt	nicotinamide phosphoribosyltransferase	0.41	0.003
H19	H19 fetal liver mRNA	0.39	0.004
Hipk2	homeodomain interacting protein kinase 2	0.38	0.002
Fbp2	fructose bisphosphatase 2	0.37	0.003
Gpx1	glutathione peroxidase 1	0.36	0.001
Sepp1	selenoprotein P, plasma, 1	0.35	0.004
Parp3	poly (ADP-ribose) polymerase family,	0.32	0.001
	member 3
Hspb8	heat shock protein 8	0.32	0.000
Musk	muscle, skeletal, receptor tyrosine kinase	0.31	0.004
Fhl3	four and a half LIM domains 3	0.31	0.005
Hsph1	heat shock 105 kDa/110 kDa protein 1	0.30	0.001
Arfgap2	ADP-ribosylation factor GTPase activating	0.30	0.001
	protein 2
Cd24a	CD24a antigen	0.28	0.002
Sepx1	selenoprotein X 1	0.28	0.003
Hk2	hexokinase 2	0.26	0.003
Ggct	gamma-glutamyl cyclotransferase	0.24	0.005
Trip10	thyroid hormone receptor interactor 10	0.23	0.000
Npc1	Niemann Pick type C1	0.22	0.001
Asb5	ankyrin repeat and SOCs box-containing 5	0.21	0.001
Vps29	vacuolar protein sorting 29 (S. pombe)	0.20	0.000
Ahsa2	AHA1, activator of heat shock protein	0.18	0.001
	ATPase homolog 2
Lsm14a	LSM14 homolog A (SCD6, S. cerevisiae)	0.18	0.004
Pdha1	pyruvate dehydrogenase E1 alpha 1	0.18	0.001
Trappc2l	trafficking protein particle complex 2-like	0.16	0.004
Ube2l3	ubiquitin-conjugating enzyme E2L 3	0.16	0.003
Ctsb	cathepsin B	0.16	0.003
D0H4S114	DNA segment, human D4S114	0.15	0.004
Psma2	proteasome (prosome, macropain)	0.15	0.005
	subunit, alpha type 2
Mrpl46	mitochondrial ribosomal protein L46	0.15	0.001
Eef1e1	eukaryotic translation elongation factor 1	0.15	0.002
	epsilon 1
Krr1	KRR1, small subunit (SSU) processome	0.15	0.005
	component, homolog
Ndufaf4	NADH dehydrogenase (ubiquinone) 1	0.14	0.005
	alpha subcomplex, assembly factor 4
Ndufs2	NADH dehydrogenase (ubiquinone) Fe—S	0.14	0.002
	protein 2
2610507B11Rik	RIKEN cDNA 2610507B11 gene	0.14	0.000
Ssr4	signal sequence receptor, delta	0.14	0.000
Ndufs4	NADH dehydrogenase (ubiquinone) Fe—S	0.14	0.003
	protein 4
Sqstm1	sequestosome 1	0.12	0.001
Gfm1	G elongation factor, mitochondrial 1	0.12	0.003
2310016M24Rik	RIKEN cDNA 2310016M24 gene	0.12	0.004
Sod2	superoxide dismutase 2, mitochondrial	0.12	0.001
Prdx5	peroxiredoxin 5	0.10	0.005
BC004004	cDNA sequence BC004004	0.06	0.001
Ghitm	growth hormone inducible transmembrane	0.05	0.005
	protein
Foxn3	forkhead box N3	−0.09	0.000
Klhl31	kelch-like 31 (Drosophila)	−0.09	0.001
Acadm	acyl-Coenzyme A dehydrogenase,	−0.11	0.001
	medium chain
Eif4g3	eukaryotic translation initiation factor 4	−0.12	0.005
	gamma, 3
Nrap	nebulin-related anchoring protein	−0.14	0.003
Golga4	golgi autoantigen, golgin subfamily a, 4	−0.14	0.003
Paip2b	poly(A) binding protein interacting protein	−0.16	0.000
	2B
Pde4dip	phosphodiesterase 4D interacting protein	−0.18	0.001
	(myomegalin)
Sfpq	splicing factor proline/glutamine rich	−0.18	0.005
Pnn	pinin	−0.18	0.002
D4Wsu53e	DNA segment, Chr 4, Wayne State	−0.18	0.003
	University 53, expressed
Mlec	malectin	−0.19	0.003
Cacna1s	calcium channel, voltage-dependent, L	−0.22	0.001
	type, alpha 1S
Sfrs5	splicing factor, arginine/serine-rich 5	−0.22	0.005
	(SRp40, HRS)
Nnt	nicotinamide nucleotide transhydrogenase	−0.24	0.002
Adprhl1	ADP-ribosylhydrolase like 1	−0.26	0.002
Ddit4l	DNA-damage-inducible transcript 4-like	−0.32	0.000
Fbxo32	F-box protein 32 (Atrogin-1)	−0.35	0.001

As discussed above, atrogin-1 and MuRF1 are transcriptionally up-regulated by atrophy-inducing stresses (see FIG. 2B and Sacheck J M, et al. (2007) Faseb J21(1):140-155), and they are required for muscle atrophy (Bodine S C, et al. (2001) Science (New York, N.Y. 294(5547):1704-1708). Moreover, in the studies of fasted mice as described herein above, ursolic acid reduced atrogin-1 and MuRF1 mRNAs (FIG. 4H). Consistent with that finding, the arrays indicated that dietary ursolic acid reduced atrogin-1 mRNA, which was the most highly repressed mRNA (FIG. 8A). The results shown in FIG. 8A represent a subset of the mRNAs from Table 4 which had the greatest increase or decrease in expression level in response to ursolic acid. Although MuRF1 mRNA was not measured by the arrays used in these experiments, qPCR analysis confirmed that dietary ursolic acid repressed both atrogin-1 and MuRF1 mRNAs (FIG. 8B; data are means±SEM). Interestingly, one of the most highly up-regulated muscle mRNAs was IGF1 (FIGS. 8A and 8B), which encodes insulin-like growth factor-I (IGF-I), a locally generated autocrine/paracrine hormone. IGF1 mRNA is known to be transcriptionally induced in hypertrophic muscle (Hameed M, et al. (2004) The Journal of physiology 555(Pt 1):231-240; Adams G R & Haddad F (1996) J Appl Physiol 81(6):2509-2516; Gentile M A, et al. (2010) Journal of molecular endocrinology 44(1):55-73). In addition, increased skeletal muscle IGF1 expression reduces denervation-induced muscle atrophy (Shavlakadze T, et al. (2005) Neuromuscul Disord 15(2):139-146), and stimulates muscle hypertrophy (Barton-Davis E R, et al. (1998) Proceedings of the National Academy of Sciences of the United States of America 95(26):15603-15607; Musaro A, et al. (2001) Nature Genetics 27(2):195-200). Moreover, by stimulating skeletal muscle insulin/IGF-I signaling, IGF-I represses atrogin-1 and MuRF mRNAs (Sacheck J M, et al. (2004) Am J Physiol Endocrinol Metab 287(4):E591-601; Frost R A, et al. (2009) J Cell Biochem 108(5):1192-1202.), as well as DDIT4L mRNA (ibid), which, after atrogin-1 mRNA, was the second most highly repressed mRNA in muscle from ursolic acid-treated mice (FIG. 8A). Thus, 5 weeks of dietary ursolic acid altered skeletal muscle gene expression in a manner known to reduce atrophy and promote hypertrophy, and muscle-specific IGF1 induction emerged as a likely contributing mechanism in ursolic acid-induced muscle hypertrophy. The effect of ursolic acid on plasma IGF-I levels was also determined, which primarily reflect growth hormone-mediated hepatic IGF-I production (Yakar S, et al. (1999) Proceedings of the National Academy of Sciences of the United States of America 96(13):7324-7329). Although diets containing 0.14% or 0.27% ursolic acid increased muscle mass (described in greater detail below; FIG. 10A), neither increased plasma IGF-I (FIG. 8C). For the data in FIG. 8C, mice were provided ad lib access to either standard chow (control diet) or standard chow supplemented with the indicated concentration of ursolic acid for 7 weeks before plasma IGF-I levels were measured. Each data point represents one mouse, and horizontal bars denote the means. P-values were determined by one-way ANOVA with Dunnett's post-test. Of note, exon expression arrays indicated that ursolic acid increased levels of all measured IGF1 exons (exons 2-6; FIG. 9A). The data in FIG. 9A are mean exon-specific log₂ hybridization signals from the arrays described in Table 2. However, ursolic acid did not alter levels of mRNAs encoding myostatin (which reduces muscle mass, for example see Lee S J (2004) Annu Rev Cell Dev Biol 20:61-86), or twist or myogenin (which are induced by IGF-I during development, for example see Dupont J, et al. (2001) The Journal of biological chemistry 276(28):26699-26707; Tureckova J, et al. (2001) The Journal of biological chemistry 276(42):39264-39270). Moreover, ursolic acid did not alter the amount of IGF1 mRNA in adipose tissue (FIG. 9B). Briefly, the data shown in FIG. 9B were obtained as follows: mice were provided ad lib access to either standard chow (control diet) or standard chow supplemented with 0.27% ursolic acid (ursolic acid diet) for 7 weeks before retroperitoneal adipose tissue was harvested for qPCR quantification of IGF1 mRNA. The data shown are means ±SEM from 5 mice per group. Without wishing to be bound by a particular theory, ursolic acid-mediated IGF1 induction may be localized to skeletal muscle.

8. Ursolic Acid Enhances Skeletal Muscle IGF-I Signaling.

Although muscle-specific IGF1 induction is characteristic of, and contributes to, muscle hypertrophy, it may be a relatively late event that promotes hypertrophy after it has been initiated by other stimuli (Adams G R, et al. (1999) J Appl Physiol 87(5):1705-1712). Without wishing to be bound by a particular theory, it is possible that ursolic acid might have a more proximal effect on insulin/IGF-I signaling. In a previous study of non-muscle cell lines (CHO/IR and 3T3-L1 cells), ursolic acid enhanced insulin-mediated Akt activation (Jung S H, et al. (2007) The Biochemical journal 403(2):243-250). To determine whether ursolic acid might have a similar effect in skeletal muscle, the level of phosphorylated Akt was assessed in quadriceps muscles of mice fed diets lacking or containing ursolic acid. Briefly, mice were provided ad lib access to either standard chow (control diet) or standard chow supplemented with 0.27% ursolic acid for 16 weeks. Total protein extracts from quadriceps muscles were subjected to SDS-PAGE, followed by immunoblot analysis for phosphorylated and total Akt, as indicated. A representative immunoblot is shown in FIG. 8D. Immunoblot data were quantitated as follows: in each mouse, the level of phospho-Akt was normalized to the level of total Akt; these ratios were then normalized to the average phospho-Akt/total Akt ratio from control mice and the results are shown in FIG. 8E (data are means±SEM from 9 mice per diet. P-value was determined by unpaired t-test). The data show that in quadriceps, ursolic acid increased Akt phosphorylation by 1.8-fold.

The effect of ursolic acid on Akt activation was examined in C2C12 skeletal myotubes, a well-established in vitro model of skeletal muscle (Sandri M, et al. (2004) Cell 117(3):399-412; Stitt T N, et al. (2004) Mol Cell 14(3):395-403). Use of an in vitro system, such as C2C12 skeletal myotubes, circumvented potentially confounding effects from non-muscle tissues, and enabled a determination of whether IGF-I or insulin was required for ursolic acid's effect. The latter consideration was important because circulating IGF-I and insulin are always present in healthy animals. Use of an in vitro system also allowed testing of a clearly defined concentration of ursolic acid (10 μM, similar what was used in the Connectivity Map (8.8 μM)) for a clearly defined time of incubation (20 min). These considerations were important because the in vivo pharmacokinetic properties of ursolic acid are not yet known.

For the data shown in FIGS. 8F-8K, serum-starved C2C12 myotubes were treated in the absence or presence of ursolic acid (10 μM) and/or IGF-I (10 nM), as indicated. For studies of the IGF-I receptor, cells were harvested 2 min later, and protein extracts were subjected to immunoprecipitation with anti-IGF-I receptor β antibody, followed by immunoblot analysis with anti-phospho-tyrosine or anti-IGF-I receptor antibodies to assess phospho- and total IGF-I receptor, respectively. For other studies, cells were harvested 20 min after addition of ursolic acid and/or IGF-I, and immunoblot analyses were performed using total cellular protein extracts and antibodies specific for the phosphorylated or total proteins indicated. Representative immunoblots showing effect of ursolic acid on IGF-I-mediated phosphorylation of Akt (FIG. 8F), S6K (FIG. 8G) and IGF-I receptor (FIG. 8H). Data from immunoblots was quantitated as follows: levels in the presence of ursolic acid and IGF-I were normalized to levels in the presence of IGF-I alone, which were set at 1 and are indicated by the dashed line. The data shown in FIG. 8I are means±SEM from ≥3 experiments.

For the data shown in FIGS. 9C-9F, serum-starved C2C12 myotubes were treated in the absence or presence of ursolic acid (10 μM), insulin (10 nM) and/or IGF-I (10 nM), as indicated. For studies of the insulin receptor, cells were harvested 2 min later, and protein extracts were subjected to immunoprecipitation with anti-insulin receptor β antibody, followed by immunoblot analysis with anti-phospho-insulin receptor β (Y1162/1163) or anti-insulin receptor β antibodies to assess phospho- and total insulin receptor, respectively. For other studies, cells were harvested 20 min after addition of ursolic acid, insulin and/or IGF-I, and immunoblot analyses were performed using total cellular protein extracts and antibodies specific for the phosphorylated or total proteins indicated.

When serum-starved myotubes were treated with ursolic acid alone, Akt phosphorylation did not increase (FIG. 8F). However, in the presence of IGF-I, ursolic acid increased Akt phosphorylation by 1.9-fold (FIGS. 8F and 8I). Ursolic acid also increased Akt phosphorylation in the presence of insulin (FIG. 9C). Thus, ursolic acid enhanced IGF-I-mediated and insulin-mediated Akt phosphorylation. The finding that ursolic acid enhanced muscle Akt activity in vivo and in vitro was consistent with the finding that ursolic acid's mRNA expression signature negatively correlated with the mRNA expression signatures of LY-294002 and wortmannin (FIGS. 4B and 5B), which inhibit insulin/IGF-I signaling upstream of Akt. However, ursolic acid's signature also negatively correlated with the signature of rapamycin, which inhibits insulin/IGF-I signaling downstream of Akt.

Although ursolic acid alone did not increase S6K phosphorylation (FIG. 9D), it enhanced IGF-I-mediated and insulin-mediated S6K phosphorylation (FIGS. 8G, 8I and 9D). To further investigate the mechanism, the effect of ursolic acid on the IGF-I receptor was examined. Ursolic acid increased IGF-I receptor phsophorylation in the presence but not the absence of IGF-I (FIGS. 8H and 8I). Similarly, ursolic acid increased insulin receptor phosphorylation in the presence but not the absence of insulin (FIG. 9E). Both of these effects were rapid, occurring within 2 minutes after the addition of ursolic acid and either IGF-I or insulin. Consistent with enhanced signaling at the level of the IGF-I and insulin receptors, ursolic acid also enhanced IGF-I-mediated and insulin-mediated ERK phosphorylation (FIGS. 8J and 9F). Moreover, ursolic acid enhanced IGF-I-mediated phosphorylation (inhibition) of FoxO transcription factors, which activate transcription of atrogin-1 and MuRF1 mRNAs (FIG. 8K; Sandri M, et al. (2004) Cell 117(3):399-412; Stitt T N, et al. (2004) Mol Cell 14(3):395-403.). Without wishing to be bound by a particular theory, ursolic acid represses atrophy-associated gene expression and promotes muscle hypertrophy by increasing activity of the IGF-I and insulin receptors.

9. Ursolic Acid Reduces Adiposity.

Mice were provided ad lib access to standard chow supplemented with the indicated concentration (weight percent in chow, either 0.14% or 0.28% as indicated in FIG. 10) of ursolic acid for 7 weeks before tissues were harvested for analysis. Data are means±SEM from 10 mice per diet. Data for the effects of ursolic acid on weights of skeletal muscle (quadriceps+triceps), epididymal fat, retroperitoneal fat and heart are shown in FIG. 10A. The P-values, determined by one-way ANOVA with post-test for linear trend, were <0.001 for muscle; 0.01 and 0.04 for epididymal and retroperitoneal fat, respectively; and 0.46 for heart. The data show that 7 weeks of dietary ursolic acid increased skeletal muscle weight in a dose-dependent manner, with a peak effect at 0.14% ursolic acid. Interestingly, although ursolic acid increased muscle weight, it did not increase total body weight (FIG. 10B; P-values were 0.71 and 0.80 for initial and final weights, respectively).

The data in FIG. 10A also show that 7 weeks of dietary ursolic acid reduced the weight of epididymal and retroperitoneal fat depots, with a peak effect at 0.14%. In another study, mice were provided ad lib access to either standard chow (control diet) or standard chow supplemented with 0.27% ursolic acid (ursolic acid diet) for 5 weeks. The relationship between skeletal muscle weight (quadriceps, triceps, biceps, TA, gastrocnemius and soleus) and retroperitoneal adipose weight is shown in FIG. 10C. Each data point in FIG. 10C represents one mouse; P<0.001 for both muscle and adipose by unpaired t-test. The data show that 5 weeks of ursolic acid administration (0.14%) also reduced adipose weight. Thus, muscle and fat weights were inversely related. Without wishing to be bound by a particular theory, ursolic acid-treated mice contain less fat because, in part, ursolic acid increases Akt activity (see FIGS. 8 and 9), and muscle-specific increases in Akt activity reduce adiposity as a secondary consequence of muscle hypertrophy (Lai K M, et al. (2004) Molecular and cellular biology 24(21):9295-9304; Izumiya Y, et al. (2008) Cell metabolism 7(2): 159-172).

Ursolic acid reduced adipose weight by reducing adipocyte size as shown by data in FIGS. 10D-10F. FIG. 10D shows a representative H&E stain of retroperitoneal fat for animals feed a control data or a chow with 0.27% ursolic acid as indicated. The data in FIG. 10D are shown quantitatively in FIG. 10E in terms of adipocyte diameter, where data point represents theaverage diameter of ≥125 retroperitoneal adipocytes from one mouse. The retroperitoneal adipocyte size distribution. Each distribution represents combined adipocyte measurements (>1000 per diet) from FIG. 10E.

The changes in adipocyte size were accompanied by a significant reduction in plasma leptin levels, which correlated closely with adipose weight (see FIGS. 10G and 10H). In FIG. 10G, each data point represents one mouse, and horizontal bars denote the means. P-values were determined by t-test. In FIG. 10H, each data point represents one mouse. Importantly, ursolic acid also significantly reduced plasma triglyceride (FIG. 10I) and cholesterol (FIG. 10J). In FIGS. 10I and 10J, each data point represents one mouse, and horizontal bars denote the means. P-values were determined by unpaired t-test. Although ursolic acid reduced leptin, it did not alter food intake (FIG. 11A). In this study, mice were provided ad lib access to either standard chow (control diet) or standard chow supplemented with 0.27% ursolic acid (ursolic acid diet) for 4 weeks. Mice were then moved to a comprehensive animal metabolic monitoring system (CLAMS; Columbus Instruments, Columbus, Ohio) and provided with ad lib access to the same diets. Food consumption was measured for 48 hours. Data are means±SEM from 6 mice per group. However, ursolic acid did not alter weights of heart (FIG. 10A), liver or kidney (FIGS. 11B and 11C), nor did it elevate plasma markers of hepatotoxicity or nephrotoxicity (alanine aminotransferase, bilirubin and creatinine; see FIGS. 11D-11F). The data in FIGS. 11B-11F were obtained as follows: mice were provided ad lib access to either standard chow (control diet) or standard chow supplemented with 0.27% ursolic acid (ursolic acid diet) for 5 weeks before tissues and plasma were harvested for the indicated measurements; each data point represents one mouse, and horizontal bars denote the means. For FIG. 11, P-values were determined with unpaired t-tests. Thus, dietary ursolic acid had two major effects: skeletal muscle hypertrophy and reduced adiposity.

10. Ursolic Acid Reduces Weight Gain and White Adipose Tissue.

The findings that ursolic acid increased skeletal muscle and decreased adiposity suggested that ursolic acid might increase energy expenditure, which would lead to obesity resistance. To test this, C57BL/6 mice were given ad libitum access to a high fat diet (HFD; Teklad TD.93075; 55% calories from fat) lacking or containing 0.27% ursolic acid. After 7 weeks, mice from each group were studied for three days in comprehensive lab animal monitoring systems (“CLAMS”; Columbus Instruments). In the CLAMS, mice were maintained on the same diet they had been eating since the beginning of the experiment. Following CLAMS, tissues were harvested for analysis. In high fat-fed mice, ursolic acid dramatically reduced weight gain, and this effect was apparent within one week (FIG. 12A). As previously observed in mice fed ursolic acid and standard chow (FIG. 6), ursolic acid increased grip strength and muscle mass (FIGS. 12B and 12C). Moreover, ursolic acid reduced retroperitoneal and epididymal fat (FIGS. 12D and 12E). Interestingly, in the scapular fat pad, which contains a mixture of white and thermogenic brown fat, ursolic acid reduced white fat (FIG. 12F), but increased brown fat (FIG. 12G). Importantly, increased skeletal muscle and brown adipose tissue would be predicted to increase energy expenditure. Indeed, CLAMS revealed that ursolic acid increased energy expenditure (FIG. 12H), providing an explanation for how ursolic acid reduces adiposity and obesity. Remarkably, CLAMS analysis revealed that ursolic acid-treated mice consumed more food (FIG. 121), even though they gained less weight (FIG. 12A). For the data shown in FIG. 12A, data are means±SEM from 12 control mice and 15 treated mice, but it should be noted that some error bars are too small to see; P<0.01 at 1 wk and each subsequent time point. In FIGS. 12B-12I, each data point represents one mouse and horizontal bars denote the means. P-values were determined with unpaired t-tests.

11. Ursolic Acid Reduces Obesity-Related Pre-Diabetes, Diabetes, Fatty Liver Disease and Hypercholesterolemia.

The study was carried out as follows: C57BL/6 mice were given ad libitum access to a high fat diet (“HFD”; Teklad TD.93075; 55% calories from fat) lacking or containing 0.27% ursolic acid. After 5 weeks, mice were fasted for 16 h before blood glucose was measured via the tail vein (FIG. 13A). Normal fasting blood glucose: ≤100 mg/dl. (B-I) After 7 weeks, liver and plasma were harvested for analysis (FIGS. 13B-13I). The data shown in FIG. 13A suggest that most mice fed HFD without ursolic acid for 6 weeks developed impaired fasting glucose (pre-diabetes) or diabetes. Importantly, this was prevented by ursolic acid (FIG. 13A). In addition, mice fed HFD without ursolic acid developed fatty liver disease, as evidenced by increased liver weight (>30% increase above normal mouse liver weight of 1500 mg; FIG. 13B), hepatocellular lipid accumulation (FIG. 13C, H&E stain at 20× magnification; FIG. 13D, lipid-staining osmium at 10× magnification), and elevated plasma liver function tests (FIG. 13E, AST; 13F, ALT; 13G, alkaline phosphatase (labeled as “Alk. Phos. in figure); and, 13H, cholesterol). However, ursolic acid prevented all of these hepatic changes (FIG. 13B-13G). In addition, ursolic acid reduced obesity-related hypercholesterolemia (FIG. 13H). In FIGS. 13A, 13B, and 13E-13H, each data point represents one mouse and horizontal bars denote the means.

12. Oleanolic Acid Does Not Increase Skeletal Muscle Mass.

The effect of ursolic acid on skeletal muscle weight and liver weight was compared to the effects by oleanolic acid and metformin. Metformin was a compound identified from atrophy signature-1, but not atrophy signature-2. Oleanolic acid, like ursolic acid is a pentacyclic acid triterpane. This is a structurally similar compound to ursolic acid. However, the two compounds are distinct: oleanolic acid has two methyl groups at position 20, whereas ursolic acid has a single methyl group at each of positions 19 and 20 (compare FIGS. 14A and 14D). Both ursolic acid and oleanolic acid reduce blood glucose, adiposity and hepatic steatosis (Wang Z H, et al. (2010) European journal of pharmacology 628(1-3):255-260; Jayaprakasam B, et al. (2006) J Agric Food Chem 54(1):243-248; de Melo C L, et al. (2010) Chem Biol Interact 185(1):59-65). In addition, both ursolic acid and oleanolic acid possess a large number of cellular effects and biochemical targets, including nearly equivalent inhibition of protein tyrosine phosphatases (“PTPs”; see Zhang W, et al. (2006) Biochimica et biophysica acta 1760(10):1505-1512; Qian S, et al. (2010) J Nat Prod 73(11):1743-1750; Zhang Y N, et al. (2008) Bioorg Med Chem 16(18):8697-8705). However, the effects of these compounds on skeletal muscle mass were not known.

Because some PTPs (particularly PTP1B) dephosphorylate (inactivate) the insulin receptor, PTP inhibition represented a potential mechanism to explain ursolic acid-mediated enhancement of insulin signaling. Thus, because oleanolic acid and ursolic acid inhibit PTP1B and other PTPs with similar efficacy and potency in vitro (Qian S, et al. (2010) J Nat Prod 73(11):1743-1750; Zhang Y N, et al. (2008) Bioorg Med Chem 16(18):8697-8705), testing oleanolic acid's effects on skeletal mass tests the potential role of PTP inhibition. It should be noted that neither ursolic acid nor oleanolic acid is known to inhibit PTPs in vivo, and neither of these compounds are known to enhance IGF-I signaling. Moreover, ursolic acid's capacity to inhibit PTPs has been disputed based on ursolic acid's failure to delay insulin receptor de-phosphorylation in cultured cells (Jung S H, et al. (2007) The Biochemical journal 403(2):243-250), and ursolic acid's capacity to act as an insulin mimetic (Jung S H, et al. (2007) The Biochemical journal 403(2):243-250). In addition, global and muscle-specific PTP1B knockout mice do not possess increased muscle mass, although they are resistant to obesity and obesity-related disorders (Delibegovic M, et al. (2007) Molecular and cellular biology 27(21):7727-7734; Klaman L D, et al. (2000) Molecular and cellular biology 20(15):5479-5489). Furthermore, ursolic acid increases pancreatic beta cell mass and serum insulin levels in vivo, perhaps via its anti-inflammatory effects (Wang Z H, et al. (2010) European journal of pharmacology 628(1-3):255-260; Jayaprakasam B, et al. (2006) J Agric Food Chem 54(1):243-248; de Melo C L, et al. (2010) Chem Biol Interact 185(1):59-65). Importantly, inflammation is now recognized as a central pathogenic mechanism in muscle atrophy, metabolic syndrome, obesity, fatty liver disease and type 2 diabetes. Thus, the existing data suggest at least four mechanisms to explain ursolic acid's capacity to increase insulin signaling in vivo: PTP inhibition, direct stimulation of the insulin receptor, increased insulin production, and reduced inflammation. Of these four potential mechanisms, only the latter three have been demonstrated in vivo.

To compare the effects of ursolic acid and oleanolic acid on skeletal muscle and liver weight, C57BL/6 mice were administered ursolic acid (200 mg/kg), oleanolic acid (200 mg/kg), or vehicle alone (corn oil) via i.p. injection. Mice were then fasted, and after 12 hours of fasting, mice received a second dose of ursolic acid, oleanolic acid, or vehicle. After 24 hours of fasting, lower hindlimb skeletal muscles and liver were harvested and weighed. As shown previously, ursolic acid increased skeletal muscle weight (FIG. 14B), but not liver weight (FIG. 14C). In contrast, oleanolic acid increased liver weight (FIG. 14F), but not skeletal muscle weight (FIG. 14E). Interestingly, metformin (250 mg/kg) resembled oleanolic acid in biological effect: it increased liver weight (FIG. 141), but not muscle weight (FIG. 14H). Without wishing to be bound by a particular theory, ursolic acid increases skeletal muscle and inhibit muscle atrophy by a pathway that does not involve PTP inhibition.

13. Targeted Inhibition of PTP1B Does Not Induce Skeletal Muscle Hypertrophy.

To further rule out the potential role of PTP1B inhibition in skeletal muscle hypertrophy, PTP1B expression was specifically reduced in mouse skeletal muscle by transfecting plasmid DNA constructed to express RNA interference constructs. Briefly, C57BL/6 mouse tibialis anterior muscles were transfected with 20 μg pCMV-miR-control (control plasmid transfected in the left TA) or either 20 μg pCMV-miR-PTP1B #1 (encoding miR-PTP1B #1; transfected in the right TA) or 20 μg pCMV-miR-PTP1B #2 (encoding miR-PTP1B #2; transfected in the right TA). miR-PTP1B #1 and miR-PTP1B #2 encode two distinct RNA interference (RNAi) constructs targeting distinct regions of PTP1B mRNA. Tissue was harvested 10 days following transfection.

Of note with regard to FIG. 15A, mRNA measurements were taken from the entire TA muscle. Because electroporation transfects only a portion of muscle fibers, the data underestimate PTP1B knockdown in transfected muscle fibers. In FIG. 15A, mRNA levels in the right TA were normalized to levels in the left TA, which were set at 1; data are means±SEM from 3 mice. In FIG. 15B, in each TA muscle, the mean diameter of >300 transfected fibers was determined; data are means±SEM from 3 TA muscles per condition. For both FIGS. 15A and 15B, P-values were determined with one-tailed paired t-tests.

Although both miR-PTP1B constructs reduced PTP1B mRNA (FIG. 15A), neither increased skeletal muscle fiber diameter (FIG. 15B). These data demonstrate that targeted PTP1B inhibition does not cause muscle fiber hypertrophy. Without wishing to be bound by a particular theory, ursolic acid does not increase skeletal muscle by inhibiting PTP1B.

14. Ursolic Acid Serum Levels Associated with Increased Muscle Mass and Decreased Adiposity.

To determine the dose-response relationship between dietary ursolic acid and muscle and adipose weight, C57BL/6 mice were fed standard chow containing varying amounts of ursolic acid for 7 weeks. Serum ursolic acid levels from mice were determined as described above. As shown previously in FIG. 10A, ursolic acid increased skeletal muscle weight and decreased weight of retroperitoneal and epididymal fat pads in a dose-dependent manner, but did not alter heart weight (FIG. 16A; data are means±SEM). These effects of ursolic acid were discernable at 0.035% ursolic acid and were maximal at doses ≥0.14% ursolic acid. Serum was collected from these same mice at the time of necropsy, and then measured random serum ursolic acid levels via ultra high performance liquid chromatography (UPLC). The data indicate that ursolic acid serum levels in the range of 0.25-0.5 μg/ml are sufficient to increase muscle mass and decrease adiposity (FIG. 16B; data are means±SEM). Of note, 0.5 μg/ml equals 1.1 μM ursolic acid, close to the dose used in the Connectivity Map (8.8 μM) and in the C2C12 experiments (10 μM) described above.

The data described herein indicate that ursolic acid reduced muscle atrophy and stimulated muscle hypertrophy in mice. Importantly, ursolic acid's effects on muscle were accompanied by reductions in adiposity, fasting blood glucose and plasma leptin, cholesterol and triglycerides, as well as increases in the ratio of skeletal muscle to fat, the amount of brown fat, the ratio of brown fat to white fat, and increased energy expenditure. Without wishing to be bound by a particular theory, ursolic acid reduced muscle atrophy and stimulated muscle hypertrophy by enhancing skeletal muscle IGF-I expression and IGF-I signaling, and inhibiting atrophy-associated skeletal muscle mRNA expression.

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention.

More specifically, certain agents which are both chemically and physiologically related can be substituted for the agents described herein while the same or similar results can be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

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Claims

1. A method for: in an animal, the method comprising administering to the animal ursolic acid, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein said animal is selected from the group consisting of a primate, domesticated fish, domesticated crustacean, domesticated mollusk, poultry, rabbit, dog, cat, and livestock.

(a) increasing skeletal muscle mass;

(b) reducing skeletal muscle atrophy;

(c) increasing muscular strength; or

(d) promoting muscle growth

2. The method of claim 1, wherein the ursolic acid or pharmaceutically acceptable salt, hydrate, or solvate thereof is administered to the animal in an amount of greater than or equal to 100 mg per day.

3. The method of claim 1, wherein the animal is a mammal.

4. The method of claim 1, wherein the animal is a human.

5. The method of claim 1, wherein the animal is selected from the group consisting of a dog, cat, pig, cow, horse, goat, bison, sheep, chicken, turkey, duck, goose, and domesticated fish.

6. The method of claim 1, wherein the ursolic acid is present as a pharmaceutically acceptable salt selected from salts derived from aluminum, ammonium, calcium, copper (-ic and -ous), ferric, ferrous, lithium, magnesium, manganese, potassium, sodium, or zinc; salts of primary, secondary, and tertiary amines; and salts derived from arginine, betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholinc, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, or tromethamine.

7. The method of claim 1, wherein the ursolic acid or pharmaceutically acceptable salt, hydrate, or solvate thereof is administered to the animal in an amount of greater than 200 mg per day.

8. The method of claim 1, wherein the ursolic acid or pharmaceutically acceptable salt, hydrate, or solvate thereof is administered to the animal in an amount of greater than 300 mg per day.

9. The method of claim 1, wherein the ursolic acid or pharmaceutically acceptable salt, hydrate, or solvate thereof is administered to the animal in an amount of greater than 400 mg per day.

10. The method of claim 1, wherein the ursolic acid or pharmaceutically acceptable salt, hydrate, or solvate thereof is administered to the animal in an amount of greater than 500 mg per day.

11. The method of claim 1, wherein the ursolic acid or pharmaceutically acceptable salt, hydrate, or solvate thereof is administered to the animal in an amount of greater than 750 mg per day.

12. The method of claim 1, wherein the ursolic acid or pharmaceutically acceptable salt, hydrate, or solvate thereof is administered to the animal in an amount of greater than 1000 mg per day.

13. The method of claim 1, wherein the ursolic acid or pharmaceutically acceptable salt, hydrate, or solvate thereof is administered to the animal in a composition that further comprises one or more adjuvants.

14. The method of claim 1, wherein the ursolic acid or pharmaceutically acceptable salt, hydrate, or solvate thereof is administered to the animal in a dosage of from about 0.1 to about 50 mg/kg per day.

15. The method of claim 4, wherein the ursolic acid or pharmaceutically acceptable salt, hydrate, or solvate thereof is administered to the animal in a dosage of from about 0.1 to about 50 mg/kg per day.

16. The method of claim 7, wherein the ursolic acid or pharmaceutically acceptable salt, hydrate, or solvate thereof is administered to the animal in a dosage of from about 0.1 to about 50 mg/kg per day.

17. The method of claim 1, wherein the ursolic acid or pharmaceutically acceptable salt, hydrate, or solvate thereof is administered to the animal in a composition that further comprises a preservative.

18. The method of claim 17, wherein the ursolic acid or pharmaceutically acceptable salt, hydrate, or solvate thereof is administered to the animal in an amount of greater than or equal to 100 mg per day, and wherein the animal is selected from a domesticated fish, poultry, pig, cow, horse, goat, bison, sheep, dog, cat, and bison.

19. The method of claim 1, wherein the ursolic acid or pharmaceutically acceptable salt, hydrate, or solvate thereof has been isolated from a natural source.

20. The method of claim 1, for:

(b) reducing skeletal muscle atrophy;

(c) increasing muscular strength; or

(d) promoting muscle growth.