# Patent

## Title
Methods for lowering blood glucose

## Abstract

In one aspect, the invention relates to methods for treating muscle atrophy by providing to an animal in need thereof an effective amount of a compound. The compound can modulate the expression levels of multiple mRNA of a muscle atrophy signature. 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.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This Application is a national phase application of International Application No. PCT/US2012/041119, filed Jun. 6, 2012, which claims the benefit of U.S. Provisional Application No. 61/493,969, filed on Jun. 6, 2011; which are incorporated herein by reference in entirety.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
This invention was made with government support under T32 GM073610, 1R01 AR059115-01 and HL007121 awarded by the National Institutes of Health, as well as support from grant IBX000976A awarded by the Department of Veterans Affairs. The 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 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 J 20(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 treat muscle atrophy. The compounds can be selected from a tacrine and analogs, naringenin and analogs, allantoin and analogs, conessine and analogs, tomatidine and analogs, ungerine/hippeastrine and analogs, and betulinic acid and analogs, or a mixture thereof.
Tacrine and analogs can have the structure:




Naringenin and analogs can have the structure:




Allantoin and analogs can have the structure:




Conessine and analogs can have the structure:




Tomatidine and analogs can have the structure:




Ungerine/hippeastrine and analogs can have the structure:




Betulinic acid and analogs can have the structure:




The disclosed compounds can treat muscle atrophy when administered in an effective amount to an animal, such as a mammal, fish or bird. For example, human.
Also disclosed in a method of lowering blood glucose in an animal by administering ursolic acid or ursolic acid analogs, such as betulininc acid analogs, and narigenin analogs, such as naringenin, in an effective amount to an animal.
Also disclosed in a method of lowering blood glucose in an animal by administering ungerine/hippeastrine analogs, such as hippeastrine, in an effective amount to an animal.
The disclosed compounds can also promote muscle health, promote normal muscle function, and/or promote healthy aging muscles by providing to a subject in need thereof an effective amount of a disclosed compound.
Also disclosed herein are pharmaceutical compositions comprising compounds used in the methods. Also disclosed herein are kits comprising compounds used in the methods.
In further aspects, In a further aspect, the invention relates to compounds identified using muscle atrophy signature-1, muscle atrophy signature-2 or both muscle atrophy signatures. In still further aspects, 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 health promote normal muscle function, and/or promote healthy aging muscles, methods to inhibit muscle atrophy, methods to increase insulin/IGF-I signaling, methods to reduce body fat; methods to reduce blood glucose, methods to reduce blood triglycerides, methods to reduce blood cholesterol, methods to reduce obesity, methods to reduce fatty liver disease, and methods to reduce diabetes, and pharmaceutical compositions comprising compounds used in the methods.
Disclosed are methods for treating muscle atrophy in a mammal, the method comprising administering to the mammal an effective amount of a compound, wherein the compound: (a) down regulates multiple induced mRNAs of a Muscle Atrophy Signature, compared to expression levels of the induced mRNAs of the Muscle Atrophy Signature in the same type of the muscle cell in the absence of the compound, and/or (b) up regulates multiple repressed mRNAs of the Muscle Atrophy Signature, compared to expression levels of the repressed mRNAs of the Muscle Atrophy Signature in the same type of the muscle cell in the absence of the compound, thereby inhibiting muscle atrophy in the mammal.
Also disclosed are methods for identifying a compound that inhibits muscle atrophy when administered in a effective amount to a animal in need of treatment thereof, the method comprising the steps of: (i) selecting a candidate compound; (ii) determining the effect of the candidate compound on a cell's expression levels of a plurality of induced mRNAs and/or repressed mRNAs of a Muscle Atrophy Signature, wherein the candidate compound is identified as suitable for muscle atrophy inhibition if: (a) more than one of the induced mRNAs of the Muscle Atrophy Signature are down regulated, compared to expression levels of the induced mRNAs of the Muscle Atrophy Signature in the same type of cell in the absence of the candidate compound; and/or (b) more than one of the repressed mRNAs of the Muscle Atrophy Signature are up regulated, compared to expression levels of the repressed mRNAs of the Muscle Atrophy Signature in the same type of cell in the absence of the candidate compound. In one aspect, the method further comprises administering the candidate compound to an animal. The candidate compound can be tacrine and analogs, naringenin and analogs, allantoin and analogs, conessine and analogs, tomatidine and analogs, ungerine/hippeastrine and analogs, and betulinic acid and analogs, or a mixture thereof.
Also disclosed are methods for manufacturing a medicament associated with muscle atrophy or the need to promote muscle health, promote normal muscle function, and/or promote healthy aging muscles 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 promote muscle health, promote normal muscle function, and/or promote healthy aging muscles.
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 human muscle atrophy signature-1.
FIG. 3 shows human muscle atrophy signature-2.
FIG. 4 shows representative data on the effect of fasting on skeletal muscle mRNA expression in healthy human adults.
FIG. 5 shows qPCR analysis of representative fasting-responsive mRNAs from human skeletal muscle.
FIG. 6 shows representative data on the identification of ursolic acid as an inhibitor of fasting-induced skeletal muscle atrophy.
FIG. 7 shows representative data on the identification of ursolic acid as an inhibitor of denervation-induced muscle atrophy.
FIG. 8 shows representative data on ursolic acid-mediated induction of muscle hypertrophy.
FIG. 9 shows representative data on the effect of ursolic acid on mouse skeletal muscle specific tetanic force.
FIG. 10 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. 11 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. 12 shows representative data on the effect of ursolic acid on adiposity and plasma lipids.
FIG. 13 shows representative data on the effect of ursolic acid on food consumption, liver weight, kidney weight, and plasma ALT, bilirubin, and creatinine concentrations.
FIG. 14 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. 15 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. 16 shows representative data that oleanolic acid and metformin do not reduce skeletal muscle atrophy.
FIG. 17 shows representative data that targeted inhibition of PTP1B does not inhibit skeletal muscle atrophy.
FIG. 18 shows representative data on the effect of ursolic acid serum concentration on muscle mass and adiposity.
FIG. 19 shows that betulinic acid, like ursolic acid, reduces immobilization-induced skeletal muscle atrophy. Mice were administered vehicle (corn oil) or the indicated concentration of ursolic acid (A) or betulinic acid (B) via intraperitoneal injection twice a day for two days. One tibialis anterior (TA) muscle was immobilized with a surgical staple, leaving the contralateral mobile TA as an intrasubject control. Vehicle, or the same dose of ursolic acid or betulinic acid was administered via i.p. injection twice daily for six days before comparing weights of the immobile and mobile TAs. Data are means±SEM from 9-10 mice per condition. A, ursolic acid dose-response relationship. B, betulinic acid dose-response relationship.
FIG. 20 shows that naringenin reduces immobilization-induced skeletal muscle atrophy. Mice were administered vehicle (corn oil), ursolic acid (200 mg/kg), naringenin (200 mg/kg) or ursolic acid plus naringenin (both at 200 mg/kg) via intraperitoneal injection twice a day for two days. One tibialis anterior (TA) muscle was immobilized with a surgical staple, leaving the contralateral mobile TA as an intrasubject control. Vehicle, or the same dose of ursolic acid and/or naringenin was administered via i.p. injection twice daily for six days before comparing weights of the immobile and mobile TAs. Data are means±SEM from 9-10 mice per condition.
FIG. 21 shows that the combination of ursolic acid and naringenin normalizes fasting blood glucose levels in a mouse model of glucose intolerance, obesity and fatty liver disease. Mice were fed standard chow, high fat diet (HFD) plus the indicated concentrations of naringenin, or HFD containing 0.15% ursolic acid (UA) plus the indicated concentrations of naringenin for 5 weeks before measurement of fasting blood glucose (A), total body weight (B), fat mass by NMR (C), liver weight (D), grip strength (E) and skeletal muscle weight (bilateral tibialis anterior, gastrcocnemius, soleus, quadriceps and triceps muscle; F). Dashed line indicates levels in control mice that were fed standard chow. Open symbols indicate levels in mice fed HFD containing the indicated concentrations of naringenin. Closed symbols indicate levels in mice fed HFD containing 0.15% UA plus the indicated concentrations of naringenin. Data are means±SEM from ≧12 mice per condition.
FIG. 22 shows that tomatidine reduces immobilization-induced muscle atrophy. Mice were administered vehicle (corn oil) or the indicated concentration of tomatidine via intraperitoneal injection twice a day for two days. One tibialis anterior (TA) muscle was immobilized with a surgical staple, leaving the contralateral mobile TA as an intrasubject control. Vehicle, or the same dose of tomatidine was administered via i.p. injection twice daily for six days before comparing weights of the immobile and mobile TAs. Data are means±SEM from 9-10 mice per condition. A, effects of 50, 100 and 200 mg/kg tomatidine. B, effects of 5, 15 and 50 mg/kg tomatidine.
FIG. 23 shows that tomatidine reduces fasting-induced muscle atrophy. Data are means±SEM from 9-12 mice per condition. Food was withdrawn from mice, and then vehicle (corn oil), or the indicated concentrations of ursolic acid or tomatidine, were administered by i.p. injection. Twelve hours later, mice received another i.p. injection of vehicle or the same dose of ursolic acid or tomatidine. Twelve hours later, skeletal muscles (bilateral tibialis anterior, gastrcocnemius, soleus, quadriceps muscles) were harvested and weighed. A, comparison of 200 mg/kg ursolic acid and 50 mg/kg tomatidine. B, effects of 5, 15 and 50 mg/kg tomatidine.
FIG. 24 shows that allantoin, tacrine, ungerine, hippeastrine and conessine reduce fasting-induced muscle atrophy. Food was withdrawn from mice, and then vehicle or the indicated dose of ursolic acid, tomatidine, allantoin, tacrine, ungerine, hippeastrine or conessine was administered by i.p. injection. Twelve hours later, mice received another i.p. injection of vehicle or the same dose of ursolic acid, tomatidine, allantoin, tacrine, ungerine, hippeastrine or conessine. Twelve hours later, skeletal muscles (bilateral tibialis anterior, gastrcocnemius and soleus muscles) were harvested and weighed. Data are means±SEM from ≧9 mice per condition and show the percent change in skeletal muscle weight relative to vehicle-treated animals in the same experiment. The vehicle for ursolic acid, tomatidine, ungerine, hippeastrine and conessine was corn oil. The vehicle for tacrine and allantoin was saline.
FIG. 25 shows that hippeastrine and conessine reduce fasting blood glucose. Food was withdrawn from mice, and then vehicle or the indicated dose of hippeastrine or conessine was administered by i.p. injection. Twelve hours later, mice received another i.p. injection of vehicle or the same dose of hippeastrine or conessine. Twelve hours later, blood glucose was measured via tail vein. Data are means±SEM from ≧9 mice per condition.



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 CHEMDRAWTM (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, the term “muscle atrophy signature-1” refers to the set of mRNAs with an altered expression pattern associated with muscle atrophy. The mRNAs comprise mRNAs that are either induced or repressed during the pathophysiology of muscle atrophy and which were identified using the methods described herein. For clarity, muscle atrophy signature-1 comprise the induced and repressed mRNAs described in FIG. 2.
As used herein, the term “muscle atrophy signature-2” refers to the set of mRNAs with an altered expression pattern associated with muscle atrophy. The mRNAs comprise mRNAs that are either induced or repressed during the pathophysiology of muscle atrophy and which were identified using the methods described herein. For clarity, muscle atrophy signature-2 comprise the induced and repressed mRNAs described in FIG. 3.
As used herein, the term “muscle atrophy signature-3” refers to the set of mRNAs with an altered expression pattern associated with muscle atrophy. The mRNAs comprise mRNAs that are either induced or repressed during the pathophysiology of muscle atrophy and which were identified using the methods described herein. For clarity, muscle atrophy signature-3 comprise the induced and repressed mRNAs described in Example 23.
As used herein, the term “muscle atrophy signature-4” refers to the set of mRNAs with an altered expression pattern associated with muscle atrophy. The mRNAs comprise mRNAs that are either induced or repressed during the pathophysiology of muscle atrophy and which were identified using the methods described herein. For clarity, muscle atrophy signature-4 comprise the induced and repressed mRNAs described in Example 24.
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, fish, bird, 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 promoting muscle health prior to the administering step. In some aspects of the disclosed method, the subject has been diagnosed with a need for promoting muscle health prior, promote normal muscle function, and/or promote healthy aging muscles 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 the use for astetic and self improvement purposes, for example, such uses include, but are not limited to, the administration of the disclosed compound in nutraceuticals, medicinal food, energy bar, energy drink, supplements (such as multivitamins). 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, fish, bird, 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 promote muscle health, promote normal muscle function, and/or promote healthy aging muscles. As a further example, “diagnosed with a need for promoting muscle health” 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 promoting muscle health, promoting normal muscle function, and/or promoting healthy aging muscles 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 measured 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 to the half maximal (50%) inhibitory concentration (IC) or inhibitory dose of a substance. The response can be measured 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)0-4N(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)0-4 SR—, 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; —(C1-4 straight or branched)alkylene)O—N(R∘)2; or —(C1-4 straight or branched)alkylene)C(O)O—N(R∘2, wherein each R∘may be substituted as defined below and is independently hydrogen, C1-6 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•, —(C1-4 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 C1-4 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, C1-6 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, C1-6 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 C1-4 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, C1-6 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 C1-4 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, and brosylate.
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 limitation, 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 α-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, Rn(a), Rn(b), Rn(c), Rn(d), Rn(e). By “independent substituents,” it is meant that each R substituent can be independently defined. For example, if in one instance Rn(a) is halogen, then Rn(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 by providing to a subject in need thereof an effective amount of a compound or an analog thereof selected from among the compounds described herein, and pharmaceutical compositions comprising compounds used in the methods. In a further aspect, the invention relates to compounds identified using muscle atrophy signature-1, muscle atrophy signature-2, or both muscle atrophy signatures. In a further aspect, the invention relates to compounds useful in methods to modulate muscle health, methods to inhibit muscle atrophy, methods to increase insulin/IGF-I signaling, methods to reduce body fat; methods to reduce blood glucose, methods to reduce blood triglycerides, methods to reduce blood cholesterol, methods to reduce obesity, methods to reduce fatty liver disease, and methods to reduce diabetes, 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, growth hormone deficiency, IGF-I deficiency, androgen deficiency, estrogen deficiency, 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. Tacrine and Analogs
In one aspect, the compound can be a tacrine analogs.
In one aspect, the tacrine analogs has a structure represented by a formula:




wherein R13a and R13b together comprise a cycle selected from:




wherein Q11 is selected from N and CR13c;
wherein Q12 is selected from N and CR13d;
wherein Q13 and Q14 are independently selected from CR13cR13d, O, S, and NR14c;
wherein Q15 is selected from CR13cR13d, O, S, and NR14c;
wherein Q16 is selected from N and CR13c;
wherein Q17 and Q18 are independently selected from CR13cR13d, O, S, and NR14c;
wherein R11 and R12 are independently selected from H and C1-C6 alkyl;
wherein R13c, R13d, R13e, and R13f are independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, halo, hydroxyl, nitro, amino, cyano, NHCOR15, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, C1-C6 dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently are substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino;
wherein each R14c is independently selected from H and C1-C6 alkyl;
wherein R14a and R14b together comprise a cycle selected from:




wherein each of Q19, Q20, Q21, Q22, Q23, Q24, and Q25 are independently selected from CR17aR17b, O, S, and NR18;
wherein R16a and R16a are independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, halo, hydroxyl, nitro, amino, cyano, NHCOR15, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, C1-C6 dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently are substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino;
wherein R17a and R17b are independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, halo, hydroxyl, nitro, amino, cyano, NHCOR15, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, C1-C6 dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently are substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino;
wherein each R18 is independently selected from H and C1-C6 alkyl;
wherein each R15 is independently selected from H and C1-C6 alkyl;
wherein each n is independently selected from 0, 1, and 2;
wherein m is selected from 1 and 2; and
wherein p is selected from 1, 2 and 3; or
a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.
In one aspect, compound (A) has the structure represented by the formula:




wherein R12 is selected from H and C1-C6 alkyl;
wherein R13c, R13d, R13e, and R13f are independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, halo, hydroxyl, nitro, amino, cyano, NHCOR15, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, C1-C6 dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently are substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino;
wherein Q19 and Q20 are independently selected from CR17aR17b, O, S, and NR18;
wherein R17a and R17b are independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, halo, hydroxyl, nitro, amino, cyano, NHCOR15, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, C1-C6 dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently are substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino;
wherein each R18 is independently selected from H and C1-C6 alkyl; and
wherein n is selected from 0, 1, and 2.
In another aspect, R12 is H; R13c, R13d and R13f are independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, halo, hydroxyl, nitro, and amino; Q19 and Q20 are independently selected from CR17aR17b, O, S, and NR18; wherein R17a and R17b are independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, halo, hydroxyl, nitro, and amino; wherein each R18 is independently H; and n is selected from 0, 1, and 2.
In another aspect, R12 is H; R13c, R13d, R13e, and R13f are independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, halo, hydroxyl, nitro, and amino; Q19 and Q20 are independently selected from C R17aR17b; wherein R17a and R17b are independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, halo, hydroxyl, nitro, and amino; and n is 1.
In another aspect, R12 is H; R13c, R13d, R13e, and R13f are independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, halo, and hydroxyl; Q19 and Q20 are independently selected from CR17aR17b; wherein R17a and R17b are independently H; and n is 1.
In another aspect, R12 is H; R13c, R13d, R13e, and R13f are independently selected from H, C1-C6 alkyl, and halo; Q19 and Q20 are independently CR17aR17b; wherein R17a and R17b independently H; and wherein n is 1.
In another aspect, the formula is:]




2. Naringenin and Analog
In one aspect, the compound can be a naringenin analog.
In one aspect, the naringenin analog has a structure represented by a formula:




wherein each ----- represents a covalent bond selected from a single or double bond;
wherein R21a, R21b, R21c, and R21e are independently selected from H, OH, O-Glucosyl, halo, cyano, amino, nitro, nitroso, NHCOR15, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, acyl, phenyl-C1-C6 alkoxy, benzyl-C1-C6 alkoxy, and C1-C6 dialkylamino;
wherein R22 is selected from H, OH, O-Glucosyl, halo, cyano, amino, nitro, nitroso, NHCOR15, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, acyl phenyl-C1-C6 alkoxy, benzyl-C1-C6 alkoxy, and C1-C6 dialkylamino;
wherein R23a, R23b, R23c, and R23d are independently selected from H, OH, O-Glucosyl halo, cyano, amino, nitro, nitroso, NHCOR15, C1-C20 alkyl, C1-C20 alkenyl, C1-C20 alkynyl, C1-C20 alkenynyl, C1-C20 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, acyl, phenyl-C1-C6 alkoxy, benzyl-C1-C6 alkoxy, and C1-C6 dialkylamino;
wherein R15 is selected from H and C1-C6 alkyl;
wherein Z is selected from O and S; and
wherein Y is selected from O and S; or
a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof;
wherein the compound does not have the structure:




In one aspect, indicates a covalent single bond. In another aspect, indicates a covalent double bond.
In another aspect, Z is O, and Y is O.
In another aspect, R21a, R21b, R21c, R21d, and R21e are independently selected from H and OH; wherein R22 is selected from H and OH; and wherein R23a, R23b, R23c, R23d, and R23e are independently selected from H and OH.
In another aspect, R21a, R21b, R21c, R21d, and R21e are independently selected from H, OH, O-Glucosyl, halo, cyano, amino, nitro, and nitroso.
In another aspect, R22 is H.
In another aspect, R21a, R21b, R21d, and R21e are H, and R21c is OH.
In another aspect, R23a and R23c are H, and R23b and R23d are OH.
In another aspect, R21a, R21b, R21d, and R21e are H, R21c is OH, R23a and R23c are H, and R23b and R23d are OH.
In another aspect, R21a, R21d, and R21e are H, R21b and R21c are OH, R23a and R23c are H, and R23b and R23d are OH.
In another aspect, the compound has the structure:




3. Allantoin and Analogs
In one aspect, the compound can be a allantoin analog.
In one aspect, the allantoin analog has a structure represented by a formula:




wherein R31a and R31b are independently selected from H, C1-C6 alkyl, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently are substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino;
wherein R32a and R32b are independently selected from H, C1-C6 alkyl, OCl(OH)4Al2, OAl(OH)2, C1-C6 alkoxy, halo, hydroxyl, nitro, amino, cyano, NHCOR15, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, C1-C6 dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl or taken together to form a double bond selected from ═O and ═S, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently are substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino;
wherein R32c and R32d are independently selected from H, C1-C6 alkyl, OCl(OH)4Al2, OAl(OH)2, C1-C6 alkoxy, halo, hydroxyl, nitro, amino, cyano, NHCOR15, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, C1-C6 dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl or taken together to form a double bond selected from ═O and ═S, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently are substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino;
wherein R33a and R33b are independently selected from H, NR34aCONR34bR34c, C1-C6 alkyl, C1-C6 alkoxy, halo, hydroxyl, nitro, amino, cyano, NHCOR15, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, C1-C6 dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently are substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino; and
wherein R34a R34b and R34c are independently selected from H, C1-C6 alkyl, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently are substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino; or
a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.
In one aspect, R31a and R31b are H.
In another aspect, R32a and R32b are taken together to form ═O.
In another aspect, R32c and R32d are taken together to form ═O.
In another aspect, R32a and R32b are taken together to form ═O, and R32c and R32d are taken together to form ═O.
In another aspect, R31a is H, R31b is H, R32a, and R32b are taken together to form ═O, and R32c and R32d are taken together to form ═O.
In another aspect, R31a is H, R31b is H, R32a and R32b are taken together to form ═O, R32c and R32d are taken together to form ═O, and one of R33a and R33b is NR34aCONR34bR34c and the other one of R33a and R33b is H.
In another aspect, one of R33a and R33b is NR34aCONR34bR34c and the other one of R33a and R33b is H.
In another aspect, one of R32a and R32b is OCl(OH)4Al2 and the other one of R32a and R32b is H.
In another aspect, one of R32c and R32d is OCl(OH)4Al2 and the other one of R32c and R32d is H.
In another aspect, one of R32a and R32b is OAl(OH)2 and the other one of R32a and R32b is H.
In another aspect, one of R32c and R32d is OAl(OH)2 and the other one of R32c and R32d is H.
In another aspect, the compound has the structure:




4. Conessine and Analogs
In one aspect, the compound can be a conessine analog.
In one aspect, the conessine analog has a structure represented by a formula:




wherein each ----- represents a covalent bond independently selected from a single or double bond, wherein valency is satisfied;
wherein R41 is selected from NR48aR48b, ═O, ═S, C1-C6 alkoxy and hydroxyl;
wherein R48a and R48b are independently selected from H, C1-C6 alkyl, C1-C6 heteroalkyl, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino;
wherein R42 is selected from H, C1-C6 alkoxy and hydroxyl;
wherein R43 is selected from H and C1-C6 alkyl;
wherein R44a and R44b are independently selected from are independently selected from H, hydroxyl, and C1-C6 alkoxy;
wherein R47a and R47b are independently selected from are independently selected from H, hydroxyl, and C1-C6 alkoxy;
wherein R45a and R45b together comprise a cycle selected from:




wherein R49a is selected from H and C1-C6 alkyl; and
wherein R49b and R49c are independently selected from H and C1-C6 alkyl, or taken together to form ═O; or
a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.
In one aspect, R47a and R47b are independently selected from H, hydroxyl, and C1-C6 alkoxy.
In another aspect, R44a and R44b are independently selected from H, hydroxyl, and C1-C6 alkoxy.
In another aspect, R42 is H.
In another aspect, R47a and R47b are selected from H, hydroxyl, and C1-C6 alkoxy; R44a and R44b are H.
In another aspect, R41 is selected from NR48aR48b and ═O, wherein R48a and R48b are independently selected from H and C1-C6 alkyl.
In another aspect, R43 is C1 alkyl.
In another aspect, the formula has the structure:




In another aspect, the formula has the structure:




In another aspect, the formula has the structure:




In another aspect, the formula has the structure:




In another aspect, the formula has the structure:




5. Tomatidine and Analogs
In one aspect, the compound can be a tomatidine analog.
In one aspect, the tomatidine analog has a structure represented by a formula:




wherein R51 is selected from H, C1-C6 alkyl, COR53, C1-C6 alkylamino, C1-C6 dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino;
wherein R53 is selected from C1-C6 alkyl, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino;
wherein Z51 is selected from O, S, and NR54;
wherein R54 is selected from H, C1-C6 alkyl, COR55, C1-C6 alkylamino, C1-C6 dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino;
wherein R55 is selected from C1-C6 alkyl, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino; or
a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.
In one aspect, R51 is selected from H, C1-C6 alkyl and COR53, wherein R53 is C1-C6 alkyl.
In another aspect, R51 is H.
In another aspect, Z51 is NR54. In another aspect, Z51 is NR54, wherein R54 is selected from H, C1-C6 alkyl, and COR55, wherein R55 is C1-C6 alkyl.
In another aspect, R51 is selected from H, C1-C6 alkyl and COR53, wherein R53 is C1-C6 alkyl; and Z51 is NR54, wherein R54 is selected from H, C1-C6 alkyl, and COR55, wherein R55 is C1-C6 alkyl.
In another aspect, R51 and R54 identical.
In another aspect, the structure is represented by the formula:




In another aspect, the structure is represented by the formula:




In another aspect, the formula has the structure:




6. Ungerine/Hippeastrine and Analogs
In one aspect, the compound can be a ungerine/hippeastrine analog.
In one aspect, the ungerine/hippeastrine has a structure represented by a formula:




wherein R61a and R61b are independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, halo, hydroxyl, nitro, amino, cyano, NHCOR15, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, C1-C6 dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino;
wherein R62a and R62b are independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, halo, hydroxyl, nitro, amino, cyano, NHCOR15, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, C1-C6 dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino;
wherein R63a and R63b are independently selected from H, C1-C6 alkyl, C1-C6 alkoxy, halo, hydroxyl, nitro, amino, cyano, NHCOR15, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, C1-C6 dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, or taken together to form a group selected from ═O and ═S, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino;
wherein R64a and R64b are independently selected from H, OR67, C1-C6 alkyl, C1-C6 alkoxy, halo, hydroxyl, nitro, amino, cyano, NHCOR15, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, C1-C6 dialkylamino, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino;
wherein R65 is selected from H, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, C1-C6 dialkylamino, COR66, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino;
wherein R66 is selected from C1-C6 alkyl, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino;
wherein R67 is selected from C1-C6 alkyl, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl, wherein C6-C10 aryl, C3-C10 cycloalkyl, C5-C9 heteroaryl, and C2-C9 heterocyclyl are independently substituted with 0, 1, 2, or 3 substituents selected from halogen, hydroxyl, cyano, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 monohaloalkyl, C1-C6 polyhaloalkyl, C1-C6 alkylamino, and C1-C6 dialkylamino;
wherein each R15 is independently selected from H and C1-C6 alkyl; or
a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof,
wherein the compound is present in an effective amount.
In one aspect, the structure is represented by a formula:




In another aspect, the structure is represented by a formula:




In another aspect, R61a, R61b, R62a, and R62b are H.
In another aspect, one of R63a and R63b is hydroxyl and the other one of R63a and R63b is H.
In another aspect, R63a and R63b are taken together and form ═O.
In another aspect, one of R64a and R64b is hydroxyl or OR67 and the other one of R64a and R64b is H.
In another aspect, one of R64a and R64b is hydroxyl or OR67 and the other one of R64a and R64b is H, wherein R67 is C1-C6 alkyl.
In another aspect, R65 is C1-C6 alkyl.
In another aspect, the structure is represented by a formula:




In another aspect, the structure is represented by a formula:




In another aspect, the structure is represented by a formula:




In another aspect, the structure is represented by a formula:




In another aspect, the structure is represented by a formula:




7. Betulinic Acid and Analogs
In one aspect, the compound can be a betulinic acid derivative.
In one aspect, has a structure represented by a formula:




wherein ----- is an covalent bond selected from a single bond and a double bond, wherein valency is satisfied, and R70 is optionally present; wherein n is 0 or 1; wherein R70, when present, is hydrogen; wherein R71a is selected from C1-C6 alkyl and —C(O)ZR82; wherein R71b is selected from C1-C6 alkyl, or wherein R71a and R71b are optionally covalently bonded and, together with the intermediate carbon, comprise an optionally substituted C3-C5 cycloalkyl or C2-C5 heterocycloalkyl; wherein one of R72a and R72b is —Z72, and the other is hydrogen, or R72a and R72b together comprise ═O; wherein each of R73a and R73b is independently selected from hydrogen, hydroxyl, C1-C6 alkyl, and C1-C6 alkoxyl, provided that R73a and R73b are not simultaneously hydroxyl, wherein R73a and R73b are optionally covalently bonded and, together with the intermediate carbon, comprise an optionally substituted C3-C5 cycloalkyl or C2-C5 heterocycloalkyl; wherein each of R74, R75, and R76 is independently selected from C1-C6 alkyl; wherein R77 is selected from C1-C6 alkyl, and —C(O)Z71R80; wherein R80 is selected from hydrogen and C1-C6 alkyl; wherein R78a and R78b are independently selected from hydrogen and C1-C6 alkyl; wherein each of R79a and R79b is independently selected from hydrogen and C1-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl, provided that R79a and R79b are not simultaneously hydrogen; or wherein R79a and R79b are covalently bonded and, along with the intermediate carbon, together comprise C3-C5 cycloalkyl or C2-C5 heterocycloalkyl; wherein R82 is selected from hydrogen and C1-C6 alkyl; wherein Z71 and Z72 are independently selected from —OR81— and —NR83—; wherein R83 and R83 are independently selected from hydrogen and C1-C4 alkyl; or, wherein Z71 and Z72 are independently N, R84 and R85 are covalently bonded and —NR84R85 comprises a moiety of the formula:





wherein Y is selected from —O—, —S—, —SO—, —SO2—, —NH—, —NCH3—, or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.
In another aspect, the formula has the structure:




In another aspect, the formula has the structure:




In another aspect, the formula has the structure:




In one aspect, ----- is a single bond. In another aspect, ----- is a double bond.
In one aspect, n is 0. In another aspect, n is 1.
In another aspect, R71a is C1-C6 alkyl; R71b is selected from C1-C6 alkyl; one of R72a is —Z72, and R72b is hydrogen; R74, R75 are independently selected from C1-C6 alkyl; wherein R79b is selected from C1-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl; Z71 is —O—; and Z72 is selected from —OR8 and —NR83—; R81 and R83 are independently selected from hydrogen and C1-C4 alkyl.
In another aspect, R71a is C1 alkyl; R71b is C1 alkyl; R72a is —Z72, and R72b is hydrogen; R74, R75 are independently selected from C1 alkyl; wherein R79b is selected from C1-C6 alkyl, C2-C6 alkenyl, and C2-C6 alkynyl; Z71 is —O—; and Z72 is selected from —OR81 and —NR83—; wherein R81 and R83 are hydrogen.
In another aspect, R71a is C1 alkyl; R71b is C1 alkyl; R72a is —Z72, and R72b is hydrogen; R74, R75 are independently selected from C1 alkyl; R79b is C2-C6 alkenyl; Z71 is —O—; and Z72 is selected from —OR81 and —NR83—; wherein R81 and R83 are hydrogen.
8. Compounds Identified by Muscle Atrophy Signature-1 and Muscle Atrophy Signature-2.
In various aspects, the invention relates to uses of one or more compounds selected from tacrine analogs, naringenin analogs, allantoin analogs, conessine analogs, tomatidine analogs, hippeastrine/ungerine analogs and betulinic acid analogs.
a. Muscle Atrophy Signature-1
In one aspect, the disclosed compounds comprise compounds identified using muscle atrophy signature-1. Such compounds include, but are not limited to, allantoin; conessine; naringenin; tacrine; tomatidine or a pharmaceutically acceptable salt, tautomer, stereoisomer, hydrate, solvate, or polymorph thereof. In a yet further aspect, the compound is an analog of one the preceding compounds as defined above.
B. Muscle Atrophy Signature-2
In a further aspect, the disclosed compounds comprise compounds identified using muscle atrophy signature-2. Such compounds include, but are not limited to, allantoin; betulinic acid; conessine; naringenin; tacrine; tomatidine or a pharmaceutically acceptable salt, tautomer, stereoisomer, hydrate, solvate, or polymorph thereof. In a yet further aspect, the compound is an analog of one the preceding compounds as defined above.
c. Muscle Atrophy Signature-1 or Muscle Atrophy Signature-2
In a further aspect, the disclosed compounds comprise compounds identified using either muscle atrophy signature-1 or muscle atrophy signature-2. Such compounds include, but are not limited to, allantoin; betulinic acid; conessine; naringenin; tacrine; tomatidine or a pharmaceutically acceptable salt, tautomer, stereoisomer, hydrate, solvate, or polymorph thereof. In a yet further aspect, the compound is an analog of one the preceding compounds as defined above.
D. Muscle Atrophy Signature-1 and Muscle Atrophy Signature-2
In a further aspect, the disclosed compounds comprise compounds identified using both muscle atrophy signature-1 and muscle atrophy signature-2, and is a compound associated with both muscle atrophy signatures. Such compounds include, but are not limited to, allantoin; conessine; naringenin; tacrine; tomatidine or a pharmaceutically acceptable salt, tautomer, stereoisomer, hydrate, solvate, or polymorph thereof. In a yet further aspect, the compound is an analog of one the preceding compounds as defined above.
9. Inhibition of Muscle Atrophy
In one aspect, the disclosed compounds inhibit muscle atrophy. In a further aspect, the disclosed compounds promoting muscle health, promoting normal muscle function, and/or promoting healthy aging muscles. In a yet further aspect, the disclosed compounds inhibit of muscle atrophy and promote muscle health, promote normal muscle function, and/or promote healthy aging muscles. In a further aspect, the inhibition of muscle atrophy is in an animal. In an even further aspect, the promoting muscle health, promoting normal muscle function, and/or promoting healthy aging muscles 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 5 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 10 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 25 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 50 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 75 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 100 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 150 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 200 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 250 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 1500 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.
It is contemplated that one or more compounds can optionally be omitted from the disclosed invention.
C. 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. In another example, a pharmaceutical composition can be provided comprising a prophylactically effective amount of at least one disclosed compound
In one aspect, the invention relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a compound, wherein the compound is present in an effective amount. The compound can be selected from a tacrine analog, allantoin analog, naringenin analog, conessine analog, tomatidine analog, ungerine/hippeastrine analog and betulinic acid analog. For example, the compound can be a tacrine analog. In another example, the compound can be a naringenin analog. In another example, the compound can be a conessine analog. In another example, the compound can be a tomatidine analog. In another example, the compound can be an ungerine/hippeastrine analog. In another example, the compound can be a betulinic acid analog.
In one aspect, the compound is present in an amount greater than about an amount selected from 5 mg, 10 mg, 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400, mg, 500 mg, 750 mg, 1000 mg, 1,500 mg, or 2,000 mg.
A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of one or more of: (a) a compound selected from a tacrine analog, allantoin analog, naringenin analog, conessine analog, tomatidine analog, ungerine/hippeastrine analog and betulinic acid analog; (b) a compound that down regulates multiple induced mRNAs of Muscle Atrophy Signature 1, compared to expression levels in the same type of the muscle cell in the absence of the compound; (c) a compound that up regulates multiple repressed mRNAs of Muscle Atrophy Signature 1, compared to expression levels in the same type of the muscle cell in the absence of the compound; (d) a compound that down regulates multiple induced mRNAs of Muscle Atrophy Signature 2, compared to expression levels in the same type of the muscle cell in the absence of the compound; and/or (e) a compound that up regulates multiple mRNAs of Muscle Atrophy Signature 2, compared to expression levels in the same type of the muscle cell in the absence of the compound.
In a further aspect, the amount is a therapeutically effective amount. In a still further aspect, the amount is a prophylactically effective amount.
In a further aspect, pharmaceutical composition is administered to an animal. In a still further aspect, the animal is a mammal, fish or bird. 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 pharmaceutical composition comprises a compound identified using muscle atrophy signature-1. In a yet further aspect, the pharmaceutical composition comprises a compound identified using muscle atrophy signature-2. In a yet further aspect, the pharmaceutical composition comprises a compound identified using both muscle atrophy signature-1 and muscle atrophy signature-2.
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 promoting muscle health, promoting normal muscle function, and/or promoting healthy aging muscles.
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 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 thereof, 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 health, muscle function and/or healthy muscle aging 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 milligrams 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 health, muscle function, and/or healthy aging muscles (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.
D. 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 bed rest, 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 excess, growth hormone deficiency, IGF-I deficiency, estrogen deficiency, 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 bed rest, 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, growth hormone deficiency, IGF-I deficiency, estrogen deficiency, 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 promote muscle health, promote normal muscle function, and/or promote healthy aging muscles 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.
In certain aspects, the disclosed compounds 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, a disclosed compound 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 one or more disclosed compounds (e.g., by parenteral injection or by oral consumption) can be used to promote muscle health, promote normal muscle function, and/or promote healthy aging muscles, and reduce muscle atrophy in all muscles, including those of the limbs and the diaphragm. Local administration of a disclosed compound (by a topical route or localized injection) can be used to promote local muscle health, 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 health including ursolic acid, 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 inhibitors, myostatin signaling inhibitors, beta-2 adrenergic agents including clenbuterol, androgens, selective androgen receptor modulator (such as GTx-024, BMS-564,929, LGD-4033, AC-262,356, JNJ-28330835, LGD-2226, LGD-3303, S-40503, or S-23), aromatase inhibitors (such as anastrozole, letrozole, exemestane, vorozole, formestane, fadrozole, 4-hydroxyandrostenedione, 1,4,6-androstatrien-3,17-dione, and 4-androstene-3,6,17-trione), growth hormone, a growth hormone analog, ghrelin, a ghrelin analog. A disclosed compound or salt thereof can be administered orally, intramuscularly, intravenously or intraarterially. A disclosed compound or salt thereof can be substantially pure. A disclosed compound 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 ursolic acid, 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 inhibitors, myostatin signaling inhibitors, beta-2 adrenergic agents including clenbuterol, androgens, selective androgen receptor modulator (such as GTx-024, BMS-564,929, LGD-4033, AC-262,356, JNJ-28330835, LGD-2226, LGD-3303, S-40503, or S-23), aromatase inhibitors (such as anastrozole, letrozole, exemestane, vorozole, formestane, fadrozole, 4-hydroxyandrostenedione, 1,4,6-androstatrien-3,17-dione, and 4-androstene-3,6,17-trione), growth hormone, a growth hormone analog, ghrelin, or a ghrelin analog. A disclosed compound or salt thereof can be administered orally, intramuscularly, intravenously or intraarterially. A disclosed compound or salt thereof can be substantially pure. A disclosed compound 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. In still further aspects, the invention is related to methods to modulate muscle health, methods to inhibit muscle atrophy.
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 promoting muscle health, promote normal muscle function, and/or promote healthy aging muscles 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.
The compounds disclosed herein are useful for treating, preventing, ameliorating, controlling or reducing the risk of a variety of metabolic disorders. In a further aspect, the disclosed compounds in treating disorders associated with a dysfunction of insulin/IGF-I signaling. Thus, are provided methods to increase insulin/IGF-I signaling, methods to reduce body fat; methods to reduce blood glucose, methods to reduce blood triglycerides, methods to reduce blood cholesterol, methods to reduce obesity, methods to reduce fatty liver disease, and methods to reduce diabetes, and pharmaceutical compositions comprising compounds used in the methods.
a. Treating Muscle Atrophy
Disclosed herein is a method of treating muscle atrophy in an animal comprising administering to the animal an effective amount of a compound. The compound can be selected from a tacrine and analogs, naringenin and analogs, allantoin and analogs, conessine and analogs, tomatidine and analogs, ungerine/hippeastrine and analogs, and betulinic acid and analogs, or a mixture thereof. For example, the compound can be a tacrine analog. In another example, the compound can be a naringenin analog. In another example, the compound can be an allantoin analog. In another example, the compound can be a conessine analog. In another example, the compound can be a tomatidine analog. In another example, the compound can be a ungerine/hippeastrine analog. In another example, the compound can be a betulinic acid analog.
In one aspect, the compound is administered in an amount between 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 milligrams 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
In one aspect, the disclosed compounds inhibit muscle atrophy. In a further aspect, the disclosed compounds promote muscle health, promote normal muscle function, and/or promote healthy aging muscles. In a yet further aspect, the disclosed compounds inhibit of muscle atrophy and promoting muscle health, promoting normal muscle function, and/or promoting healthy aging muscles. In an even further aspect, the disclosed compounds inhibit of muscle atrophy.
In a further aspect, the compound administered is a disclosed compound or a product of a disclosed method of making a compound. In a yet further aspect, the invention relates to a pharmaceutical composition comprising at least one compound as disclosed herein.
In a further aspect, the compound is co-administered with an anabolic agent. In a further aspect, wherein the compound is co-administered with ursolic acid or a ursolic acid derivative.
In a further aspect, the animal is a mammal, fish or bird. 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 Muscle Atrophy Signature is Muscle Atrophy Signature 1. In a still further aspect, the Muscle Atrophy Signature is Muscle Atrophy Signature 2.
In a further aspect, prior to the administering step the mammal has been diagnosed with a need for treatment of a disorder selected muscle atrophy, diabetes, obesity, and fatty liver disease. In a yet further aspect, the disorder is muscle atrophy.
In a further aspect, prior to the administering step the mammal has been diagnosed with a need for treatment of a disorder associated with a dysfunction in insulin/IGF-I signaling.
In a further aspect, the treatment of the disorder increases muscle IGF-I signaling. In a still further aspect, the treatment of the disorder increases muscle IGF-I production.
In a further aspect, prior to the administering step the mammal has been diagnosed with a need for treatment of a disorder associated with circulating levels of leptin. In a still further aspect, the treatment decreases the circulating levels of leptin.
In a further aspect, administration the methods are promoting muscle health, normal muscle function, and/or promoting healthy aging muscles in the mammal. In a yet further aspect, administration increases energy expenditure. In a still further aspect, increases brown fat. In an even further aspect, administration increases the ratio of brown fat to white fat. In a still further aspect, administration increases the ratio of skeletal muscle to fat. In a yet further aspect, the compound is co-administered with a disclosed compound or a derivative thereof.
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.
B. Promoting Muscle Health
In one aspect, the invention relates to a method for promoting muscle health, promoting normal muscle function, and/or promoting healthy aging muscles in an animal, the method comprising administering to the animal an effective amount of a compound selected from a tacrine and analogs, naringenin and analogs, allantoin and analogs, conessine and analogs, tomatidine and analogs, ungerine/hippeastrine and analogs, and betulinic acid and analogs, or a mixture thereof, thereby promoting muscle health in the animal. For example, the compound can be a tacrine analog. In another example, the compound can be a naringenin analog. In another example, the compound can be an allantoin analog. In another example, the compound can be a conessine analog. In another example, the compound can be a tomatidine analog. In another example, the compound can be a ungerine/hippeastrine analog. In another example, the compound can be a betulinic acid analog. In one aspect, the invention relates to a method for promoting muscle health. In another aspect, the invention relates to a method for promoting normal muscle function. In another aspect, the invention relates to a method for promoting healthy aging muscles.
In one aspect, the invention relates to a method for promoting muscle health, promoting normal muscle function, and/or promoting healthy aging muscles in an animal, the method comprising administering to the animal an effective amount of a compound, wherein the compound down regulates at least one of the induced mRNAs of Muscle Atrophy Signature 1 or Muscle Atrophy Signature 2, compared to expression levels in the same type of the muscle cell in the absence of the compound, and/or wherein the compound up regulates at least one of the repressed mRNAs of Muscle Atrophy Signature 1 or Muscle Atrophy Signature 2, compared to expression levels in the same type of the muscle cell in the absence of the compound, thereby promoting muscle health, promoting normal muscle function, and/or promoting healthy aging muscles in the animal.
In a further aspect, the animal is a mammal, fish or bird. 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 Muscle Atrophy Signature is Muscle Atrophy Signature 1. In a still further aspect, the Muscle Atrophy Signature is Muscle Atrophy Signature 2.
In a further aspect, prior to the administering step the mammal has been diagnosed with a need for treatment of a disorder selected muscle atrophy, diabetes, obesity, and fatty liver disease. In a yet further aspect, the disorder is muscle atrophy.
In a further aspect, prior to the administering step the mammal has been diagnosed with a need for treatment of a disorder associated with a dysfunction in insulin/IGF-I signaling.
In a further aspect, the treatment of the disorder increases muscle IGF-I signaling. In a still further aspect, the treatment of the disorder increases muscle IGF-I production.
In a further aspect, prior to the administering step the mammal has been diagnosed with a need for treatment of a disorder associated with circulating levels of leptin. In a still further aspect, the treatment decreases the circulating levels of leptin.
In a further aspect, administration promoting muscle health, promoting normal muscle function, and/or promoting healthy aging muscles in the mammal. In a yet further aspect, administration increases energy expenditure. In a still further aspect, increases brown fat. In an even further aspect, administration increases the ratio of brown fat to white fat. In a still further aspect, administration increases the ratio of skeletal muscle to fat. In a yet further aspect, the compound is co-administered with a disclosed compound or a derivative thereof.
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.
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 animal an effective amount of a compound selected from a tacrine and analogs, naringenin and analogs, allantoin and analogs, conessine and analogs, tomatidine and analogs, ungerine/hippeastrine and analogs, and betulinic acid and analogs, or a mixture thereof, thereby promoting muscle health, promoting normal muscle function, and/or promoting healthy aging muscles in the animal. For example, the compound can be a tacrine analog. In another example, the compound can be a naringenin analog. In another example, the compound can be an allantoin analog. In another example, the compound can be a conessine analog. In another example, the compound can be a tomatidine analog. In another example, the compound can be a ungerine/hippeastrine analog. In another example, the compound can be a betulinic acid analog.
In a further aspect, the invention relates to a method of enhancing muscle formation in a mammal, the method comprising administering to the animal an effective amount of a compound, wherein the compound down regulates at least one of the induced mRNAs of Muscle Atrophy Signature 1 or Muscle Atrophy Signature 2, compared to expression levels in the same type of the muscle cell in the absence of the compound, and/or wherein the compound up regulates at least one of the repressed mRNAs of Muscle Atrophy Signature 1 or Muscle Atrophy Signature 2, compared to expression levels in the same type of the muscle cell in the absence of the compound, thereby promoting muscle health, promoting normal muscle function, and/or promoting healthy aging muscles in the animal.
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 the compound promote muscle health, promote normal muscle function, and/or promote healthy aging muscles 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.
3. Facilitating Tissue Formation In Vitro
In one aspect, the invention relates to a method of enhancing tissue health in vitro, the method comprising administering to the tissue an effective amount of a compound wherein the compound down regulates at least one of the induced mRNAs of Muscle Atrophy Signature 1 or Muscle Atrophy Signature 2, compared to expression levels in the same type of the muscle cell in the absence of the compound, and/or wherein the compound up regulates at least one of the repressed mRNAs of Muscle Atrophy Signature 1 or Muscle Atrophy Signature 2, compared to expression levels in the same type of the muscle cell in the absence of the compound, thereby promoting muscle health, promoting normal muscle function, and/or promoting healthy aging muscles.
In a further aspect, the compound administered is a disclosed compound. In a further aspect, the compound is selected from a tacrine and analogs, naringenin and analogs, allantoin and analogs, conessine and analogs, tomatidine and analogs, ungerine/hippeastrine and analogs, and betulinic acid and analogs, or a mixture thereof, thereby facilitating tissue formation in vitro. For example, the compound can be a tacrine analog. In another example, the compound can be a naringenin analog. In another example, the compound can be an allantoin analog. In another example, the compound can be a conessine analog. In another example, the compound can be a tomatidine analog. In another example, the compound can be a ungerine/hippeastrine analog. In another example, the compound can be a betulinic acid analog.
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 skeletal muscle stem or progenitor cells. In an even further aspect, the skeletal muscle stem or progenitor 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 promoting muscle health, promoting normal muscle function, and/or promoting healthy aging muscles 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 one aspect, the invention relates to a method for manufacturing a medicament associated with muscle atrophy or the need to promote muscle health, promote normal muscle function, and/or promote healthy aging muscles, the method comprising the step of combining an effective amount of one or more of: (a) a compound selected from tacrine analog, naringenin analog, allantoin analog, conessine analog, tomatidine analog, ungerine/hippeastrine analog and betulinic acid analog, or a mixture thereof; (b) a compound that down regulates multiple induced mRNAs of Muscle Atrophy Signature 1, compared to expression levels in the same type of the muscle cell in the absence of the compound; (c) a compound that up multiple repressed mRNAs of Muscle Atrophy Signature 1, compared to expression levels in the same type of the muscle cell in the absence of the compound; (d) a compound that down regulates multiple induced mRNAs of Muscle Atrophy Signature 2, compared to expression levels in the same type of the muscle cell in the absence of the compound; and/or (e) a compound that up regulates at least one of the repressed mRNAs of Muscle Atrophy Signature 2, compared to expression levels in the same type of the muscle cell in the absence of the compound, with a pharmaceutically acceptable carrier or diluent.
In a further aspect, the medicament comprises a disclosed compound. In a still further aspect, the compound is selected from a tacrine and analogs, naringenin and analogs, allantoin and analogs, conessine and analogs, tomatidine and analogs, ungerine/hippeastrine and analogs, and betulinic acid and analogs, or a mixture thereof. For example, the compound can be a tacrine analog. In another example, the compound can be a naringenin analog. In another example, the compound can be an allantoin analog. In another example, the compound can be a conessine analog. In another example, the compound can be a tomatidine analog. In another example, the compound can be a ungerine/hippeastrine analog. In another example, the compound can be a betulinic acid analog.
In a further aspect, the medicament is modulates muscle health. In a still further aspect, the medicament inhibits muscle atrophy. In a yet further aspect, the medicament promote muscle health, promote normal muscle function, and/or promote healthy aging muscles.
5. Kits
Also disclosed herein are kit comprising a tacrine analog, naringenin analog, allantoin analog, conessine analog, tomatidine analog, ungerine/hippeastrine analog and betulinic acid analog, or a mixture thereof, and one or more of: a) at least one agent known to treat muscle atrophy in an animal; b) at least one agent known to decrease the risk of obtaining muscle atrophy in an animal; c) at least one agent known to have a side effect of muscle atrophy; d) instructions for treating muscle atrophy; or e) at least one anabolic agent. For example, the compound can be a tacrine analog. In another example, the compound can be a naringenin analog. In another example, the compound can be a allantoin analog. In another example, the compound can be a conessine analog. In another example, the compound can be a tomatidine analog. In another example, the compound can be a ungerine/hippeastrine analog. In another example, the compound can be a betulinic acid analog.
In one aspect, the kit further comprises at least one agent, wherein the compound and the agent are co-formulated.
In another aspect, the compound and the agent are co-packaged. The agent can be any agent as disclosed herein, such as anabolic agent, agent known to have a side effect of muscle atrophy, agent known to decrease the risk of obtaining muscle atrophy in an animal, or agent known to treat muscle atrophy in an animal.
In one aspect, the invention relates to a kit comprising an effective amount of one or more of: (a) a compound selected from a tacrine analog, naringenin analog, allantoin analog, conessine analog, tomatidine analog, ungerine/hippeastrine analog and betulinic acid analog; (b) a compound that down regulates multiple induced mRNAs of Muscle Atrophy Signature 1, compared to expression levels in the same type of the muscle cell in the absence of the compound; (c) a compound that up regulates multiple repressed mRNAs of Muscle Atrophy Signature 1, compared to expression levels in the same type of the muscle cell in the absence of the compound; (d) a compound that down regulates multiple induced mRNAs of Muscle Atrophy Signature 2, compared to expression levels in the same type of the muscle cell in the absence of the compound; and/or (e) a compound that up regulates multiple repressed mRNAs of Muscle Atrophy Signature 2, compared to expression levels in the same type of the muscle cell in the absence of the compound, (f) and one or more of: (i) a protein supplement; (ii) an anabolic agent; (iii) a catabolic agent; (iv) a dietary supplement; (v) at least one agent known to treat a disorder associated with muscle wasting; (vi) instructions for treating a disorder associated with cholinergic activity; or (vii) instructions for using the compound to promote muscle health, promote normal muscle function, and/or promote healthy aging muscles.
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.
6. Method of Lowering Blood Glucose
In one aspect, the invention relates to a method of lowering blood glucose in an animal comprising administering to the animal an effective amount of a composition comprising ursolic acid and a naringenin analog, thereby lowering the blood glucose in the animal. In one aspect, the naringenin analog can be naringenin. In one aspect, the ursolic acid can be a ursolic acid derivative.
In another aspect, invention relates to a method of lowering blood glucose in an animal comprising administering to the animal an effective amount of a hippeastrine analog, thereby lowering the blood glucose in the animal. In one aspect, the hippeastrine analog can be hippeastrine.
In another aspect, invention relates to a method of lowering blood glucose in an animal comprising administering to the animal an effective amount of a conessine analog, thereby lowering the blood glucose in the animal. In one aspect, the conessine analog can be conessine.
In a further aspect, the animal is a mammal, fish or bird. 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, prior to the administering step the mammal has been diagnosed with a need for treatment of a disorder associated with the need of lowering blood glucose.
In a further aspect, prior to the administering step the mammal has been diagnosed with a need for treatment of a disorder associated with a dysfunction in insulin/IGF-I signaling.
In a further aspect, the treatment of the disorder increases muscle IGF-I signaling. In a still further aspect, the treatment of the disorder increases muscle IGF-I production.
In a further aspect, prior to the administering step the mammal has been diagnosed with a need for treatment of a disorder associated with circulating levels of leptin. In a still further aspect, the treatment decreases the circulating levels of leptin.
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, high blood glucose is prevented 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 lowering of blood glucose. In a yet further aspect, the method further comprises the step of identifying the mammal in a need of prevention the need of lowering blood glucose. In an even further aspect, the mammal has been diagnosed with a need for lowering of blood glucose prior to the administering step.
7. Identification of Compounds that Inhibit Muscle Atrophy
Also disclosed are methods for identifying a compound that inhibits muscle atrophy when administered in a effective amount to a animal in need of treatment thereof, the method comprising the steps of: (i) selecting a candidate compound; (ii) determining the effect of the candidate compound on a cell's expression levels of a plurality of induced mRNAs and/or repressed mRNAs of a Muscle Atrophy Signature, wherein the candidate compound is identified as suitable for muscle atrophy inhibition if: (a) more than one of the induced mRNAs of the Muscle Atrophy Signature are down regulated, compared to expression levels of the induced mRNAs of the Muscle Atrophy Signature in the same type of cell in the absence of the candidate compound; and/or (b) more than one of the repressed mRNAs of the Muscle Atrophy Signature are up regulated, compared to expression levels of the repressed mRNAs of the Muscle Atrophy Signature in the same type of cell in the absence of the candidate compound. In one aspect, the method further comprises administering the candidate compound to an animal. In yet another aspect, the method further comprises writing a report. In yet another aspect, the method further comprises reporting the results. In yet another aspect, the method further comprises performing further tests on the candidate compound, such as confirmatory tests. In yet another aspect, the method further comprises performing toxicity studies on the candidate compound.
In a further aspect, the candidate compound comprises a disclosed compound. In a still further aspect, the compound is selected from a tacrine analog, naringenin analog, allantoin analog, conessine analog, tomatidine analog, ungerine/hippeastrine analog and betulinic acid analog, as defined elsewhere herein. For example, the compound can be a tacrine analog. In another example, the compound can be a naringenin analog. In another example, the compound can be an allantoin analog. In another example, the compound can be a conessine analog. In another example, the compound can be a tomatidine analog. In another example, the compound can be a ungerine/hippeastrine analog. In another example, the compound can be a betulinic acid analog.
In a further aspect, the animal is a mammal, fish or bird. 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 Muscle Atrophy Signature is Muscle Atrophy Signature 1. In a still further aspect, the Muscle Atrophy Signature is Muscle Atrophy Signature 2.
In a further aspect, the Muscle Atrophy Signature is determined according to steps comprising: a) determining mRNA expression levels in a muscle cell undergoing muscle atrophy, b) determining mRNA expression levels in a muscle cell not undergoing muscle atrophy, wherein an mRNA is determined to be part of the Muscle Atrophy Signature if: (a0 the mRNA is up regulated in the muscle cell undergoing muscle atrophy compared to the muscle cell not undergoing muscle atrophy, or (b) the mRNA is down regulated in the muscle cell undergoing muscle atrophy compared to the muscle cell not undergoing muscle atrophy.
In one aspect, the muscle cell undergoing atrophy and the muscle cell not undergoing atrophy are harvested from an animal. In another aspect, the muscle cell undergoing atrophy is harvested while the animal is in a state of fasting and the muscle cell not undergoing atrophy is harvested prior to the state of fasting. In yet another aspect, the muscle cell undergoing atrophy is harvested from an immobilized muscle and the muscle cell not undergoing atrophy is harvested from a mobile muscle. In yet another aspect, the muscle cell undergoing atrophy is harvested from an animal with spinal cord injury and the muscle cell not undergoing atrophy is harvested from a muscle that has received electrical stimulation. In yet another aspect, the Muscle Atrophy Signature is determined by selecting mRNAs commonly up regulated or commonly down regulated between two or more of the Muscle Atrophy Signatures of the methods described herein.
In a further aspect, the invention relates to a method for inhibiting muscle atrophy in a mammal, the method comprising administering to the mammal a therapeutically effective amount of a compound of identified using the method described above.
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 inhibitors of muscle atrophy related activity in laboratory animals such as cats, dogs, rabbits, monkeys, rats, fish, birds, and mice, as part of the search for new therapeutic agents of promoting muscle health, promoting normal muscle function, and/or promoting healthy aging muscles.
E. 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, Massachussetts, 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, Massachussetts, 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”) A1c 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 log2 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 log2 signals. To determine which mouse skeletal muscle mRNAs were significantly altered by ursolic acid (P≦0.005), unpaired t-tests were used to compare log2 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 log2 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 ACt 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. 18779) or 10 nM bovine insulin (Sigma; Cat. No. 16634) 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/mlleupeptin, 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)/Fox03a(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 plasmids pCMV-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 ACt 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 out. These three studies determined the effects of: A) fasting on human skeletal muscle mRNA levels as described herein, 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 to quantitate levels of more than 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 “muscle atrophy signature-1” (see FIG. 2). Next, the effects of fasting and SCI on human skeletal muscle were determined and two sets of mRNAs were identified: 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 “muscle atrophy signature-2” (see FIG. 3). Almost all of the mRNAs in muscle 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 muscle 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 muscle 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. The predicted inhibitors of human skeletal muscle atrophy, i.e. compounds with negative connectivity with the muscle atrophy signatures, are shown in Tables 2 and 3 below. Table 2 shows compounds with negative connectivity to human muscle atrophy signature-1 (see FIG. 2 for mRNAs in the signature), whereas Table 3 shows compounds with negative connectivity to human muscle atrophy signature-2 (see FIG. 3 for mRNAs in the signature).
As a proof-of-concept of the utility of muscle 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.







TABLE 2







Compounds with negative connectivity to human muscle atrophy


signature-1.














Connect-




%


Cmap name/cell
ivity

Enrich-

Specif-
Non-


line
score
n
ment
p
icity
null
















conessine - HL60
−0.752
1
−0.991
—
—
100


allantoin - HL60
−0.622
1
−0.954
—
—
100


conessine - PC3
−0.598
1
−0.941
—
—
100


tacrine - HL60
−0.551
1
−0.91
—
—
100


tomatidine - HL60
−0.497
1
−0.873
—
—
100


tomatidine - PC3
−0.483
1
−0.861
—
—
100


naringenin - PC3
−0.462
1
−0.846
—
—
100


allantoin-MCF7
−0.347
2
−0.735
0.13873
0.1118
50


tomatidine - MCF7
−0.343
2
−0.78
0.09489
0.2263
50


naringenin - MCF7
−0.219
2
−0.546
0.4127 
0.6589
50


allantoin - PC3
−0.077
2
−0.414
0.78446
0.7654
50
















TABLE 3







Compounds with negative connectivity to human muscle atrophy


signature-2.














Connect-




%



ivity

Enrich-

Specif-
Non-


Cmap name/cell line
score
n
ment
p
icity
null
















tacrine - HL60
−0.870
1
−0.998
—
—
100


tomatidine - PC3
−0.861
1
−0.998
—
—
100


naringenin - PC3
−0.754
1
−0.990
—
—
100


betulinic acid -
−0.569
1
−0.929
—
—
100


HL60


conessine - HL60
−0.543
1
−0.915
—
—
100


allantoin - MCF7
−0.486
2
−0.840
0.05114
0.04710
100


naringenin - MCF7
−0.314
2
−0.460
0.64871
0.84500
50


tomatidine - MCF7
−0.281
2
−0.611
0.30586
0.65260
50









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. 4A. 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. 4A). The table (FIG. 4A, insert) shows baseline circulating metabolic and inflammatory markers. The graph shows plasma glucose and insulin levels (FIG. 4A). 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. 4A). 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. 4B). A complete list of these fasting-responsive mRNAs is shown below in Table X1 (“Change” is the mean log2 change or difference between fasting and fed states). The data in Table X1 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 log 2 hybridization signals, as assessed by Affymetrix Human Exon 1.0 ST arrays are shown in FIG. 4B. 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 X1. 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. 4B). 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. 4B). 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 analyze RNA from paired fed and fasted skeletal muscle biopsy samples obtained from seven healthy human subjects (see FIG. 5; 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 X1







Fasting-responsive human mRNAs.









Change













Affymetrix


Accession
(Fasting-




ID
mRNA
Gene 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 (Xenopuslae


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 // hemeoxygenase




(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 11 847 ///




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-
NM_019593 //
NM_019593
0.34
0.10
0.007



1022P6.2
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)-box 6 // 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 //




BAIL-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 //




phosphatidylethanolamine




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 //




SUMO 1/sentrin/SMT3




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 //




methylenetetrahydrofolate




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 (muscle 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 X2; the data in column labeled “Change” show mean changes in log2 hybridization signals between fasting and fed states for the species indicated, [Mean log2 mRNA levels for fasted] minus [Mean log2 mRNA levels in unfasted]; P-values were determined with paired t-tests). The data shown in Table X2 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 X2, 63 mRNAs were represented on the HG-U133A arrays used in the Connectivity Map (FIG. 6A). 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 X2







Fasting-regulated mRNAs common to human and mouse skeletal muscle.










Human
Mouse



Mean Log2 Change
Mean Log2 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,
0.37
0.016
0.26
0.018



sub-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
0.29
0.013
0.53
0.006



oxidase 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
0.23
0.013
0.40
0.001



1 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
0.21
0.009
0.40
0.008



71






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
0.18
0.000
0.23
0.011



subunit 13-like






CALCOCO1
calcium binding and
0.16
0.010
0.31
0.028



coiled coil domain 1






MYO5A
myosin VA (heavy chain
0.14
0.006
0.36
0.012



12, 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
0.11
0.005
0.26
0.036



homolog (S. cerevisiae)






SFRS8
splicing factor,
0.08
0.016
0.13
0.011



arginine/serine-rich 8






NFYA
nuclear transcription
−0.07
0.011
−0.31
0.045



factor 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-
−0.16
0.015
−0.26
0.015



twitch, 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-
−0.18
0.001
−0.29
0.043



like family, member 1






SUV39H2
suppressor of variegation
−0.20
0.011
−0.26
0.014



3-9 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
−0.21
0.000
−0.50
0.019



elongation 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
−0.26
0.015
−0.54
0.002



regulated 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
−0.34
0.019
−0.20
0.043



105 kDa/110 kDa protein 1






VEGFA
vascular endothelial
−0.35
0.016
−0.26
0.002



growth factor A






PMP22
peripheral myelin protein
−0.36
0.004
−0.13
0.012



22






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
−0.44
0.017
−0.30
0.003



factor, 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. 6B 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. 6B 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. 6E). Although metformin did not alter muscle weight in fasted mice (FIG. 6F), ursolic acid increased it by 7±2% (FIG. 6G). 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. 6H; 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, muscle atrophy signature-2 (described above), to determine if this muscle atrophy signature would also correlate with ursolic acid, among other compounds. As described above, muscle 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 X3 (“Change” represents mean changes in log2 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 X3 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 X3 were determined with paired t-tests.







TABLE X3







Human skeletal muscle mRNAs induced or repressed by fasting and SCI.










EFFECT OF
EFFECT OF



FASTING
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
0.39
0.019
0.68
0.020



FLJ33996






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
0.30
0.011
0.39
0.042



C2






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
0.22
0.005
0.30
0.021



FLJ14154






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
−0.11
0.003
−0.33
0.001



protein 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 X3, 29 were represented on the HG-U133A arrays used in the Connectivity Map (FIG. 7A), but only 10 were common to the 63 mRNAs used in the first Connectivity Map query described above for muscle atrophy signature-1 (IGF-IR, NOX4, SUPT6H, MRPS15, PDE7B, PGC-1α, TSPAN13, TTLL1, VEGFA and ZNF280B). The mRNAs listed in FIG. 7A 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. 7B. 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. 7B).
Because muscle 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. 7C). In FIG. 7C, 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, respectively). 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. 8A), quadriceps weight (FIG. 8B), and upper forelimb muscle (triceps and biceps) weight (FIG. 8C). 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. 8D. 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. 8E. 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. 9). 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% 02 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 log2 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 X4 (“Change” is the meang log2 change or difference between mice on ursolic acid diet and control diet, i.e. [Mean log2 mRNA levels in ursolic acid diet] minus [Mean log2 mRNA levels in control diet]).







TABLE X4







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 protein
0.69
0.001


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, member 3
0.32
0.001


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 ATPase
0.18
0.001



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) subunit,
0.15
0.005



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 alpha
0.14
0.005



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, medium
−0.11
0.001



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 2B
−0.16
0.000


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 University
−0.18
0.003



53, expressed




Mlec
malectin
−0.19
0.003


Cacna1s
calcium channel, voltage-dependent, L type,
−0.22
0.001



alpha 1S




Sfrs5
splicing factor, arginine/serine-rich 5 (SRp40,
−0.22
0.005



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. 4B and Sacheck J M, et al. (2007) Faseb J 21(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. 6H). Consistent with that finding, the arrays indicated that dietary ursolic acid reduced atrogin-1 mRNA, which was the most highly repressed mRNA (FIG. 10A). The results shown in FIG. 10A represent a subset of the mRNAs from Table X4 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. 10B; 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; Musarò 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. 10A). 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. 12A), neither increased plasma IGF-I (FIG. 10C). For the data in FIG. 10C, 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. 11A). The data in FIG. 11A are mean exon-specific log2 hybridization signals from the arrays described in Table X2. 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. 11B). Briefly, the data shown in FIG. 11B 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. 10D. 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. 10E (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 β 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. 10F), S6K (FIG. 10G) and IGF-I receptor (FIG. 10H). 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. 10I 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 13 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. 10F). 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. 11C). 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. 11D), 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. 11E). 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. 10K; 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. 12) 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. 12A. 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. 12B; P-values were 0.71 and 0.80 for initial and final weights, respectively).
The data in FIG. 12A 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. 12C. Each data point in FIG. 12C 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. 12D 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. 12D are shown quantitatively in FIG. 12E in terms of adipocyte diameter, where data point represents the average diameter of ≧125 retroperitoneal adipocytes from one mouse. The retroperitoneal adipocyte size distribution. Each distribution represents combined adipocyte measurements (>1000 per diet) from FIG. 12E.
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. 12G, each data point represents one mouse, and horizontal bars denote the means. P-values were determined by t-test. In FIG. 12H, each data point represents one mouse. Importantly, ursolic acid also significantly reduced plasma triglyceride (FIG. 12I) and cholesterol (FIG. 12J). 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. 13A). 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. 12A), 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. 13, 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. 14A). As previously observed in mice fed ursolic acid and standard chow (FIG. 8), 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. 14F), but increased brown fat (FIG. 14G). 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. 14H), providing an explanation for how ursolic acid reduces adiposity and obesity. Remarkably, CLAMS analysis revealed that ursolic acid-treated mice consumed more food (FIG. 14I), even though they gained less weight (FIG. 14A). For the data shown in FIG. 14A, 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. 15A). 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. 15A 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. 15A). 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. 15B), hepatocellular lipid accumulation (FIG. 15C, H&E stain at 20× magnification; FIG. 15D, lipid-staining osmium at 10× magnification), and elevated plasma liver function tests (FIG. 15E, 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. 15B-13G). In addition, ursolic acid reduced obesity-related hypercholesterolemia (FIG. 15H). 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 muscle atrophy signature-1, but not muscle 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. 16B), but not liver weight (FIG. 16C). In contrast, oleanolic acid increased liver weight (FIG. 14F), but not skeletal muscle weight (FIG. 16E). Interestingly, metformin (250 mg/kg) resembled oleanolic acid in biological effect: it increased liver weight (FIG. 16I), but not muscle weight (FIG. 16H). 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. 17A, 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. 17A, 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. 17B, 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. 17A), neither increased skeletal muscle fiber diameter (FIG. 17B). 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. 12A, 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. 18A; 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. 18B; 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.
15. Treatment of Muscle Atrophy
Several compounds have been shown to treat muscle atrophy as shown below.
a. Betulinic Acid
Betulinic acid has the following structure:




The mRNA expression signature of betulinic acid negatively correlated to human muscle atrophy signature-2. Therefore betulinic acid, like ursolic acid, could inhibit skeletal muscle atrophy. To test this, a mouse model of immobilization-induced skeletal muscle atrophy was used: mice were administered vehicle (corn oil) or varying doses of ursolic acid (positive control) or betulinic acid via intraperitoneal injection twice a day for two days. One tibialis anterior (TA) muscle was immobilized with a surgical staple, leaving the contralateral mobile TA as an intra-subject control. The vehicle or the same dose of ursolic acid or betulinic acid was continuously administered via i.p. injection twice daily for six days before comparing weights of the immobile and mobile TAs. As expected, immobilization caused muscle atrophy, and ursolic acid reduced muscle atrophy in a dose-dependent manner, with maximal inhibition at 200 mg/kg (FIG. 19A). Betulinic acid also reduced muscle atrophy in a dose-dependent manner, with maximal inhibition at ≦50 mg/kg (FIG. 19B). These data indicate that betulinic acid reduces immobilization-induced muscle atrophy, and it is more potent than ursolic acid.
B. Naringenin
Naringenin has the following structure:




The mRNA expression signature of naringenin negatively correlated to human muscle atrophy signatures-1 and -2. Therefore naringenin could inhibit skeletal muscle atrophy. To test this, mice were administered vehicle (corn oil), ursolic acid (200 mg/kg), naringenin (200 mg/kg), or the combination of ursolic acid and naringenin (each at 200 mg/kg) via i.p injection twice a day for two days. One tibialis anterior (TA) muscle was immobilized with a surgical staple, leaving the contralateral mobile TA as an intrasubject control. Vehicle or the same doses of ursolic acid and/or naringenin was continuously administered via i.p. injection twice daily for six days before comparing weights of the immobile and mobile TAs. Like ursolic acid, naringenin reduced muscle atrophy (FIG. 20). The combination of ursolic acid and naringenin also reduced muscle atrophy, but not more than either compound alone (FIG. 20). These data indicate that naringenin reduces skeletal muscle atrophy.
Like ursolic acid, naringenin reduces blood glucose, as well as obesity and fatty liver disease. Therefore ursolic acid and naringenin could have additive effects. To determine this, weight-matched mice were provided ad libitum access to standard (Harlan Teklad formula 7013), high fat diet (HFD; Harlan Teklad formula TD93075), or HFD containing varying concentrations of ursolic acid (0.15%) and/or naringenin (0.5% or 1.5%). After the mice consumed these diets for 5 weeks, fasting blood glucose, total body weight, fat mass, liver weight, grip strength, and skeletal muscle weight was measured. As expected, HFD increased blood glucose, and this increase in blood glucose was partially prevented by ursolic acid and naringenin (FIG. 21A). The combination of ursolic acid plus either dose of naringenin reduced blood glucose more than either compound alone, and it restored blood glucose to normal levels (FIG. 21A). Importantly, ursolic acid and naringenin did not have additive effects on total body weight (FIG. 21B), fat mass (FIG. 21C), liver weight (FIG. 21D), grip strength (FIG. 21E), or skeletal muscle weight (FIG. 21F). In addition, ursolic acid increased strength to a greater extent than naringenin (FIG. 21E), and ursolic acid, but not naringenin, increased muscle weight (FIG. 21F). These differences between ursolic acid and naringenin in high fat fed mice indicates that ursolic acid and naringenin have differences in their mechanisms of action, which could explain their additive effects on fasting blood glucose.
c. Tomatidine
Tomatidine has the following structure:




The mRNA expression signature of tomatidine negatively correlated to human muscle atrophy signatures-1 and -2. Therefore tomatidine could inhibit skeletal muscle atrophy. To test this, mice were administered vehicle (corn oil) or tomatidine (50, 100 or 200 mg/kg) via i.p injection twice a day for two days. One tibialis anterior (TA) muscle was immobilized with a surgical staple, leaving the contralateral mobile TA as an intrasubject control. Vehicle or the same doses of tomatidine was administered via i.p. injection twice daily for six days before comparing weights of the immobile and mobile TAs. All 3 doses of tomatidine reduced muscle atrophy, and the effect was maximal at 50 mg/kg (FIG. 22A). The same protocol was used to compare the effects of vehicle (corn oil) and tomatidine (5, 15 or 50 mg/kg) on immobilization-induced muscle atrophy. Tomatidine reduced muscle atrophy in dose-dependent manner, with maximal effect at 50 mg/kg and EC50<5 mg/kg (FIG. 22B). These data indicate that tomatidine reduces immobilization-induced muscle atrophy, and it is more potent than ursolic acid.
Tomatidine could also inhibit skeletal muscle atrophy induced by fasting. To test this, food was withdrawn from mice, and then vehicle, ursolic acid (200 mg/kg) or tomatidine (50 mg/kg) were administered by i.p. injection. Twelve hours later, mice received another i.p. injection of vehicle or the same dose of ursolic acid or tomatidine. Twelve hours later, skeletal muscles were harvested and weighed. Both ursolic acid and tomatidine increased skeletal muscle, indicating decreased fasting-induced skeletal muscle atrophy (FIG. 23A). We next used the same protocol to compare the effects of vehicle (corn oil) and tomatidine (5, 15 and 50 mg/kg). Tomatidine reduced muscle atrophy in dose-dependent manner, with maximal effect at 50 mg/kg and EC50 between 5 and 15 mg/kg (FIG. 23B).
d. Allantoin, Tacrine, Ungerine, Hippeastrine and Conessine
Allantoin has the following structure:




Tacrine has the following structure:




Ungerine has the following structure:




Hippeastrine has the following structure:




Conessine has the following structure:




The mRNA expression signatures of allantoin, tacrine, ungerine (Prestwick-689), hippeastrine (Prestwick-675) and conessine also negatively correlated to human muscle atrophy signatures-1 and -2. Therefore these compounds could inhibit skeletal muscle atrophy. To test this, the fasting-induced muscle atrophy model described above was used to compare the effects of ursolic acid (200 mg/kg), tomatidine (50 mg/kg), allantoin (2 mg/kg), tacrine (4 mg/kg), ungerine (2 mg/kg), hippeastrine (2 mg/kg) and conessine (2 mg/kg) Like ursolic acid and tomatidine, allantoin, tacrine, ungerine, hippeastrine and conessine increased muscle weight in fasted mice (FIG. 24), indicating that these compounds decrease skeletal muscle atrophy.
Since ursolic acid and naringenin reduced fasting blood glucose, hippeastrine (2 mg/kg) and conessine (2 mg/kg) could have a similar effect. Hippeastrine and conessine reduced fasting blood glucose (FIG. 25).
16. Prophetic Synthesis of Tacrine and Analogs
The formulas disclosed herein could be synthesized by reacting an anthranilonitrile derivative with a cyclohexanone derivative in the presence of zinc chloride (Proctor et al., Curr Medici. Chem., 2000, 7, 295-302). Such reaction is shown in Scheme 1A.




Thus, tacrine can be synthesized as shown in scheme 1B.




The formulas disclosed herein could also be synthesized by reacting an α-cyanocyclonones with a vide variety of anilines using either TiCl4 or AlCl3 as reagents (Proctor et al., Curr Medici. Chem., 2000, 7, 295-302). An example of such reaction is shown in Scheme 1C.




Thus, tacrine could be synthesized as shown in scheme 1D.




17. Prophetic Synthesis of Naringenin and Analogs
The disclosed formulas could be synthesized as described in PCT application WO 2007/053915 by De Keukkeleire et al. which is hereby incorporated in its entirety by reference. In another example, Glucoyl substituted naringenin could be extracted as described in U.S. Pat. No. 6,770,630 by Kashiwaba et al. which is hereby incorporated in its entirety by reference. As described by De Keukkeleire et al. the disclosed formulas could be synthesized as shown in Scheme 2A:




The formation of the thioketone was described by Pathak, et al. (J. Org. Chem., 2008, 73, 2890-2893). The * in the scheme denotes moieties that is or can be converted, using known chemistry, into the disclosed R moieties. For example, the synthesis of naringenin is shown in Scheme 2B.




18. Prophetic Synthesis of Allantoin and Analogs
The disclosed formulas could be made using a variety of chemistry known in the art. For example, one set of the disclosed formulas could be made as shown in Scheme 3A and as described in U.S. Pat. No. 4,647,574 by Ineaga et al, which is hereby incorporated herein by reference in its entirety.




Allantoin could be prepared as described in U.S. Pat. No. 5,196,545 by Schermanz, which is hereby incorporated herein by reference in its entirety, and as shown in Scheme 3B.




A comprehensive guide for how to make the disclosed formulas can be found in Kirk-Othmer Encyclopedia of Chemical Technology under the chapter Hydantoin and Its Derivatives by Avendaño et al (2000), which is hereby incorporated herein by reference in its entirety.
19. Prophetic Synthesis of Conessine and Analogs
Conessine is a steroid alkaloid found in plant species from the Apocynaceae family, for example in Holarrhena floribunda. Conessine derivatives could be prepared as described in U.S. Pat. Nos. 3,539,449, 3,466,279, and 3,485,825 by Marx, which are hereby incorporated by reference in their entirety. As described in U.S. Pat. Nos. 3,539,449, 3,466,279, and 3,485,825 by Marx, conessine derivatives could be prepared using microorganisms such as the fungus Stachybotrys parvispora and enzymes from Gloeosporium, Colletotrichum, and Myrothecium. For example, see Scheme 4A.




The conessine oxo derivatives could be further modified via a reduction and subsequent chemistry known to one skilled in the art, as shown in Scheme 4B.




The hydroxyl functionality could undergo a number of chemical reactions known in the art. One example, as shown in Scheme 4B, is a Williamson ether synthesis.
Conessine derivatives could be prepared synthetically as described in U.S. Pat. No. 2,910,470, which is hereby incorporated by reference in its entirety. Conessine derivatives are also described in WO 2011/046978 by Orlow, which is hereby incorporated by reference in its entirety. Synthesis of the disclosed formulas is also described in U.S. Pat. No. 3,625,941 by Pappo, which is hereby incorporated in its entirety by reference.
20. Prophetic Synthesis of Tomatidine and Analogs
The formulas disclosed herein could be synthesized by the method disclosed by Uhle, and Moore, J. Am. Chem. Soc. 76, 6412 (1954); Uhle, J. Am. Chem. Soc. 83, 1460 (1961); and Kessar et al., Tetrahedron 27, 2869 (1971), which are all hereby incorporated by reference in their entirety. The disclosed compounds can also be made as shown in Scheme 5A.




21. Prophetic Synthesis of Hippeastrine/Ungerine and Analogs
The disclosed formulas can be synthesized by method disclosed by Mañas et al. (J. Am. Chem. Soc. 2010, 132, 5176-78), which is hereby incorporated by reference in its entirety. Thus, disclosed formulas can be synthesized as shown in Scheme 6A.




Thus, for example, Hippeastrine can be made as shown in Scheme 6B.





Another route to make the disclosed formulas is shown in Scheme 6C, as demonstrated by Mañas et al.




The disclosed derivatives can also be made using methods disclosed by Haning et al (Org. Biomolec. Chem. 2011, 9, 2809-2820).
22. Prophetic Synthesis of Betulinic Acid and Analogs
Betulininc acid analogs are also described in International Published application WO 2011/153315 by Regueiro-Ren et al. and in International Published application WO 2008/063318 by Safe et al. which are hereby incorporated by reference in its entirety. Betulinic acid analogs of the present invention of the present invention could be prepared generically as shown below in scheme 7A. The starting materials could be made with methods known in the art.




Compounds are represented in generic form, with substituents as noted in compound descriptions elsewhere herein. A more specific example is set forth below in scheme 7B.




23. Muscle Atrophy Signature-3
Induced and repressed mRNA were evaluated for muscle atrophy signature-3. The statisitical significance for the identified mRNAs was defined as P≦0.01.
For induced mRNAs: mouse tibialis anterior mRNAs significantly induced by 1 week of denervation and significantly induced by 24 h fasting.
For repressed mRNAs: mouse tibialis anterior mRNAs significantly repressed by 1 week of denervation and significantly repressed by 24 h fasting.
The identified induced mRNAs included 1200011I18Rik, 2310004I24Rik, Akap81, Als2, Anapc7, Apod, Arrdc3, Atp6v1h, BC027231, Bsdc1, Ccdc77, Cd68, Cdkn1a, Ctps2, Cts1, D930016D06Rik, Ddx21, Depdc7, Dido1, nttip2, Ece1, Eda2r, Egln3, Elk4, Erbb2ip, Errfi1, Fbxo30, Fbxo32, Fip111, Frg1, Gabarap11, Gadd45a, Gnl2, Gnl3, Herpud2, Hpgd, Hspb7, Htatip2, Impact, Kdm3a, K1h15, Lpin2, Med12, Mfap1b, Mgea5, Mknk2, Nmd3, Nup93, ORF19, Pacrg1, Parp4, Pdk4, Phc3, Plaa, Ppfibp1, Psma2, Ranbp10, Ranbp9, Rassf4, Riok1, Rlim, Sf3b1, Sik1, Slc20a1, Sln, Spag5, Srsf2ip, Syf2, Tbc1d15, Tbk1, Tekt1, Tgif1, Tmem140, Tmem71, Tnks, Trim25, Trmt1, Tspyl2, Tsr1, Tulp3, Txing, Ubfd1, Ubxn4, Utp14a, Wdr3, and Xpo4.
The identified repressed mRNAs included 1600014C10Rik, 1700021F05Rik, 2310003L22Rik, 2310010M20Rik, 2310028O11Rik, 2310061C15Rik, 2610528E23Rik, 2810432L12Rik, Abcd2, Acvr1, Aimp2, Ank1, Aqp4, Ar13, Asb10, Aurka, Bhlhe41, Bpnt1, Camk2a, Cby1, Cc2d2a, Cdc14a, Cdc42ep2, Clcn1, Cntfr, Col15a1, Col6a3, Cox11, Cox7b, Crhr2, D0H4S114, Ddit3, Deb1, Dexi, Dhrs7c, Eif4e, Endog, Epha7, Exd2, Fam69a, Fhod3, Fn3k, Fndc5, Fsd2, Gcom1, Gdap1, Gm4841, Gm5105, Gm9909, Gnb5, Gpd2, Grtp1, Heatr5a, Hlf, Homer1, Ikzf2, Inppl1, Irx3, Itgb6, Jarid2, Jph2, Khdrbs3, Klf7, K1h123, Ky, Lrp2 bp, Lrrfip1, Map2k6, Map3k4, Mat2a, Mkks, Mk11, Mrc2, Mreg, Mrp139, Narf, Ntf5, Nudt3, Osbp16, Ostc, Parp8, Pkia, Plcd4, Podx1, Polk, Polr3k, Ppm11, Pppde2, Prss23, Psd3, Psph, Ptpmt1, Ptx3, Qrsl1, Rasgrp3, Rhobtb3, Ric8b, Rnf150, Rsph1, Rundc1, Rxrg, Sell13, Sema3a, Sgcd, Shisa2, Sirt5, Slc25a19, Slc41a3, Slc4a4, SlcoSa1, Snrnp35, Stac3, Ston2, Stradb, Stxbp4, Tfrc, Tmc7, Tmem218, Tmtc1, Tnfaip2, Tob1, Trim35, Tt1, Vegfa, and Vgll4.
24. Muscle Atrophy Signature-4
Induced and repressed mRNA were evaluated for muscle atrophy signature-4. The statistical significance for the identified mRNAs was defined as P≦0.01.
For induced mRNAs: mouse tibialis anterior mRNAs significantly induced by 1 week of denervation and significantly induced by 1 week of Gadd45a overexpression.
For repressed mRNAs: mouse tibialis anterior mRNAs significantly repressed by 1 week of denervation and significantly repressed by 1 week of Gadd45a overexpression.
The identified induced mRNAs included 2410089E03Rik, 6720456H20Rik, Abca1, Abhd2, Abr, Aif11, Akap6, Alg8, Alox5ap, mpd3, Ankrd1, Anxa4, Aoah, App, Araf, Arfgap3, Arhgef2, Arpc3, Arpp21, Atf7ip, Atp6ap2, Atp6v1h, Atp7a, Atp8b1, B4galt5, Bax, Baz2a, Bhlhb9, Bmp2k, C3ar1, Canx, Casp3, Ccdc111, Ccdc122, Ccdc93, Ccndbpl, Cct4, Cd68, Cd82, Cdkn1a, Cep192, Cgref1, Chd4, Chrna1, Chrnb1, Chrng, Chuk, Clec12a, Clec4a3, Col19a1, Copb2, Cpne2, Cstb, Ctnna1, Ctps2, Ctsd, Ctsl, Ctss, Ctsz, Cyb5r3, Cybb, Cyr61, D10Wsu52e, D930016D06Rik, Dcaf13, Dclre1c, Dctn5, Ddb1, Ddhd1, Decr2, Derl1, Dhx9, Dido1, Dnajc1, Eda2r, Eef1b2, Eef2, Emr1, Epb4.113, Erbb2ipm, Erlin1, Esyt1, Fam108c, Fam115a, Fbxo30, Frrs1, Fst, Fubp1, Fyb, Gab2, Gabarap, Gadd45a, Galc, Galnt7, Ganab, Gigyf2, Gm3435, Gnb211, Gng2, Gn12, Gn13, Gpraspl, Gpsm2, Gramdlb, H19, H2-Aa, Hmgn3, Hn1, Hnrnpu, Hprt, Hsp90ab1, Hsp90b1, Hspa2, Hspa4, Hspb8, Htatip2, Id2, Ifi30, Igbp1, Igdcc4, Ilf3, Imp4, Impact, Irak4, Itm2b, Ivns1abp, Kcnn3, Kdm3a, Khdrbs1, Kif5b, Klh15, Krt18, Lbh, Lgals3, Lgmn, Lpar6, Lpin2, Lyz2, Macf1, Map11c3a, Map3k1, Map4k4, Marveld2, Matr3, Mcm6, Mdm2, Mdm4, Me2, Med12, Mgea5, Mica111, Mppl, Mrc1, Mtaplb, Myf6, My14, Myo5a, Ncaml, Nip7, Nln, Nop58, Nrcam, Nup93, Nvl, Obfc2a, Osbp18, Palm2, Parp4, Pcbdl, Pcgf3, Pdlim3, Pfn1, Pgd, Pik3r3, Plaa, Plekha5, Plxdc2, Plxnal, Polr2a, Polr3b, Ppfibp1, Ppib, Prep, Prkdc, Prmt1, Prss48, Prune2, Psmb1, Psmd5, Rad50, Rassf4, Rb1, Rbm45, Reep5, Rgs2, Riok3, Mina, Rnasel, Rpl31, Rps3, Rps9, Rrad, Rras2, Rspry1, Runx1, Sap30 bp, Sema4d, Sema6a, Serf1, Serpinb6a, Sesn3, Sf3b1, Sf3b3, Sgpll, Sh3d19, Sh3pxd2a, Sh3rf1, Sik1, Sirpa, Slc20a1, Slc25a24, Slc9a7, Slc9a9, Sln, Smarcad1, Smc1a, Smc5, Snd1, Snx5, Spin1, Srp14, Ssu72, Stam, Supt5h, Tbc1d8, Tbcd, Tbxas1, Tec, Tgfbrl, Tgs1, Thoc5, Thumpd3, Tiam2, Tlr4, Tlr6, Tmeff1, Tmem176b, Tmem179b, Tmem209, Tmem38b, Tnc, Tnfrsf22, Tnfrsf23, Tnnt2, Trim25, Trp63, Tubb5, Tubb6, Tyrobp, Uchl1, Ugcg, Usp11, Usp5, Wasf2, Wbp5, Wbscr27, Wdr36, Wdr61, Wdr67, Wdr77, Wdyhvl, Wsbl, Ylpml, Ype12, Ywhab, Zfp280d, Zfp318, Zfp346, Zfp3611, and Zmynd8.
The identified repressed mRNAs included 0610012G03Rik, 1110001J03Rik, 1110067D22Rik, 2010106G01Rik, 2310002L09Rik, 2310003L22Rik, 2310010M20Rik, 2610507B11Rik, 2610528E23Rik, 2810407CO2Rik, 4931409K22Rik, 4933403F05Rik, 5730437N04Rik, 9630033F20Rik, A21d1, A930018M24Rik, Abcb1a, Abcb4, Abcd2, Abi3 bp, Acaa2, Acadm, Acadv1, Acat1, Acot13, Ada1, Adcyl0, Adk, Adss11, Aes, AI317395, Aimp2, Ak1, Alas2, Aldh1a1, Ank, Ank1, Ankrd9, Ano2, Ano5, Ap1p2, Apobec2, Aqp4, Ar, Arhgapl9, Arhgap20, Arhgap31, Ar13, Asb10, Asb11, Asb12, Asb14, Asb15, Atp11a, Atp13a5, Atp1b1, Atp5a1, Atp5e, Atp8a1, Atxn1, B4galt4, Bckdk, Bhlhe41, Bpgm, Bpi11, Brp44, Btbd1, C2cd2, Camk2a, Camk2g, Capn5, Car8, Cast, Cc2d2a, Ccngl, Ccnk mCd34, Cd36, Cdcl4a, Cdc42ep3, Cdh5, Cdnf, Ces1d, Chchd10, Chchd3, Cib2, Ckm, Clcn1, Clic5, Cmb1, Cntfr, Coll1a1, Coq9, Cox11, Cox6a2, Cox8b, Cpt1b, Csrp2 bp, Cuedc1, Cyb5b, Cyyrl, DOH4S114, D1Ertd622e, Dab2ip, Dcunld2, Deb1, Decr1, Dgkb, Dhrs7c, Dlat, Dlc1, Dlg1, Dlst, Dnajb5, Dusp28, Ecsit, Eef1a2, Eepd1, Efcab2, Eif4e, Endog, Eno3, Epas1, Epha7, Etfb, Exd2, Eya1, Fam132a, Fastkd3, Fbp2, Fbxo3, Fdx1, Fez2, Fgfbp1, Fh1, Fitm2, Flt1, Fmo5, Fsd2, Fxyd1, Fzd4, G3bp2, Ganc, Gbas, Gcom1, Gdap1, Ghr, Gjc3, G1b112, Gm4841, Gm4861, Gm4951, Gm5105, Gmpr, Gpcpdl, Gpd1, Gpd2, Gpt2, Grsfl, Gucyla3, Gysl, Hadh, Hfe2, Hivep2, Hk2, Hlf, Homer1, Hsd12, Idh3a, Idh3g, Il15ra, Inpp5a, Irx3, Jak2, Jam2, Jph1, Kcna7, Kcnj2, Kcnn2, Kdr, Khdrbs3, Kif1b, Kif1c, Kit1, Klf12, Klhl23, Klhl31, Klh31, Klhl7, Ky, Ldb3, Lifr, Lmbr1, Lphn1, Lpin1, Lpl, Lrig1, Lrrc39, Lynx1, Man2a2, Maob, Map2k6, Map2k7, Map3k4, Mapkapk2, Mbn11, Mccc1, Mdh1, Mdh2, Me3, Mfn1, Mfn2, Mgst3, Mlfl, Mpnd, Mpz, Mr1, Mreg, Mtus1, Mybpc2, Myo5c, Myom2, Myozl, Narf, Ndrg2, Ndufa3, Ndufa5, Ndufa8, Ndufb8, Ndufb9, Ndufs1, Ndufs2, Ndufs6, Ndufs8, Ndufv1, Nf2, Nos1, Nr1d1, Nudt3, Oat, Ociad2, Ocr1, Osbp16, Osgep11, Ostn, Paqr9, Parp3, Pcmtd1, Pcnt, Pcnx, Pdgfd, Pdha1, Pdpr, Pfkfb3, Pfkm, Pfn2, Pgam2, Phb, Phka1, Phkg1, Phtf2, Phyh, Pitpnc1, Pkdcc, Pkia, Pla2g12a, Pla2g4e, Plcb1, Plcd4, Pln, Pmp22, Ppara, Ppargc1a, Ppat, Ppm1a, Ppm11, Ppp1cb, Ppp1r1a, Ppp2r2a, Ppp3cb, Pre1p, Prkab2, Prkca, Prkgl, Ptp4a3, Ptprb, Pttg1, Pxmp2, Pygm, Rab28, Rasgrp3, Rcan2, Rgs5, Rhot2, Rnf123, Rpa1, Rpl31, Rtn4ip1, Samd12, Samd5, Satb1, Scn4a, Scn4b, Sdha, Sdhb, Sdr39u1, Sell13, Sematic, Serpine2, Shisa2, Slc15a5, Slc16a3, Slc19a2, Slc24a2, Slc25a11, Slc25a12, Slc25a3, Slc25a4, Slc2a12, Slc2a4, Slc35f1, Slc37a4, Slc43a3, Slc4a4, Slc6a13, Slc6a8, Slc9a3r2, Slco3a1, Smarca1, Smox, Smyd1, Snrk, Sorbs2, Spop, Srl, St3ga13, St3ga16, St6galnac6, Stk381, Stradb, Strbp, Strbp, Stxbp4, Suclgl, Tab2, Taf3, Tars12, Tcea3, Thra, Tiam1, Timp4, Tln2, Tmem126a, Tmem126b, Tmem65, Tnfaip2, Tnmd, Tnnc2, Tnni2, Tnxb, Tomm401, Trak1, Trak2, Trim24, Trpc3, Tuba8, Txlnb, Txnip, U05342, Uaca, Ulk2, Uqcrc1, Uqcrfs1, Uqcrq, Vamp5, Vdac1, Vegfa, Vegfb, Xpr1, Yipf7, Zfand5, Zfp191, and Zfp238.
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The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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## Claims

1. A method of lowering blood glucose in an animal comprising administering to the animal a composition comprising from 25 to 500 mg of ursolic acid, and a compound having a structure represented by a formula:



wherein each ----- represents a covalent bond selected from a single or double bond;
wherein each of R21a, R21b, R21c, R21d, R21e, R22, R23a, R23b, R23c and R23d in said formula is chosen independently from H and OH or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof;
thereby lowering the blood glucose in the animal.

2. A method of lowering blood glucose in an animal comprising administering to the animal a composition comprising from 25 to 500 mg of ursolic acid, and a compound of formula: