WO2021041495A2 - Method to maintain or enhance tissue - Google Patents

Abstract

Disclosed herein is a method for maintaining or enhancing tissue mass comprising administering a composition comprising L-BIABA.

Description

METHOD TO MAINTAIN OR ENHANCE TISSUE

GOVERNMENT LICENSE RIGHTS

This invention was made with government support under AG039355 awarded by the National Institutes of Health. The government has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS:

This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Serial No. 62/891628 filed August 26, 2019, U.S. Provisional Application Serial No. 62/932766 filed November 8, 2019, and U.S. Provisional Application Serial No. 62/957398 filed January 6, 2020, the entirety of each is hereby incorporated by reference.

BACKGROUND

A need exists to identify a means to promote musculoskeletal tissue, both bone and muscle, function and formation under conditions of suboptimal loading such as space flight, immobilization, and aging. Muscle is a source of signaling molecules that can have both positive and negative effects on bone. The body produces a racemic mixture of isomers L- and D-BAIBA.

The L isomer of beta-aminoisobutyric acid (L-BAIBA) is produced by skeletal muscle from valine in response to muscle contraction.

Previously it was reported that L-BAIBA, a metabolite produced by contracting muscle, attenuates both bone and muscle loss due to hindlimb unloading. Interestingly, the L(S) enantiomer of BAIBA was 100-1000 fold more potent than the D form in preventing osteocyte cell death due to reactive oxygen species.

SUMMARY

The disclosure relies upon the discovery that L-BAIBA has a protective effect on osteocytes exposed to reactive oxygen species. In some embodiments, the disclosure provides a method comprising administering an effective amount of a composition to a subject to enhance tissue mass, wherein the composition is L-BAIBA.

In some embodiments, the disclosure provides a method comprising administering an effective amount of a composition to a subject to enhance or maintain tissue, wherein the composition is L-BAIBA. In some embodiments, the method comprises administering an effective amount of a composition to a subject to enhance or maintain tissue mass. In some embodiments, the result of enhancing tissue is shown by an increase in metabolites and/ or proteins indicative of promoting tissue growth.

In some embodiments, the methods further comprise applying a mechanical load. In some embodiments, the mechanical load is a suboptimal mechanical loading. In some embodiments, the mechanical load is an optimal mechanical load.

Embodiments of the invention are further described by the following enumerated clauses. It will be understood that any of the embodiments described herein can be used in connection with any other embodiments described herein to the extent that the embodiments do not contradict one another.

Clause 1. A method comprising: administering an effective amount of a composition to a subject to enhance tissue growth, wherein the composition is L- BAIBA.

Clause 2. The method of clause 1, wherein the composition comprises a purity of at least about 90% L-BAIBA.

Clause 3. The method of clause 1-2, wherein the tissue is selected from the group consisting of bone, adipose, brain, liver, or muscle.

Clause 4. The method of clause 1-3, wherein enhance includes an increase in tissue growth by at least about 3%.

Clause 5. The method of clause 1-4, wherein the step of administering occurs through oral administration.

Clause 6. The method of clause 1-5, wherein the enhance includes an increase in tissue function. Clause 7. The method of clause 1-6, wherein the subject is not exposed to a mechanical load.

Clause 8. A method comprising: administering an effective amount of a composition to a subject to maintain or enhance tissue growth; wherein the composition is L-BAIBA.

Clause 9. The method of clause 8 further comprising applying a mechanical load to the tissue.

Clause 10. The method of clause 9, wherein the L-BAIBA is administered prior to the application of the mechanical load. Clause 11. The method of clause 9, wherein the L-BAIBA is administered during the application of the mechanical load.

Clause 12. The method of clause 9, wherein the L-BAIBA is administered subsequent to the application of the mechanical load.

Clause 13. The method of clause 8-12, wherein the composition is at least about 90% L-BAIBA.

Clause 14. The method of clause 8-13, wherein the tissue is selected from the group consisting of bone, adipose, brain, liver, or muscle.

Clause 15. The method of clause 8-14, wherein the step of administering occurs through oral administration. Clause 16. The method of clause 8-15, wherein the effective amount enhances the tissue mass.

Clause 17. The method of clause 9-16, wherein the mechanical load is suboptimal.

Clause 18. The method of clause 9-16, wherein the mechanical load is optimal.

Clause 19. The method of clause 8-18, wherein the subject is exposed to an environment of mechanical unloading.

Clause 20. The method of clause 8-15, and 17-19 wherein the therapeutic effect results in maintaining the tissue.

Clause 21. The method of clause 9-19, wherein the tissue mass is enhanced by at least about 3%. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a graph showing water intake by the male mouse groups.

Fig. 2 is a graph showing the weight of the male mouse groups.

Fig. 3 is a graph showing the water intake by the female mouse groups.

Fig. 4 is a graph showing the weight of the female mouse groups.

Fig. 5 is a graph showing the grip strength normalized to weight divided by groups in male mouse groups.

Fig. 6 is a graph showing the grip strength normalized to weight divided by groups in female mouse groups.

Fig. 7 shows two graphs of increased grip strength normalized by weight in male mice receiving F-BAIBA vs. No F-BAIBA.

Fig. 8 shows the effects of various conditions in male mice on Mineralizing Surface/ Bone Surface, Mineral Apposition Rate, and Bone Formation Rate Fig. 9 shows the effects of various conditions in female mice on Mineralizing Surface/Bone Surface, Mineral Apposition Rate, and Bone Formation Rate. Fig. 10 are graphs showing the MS/BS ratio and the relative MS/BS in male mice.

Fig. 11 are graphs showing the MAR ratio and relative MAR in male mice.

Fig. 12 are graphs showing the BFR ratio and relative BFR in male mice.

Fig. 13 are graphs showing the MS/BS ratio and the relative MS/BS in female mice. Fig. 14 are graphs showing the MAR ratio and relative MAR in female mice.

Fig. 15 are graphs showing the BFR ratio and relative BFR in female mice.

Fig. 16 is a graph showing adenosine monophosphate-activated protein kinase (pAMPK/ AMPK) protein expression in C2C12 myotubes treated with F-BAIBA or D-BAIBA at 10 mM, 20 mM and 30 mM for 48 h.

Fig. 17 are graphs showing muscle size in three types of skeletal muscle from mice receiving either F-BAIBA or D-BAIBA for four weeks.

Fig. 18 is a graph quantifying succinate dehydrogenase (SDH) staining and Integrity density (IntDen) quantification were performed in the gastrocnemius muscle of control mice and mice treated with either F-BAIBA or D-BAIBA.

Fig. 19 shows two graphs that quantify the results of a western blot measuring peroxisome proliferator-activated receptor gamma coactivator-l-alpha (PGCla) and cytochrome C (Cyt C), markers of mitochondrial homeostatsis and respiration, in whole muscle protein extracts from mice receiving either L-BAIBA or D-BAIBA.

Fig. 20 shows representative Western blotting and quantification for optic atrophy 1 (OPA1), mitofusin, and pyruvate dehydrogenase kinase 4 (PDK4) in whole muscle protein extracts from mice receiving L-BAIBA or D-BAIBA Fig. 21 are graphs showing the quantification are shown for pAMPK/ AMPK and pAKT / AKT in whole muscle protein extracts from mice receiving L-BAIBA or D- BAIBA. Tubulin was used as loading control.

DETAILED DESCRIPTION ABBREVIATIONS

L-BIABA stands for L-beta-(P-)aminoisobutyric acid.

D-BAIBA stands for D-beta-(P-)aminoisobutyric acid.

MS stands for mineralizing surface.

MAR stands for mineral apposition rate.

BFR stands for bone-formation rate.

AKT stands for serine/ threonine-protein kinase AMPK stands for adenosine monophosphate-activated protein kinase OPA1 stands for optic atrophy 1 GSN stands for gastrocnemius PDK4 stands for pyruvate dehydrogenase kinase 4 PGCla stands for peroxisome proliferator-activated receptor gamma coactivator-l-alpha

DEFINITIONS

In describing and claiming the methods, the following terminology will be used in accordance with the definitions set forth below.

The term "about" as used herein means greater or lesser than the value or range of values stated by 10 percent, but is not intended to designate any value or range of values to only this broader definition. Each value or range of values preceded by the term "about" is also intended to encompass the embodiment of the stated absolute value or range of values.

As used herein the term "effective" amount or a "therapeutically effective amount" of a compound refers to a nontoxic but sufficient amount of the compound to provide the desired effect. The amount that is "effective" will vary from subject to subject, depending on the age and general condition of the individual, mode of administration, and the like. Thus, it is not always possible to specify an exact "effective amount." However, an appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

The term "parenteral" means not through the alimentary canal but by some other route such as intranasal, inhalation, subcutaneous, intramuscular, intraspinal, or intravenous.

As used herein, the term "purified" relates to the isolation of a molecule or compound in a form that is substantially free of contaminants normally associated with the molecule or compound in a native or natural environment and means having been increased in purity as a result of being separate from other components of the original composition. For example, the L-isomer is separated from the D- isomer. "Substantially pure" means the particular form makes up at least 75%, at least 85%, at least 90%, at least 95%, or about 94%-99% of the total amount of L- BAIBA present.

As used herein the term "enhance" means to promote the growth or gain tissue mass by at least about 2.5%. Additionally, enhancement of tissue may be measured by an increase in anabolic effect compared to a control (wherein the control is an earlier time point or a control subject or group not receiving L-BAIBA or L-BAIBA + a mechanical loading). Finally, enhancement of tissue may be measured by a gain in functional ability. For example an enhancement of musculoskeletal tissue may be demonstrated by an increase in strength.

As used herein the term "maintain" means to not lose up to about 2.5% or gain up to about 2.5% of tissue mass. As used herein, "anabolic effect" means proteins and metabolites that are indicative of tissue growth promotion.

As used herein the term "mechanical unloading" means a lack of force applied similar to the effects of disuse or lack of exercise. In some instances, mechanical unloading results in tissue loss. For example muscle or bone tissue loss while sedentary or experience a condition of mechanical unloading (e.g., time spent in microgravity during space flight).

As used herein the term "mechanical loading" means a force applied to a tissue generating a mechanosensory response from the cells within the tissue.

As used herein the term "suboptimal mechanical loading" or "suboptimal anabolic loading" means at most the maximum force applied to tissue that does not, by itself, result in the growth of new tissue. The amount of force will vary depending on the subject and environmental condition. However, an appropriate "suboptimal mechanical loading" in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

As used herein the term "optimal mechanical loading" or "optimal anabolic loading" means at least the minimum amount of force applied to a tissue, by itself, to result in the growth of new tissue. The amount of force will vary depending on the subject and environmental condition. However, an appropriate "optimal mechanical loading" in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

As used herein the term "subject" means an animal including but not limited to, humans, domesticated animals including horses, dogs, cats, cattle, and the like, rodents, reptiles, and amphibians.

EMBODIMENTS

Embodiments of the disclosure include a method of maintaining or enhancing tissue growth.

In some embodiments, a method is provided comprising administering an effective amount of a composition to a subject to enhance tissue mass. In some embodiments, a method is provided for enhancing or maintaining tissue mass in a subject. In some embodiments, the composition is L-BAIBA. L-BAIBA has the chemical structure

In some embodiments, the subject receives about 50 mg of L-BAIBA. In some embodiments, the subject receives about 100 mg of L-BAIBA. In some embodiments, the subject receives about 150 mg of L-BAIBA. In some embodiments, the subject receives about 500 mg of L-BAIBA. In some embodiments, the subject receives about 1000 mg of L-BAIBA. In some embodiments, the subject receives at least about 50 mg, at least about 100 mg, at least about 150 mg, at least about 200 mg, at least about 250 mg, at least about 300 mg, at least about 350 mg, at least about 400 mg, at least about 450 mg, at least 500 mg, at least about 550, at least about 600 mg, at least about 650 mg, at least about 700 mg, at least about 750 mg, at least about 800 mg, at least about 850 mg, at least about 900 mg, at least about 950 mg, or at least about 1,000 mg per kg of subject's weight. In some embodiments, the subject receives about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900, about 950 mg, or about 1,000 mg per kg of subject's weight.

In some embodiments, the composition comprises purified L-BAIBA. In some embodiments, the composition comprises substantially pure L-BAIBA. In some embodiments, the composition comprises a purity of at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about, or at least about 97% L-BAIBA. In some embodiments, the composition comprises a purity of up to about 99%, up to about 98%, up to about 97%, up to about 96%, up to about 95%, up to about 94%, up to about 93%, up to about 92%, up to about 91%, or up to about 90% L-BAIBA. In some embodiments, the composition comprises a purity of about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% L-BAIBA. In some embodiments, the composition comprises a purity of between about 90% to about 99%, about 91% to about 99%, about 92% to about 99%, about 93% to about 99%, about 94% to about 99%, about 95% to about 99%, or about 96% to about 99% of L-BAIBA. In some embodiments, the composition comprises a purity of between about 94% to about 98%, about 95% to about 98%, about 96% to about 98%, or about 97% to about 98%. In some embodiments, the composition comprises a purity of between about 94% to about 97%, about 95% to about 98%, or about 96% to about 99% L-BAIBA.

In some embodiments, the D-enantiomer b-aminoisobutyric acid (D-BAIBA) is not present in the composition. Further embodiments include a pharmaceutical composition that includes an effective amount of an L-enantiomer of b- aminoisobutyric acid (L-BAIBA) and a pharmaceutically acceptable carrier. The L- BAIBA in the pharmaceutical composition may be substantially pure. The pharmaceutical composition may be substantially free of a D-enantiomer of b- aminoisobutyric acid (D-BAIBA).

In some embodiments, the composition maintains or enhances the mass of a tissue. In some embodiments, the tissue is musculoskeletal tissue. In some embodiments, the musculoskeletal tissue is bone. Exemplary bones include a hip, a femur, a vertebrae, a radius, an ulna, a humerus, a tibia, and a fibula. In some embodiments, the musculoskeletal tissue is muscle. Exemplary muscles include an extensor digitorum longus muscle, soleus muscle, a gastrocnemius muscle, a bicep muscle and a tricep muscle. In some embodiments, the musculoskeletal tissue includes both bone and muscle tissue. In some embodiments, the method comprises administering a composition to a subject to enhance the subject's musculoskeletal tissue without the application of a mechanical load. In some embodiments, the tissue comprises brain, liver, muscle, bone, adipose, or a combination thereof. In some embodiments, the tissue is selected from the group consisting of brain, liver, muscle, bone, or adipose.

In some embodiments, the therapeutic effective amount results in the desired effect of maintaining the tissue mass. In some embodiments, the therapeutic effective amount results in the desired effect of enhancing the tissue. In some embodiments, the enhancement of the tissue results in an increase in tissue mass. In some embodiments, the enhancement of the tissue results in an increase in proteins and/ or metabolites that are indicative of tissue growth. In some embodiments, tissue enhancement is measured by an increase in functional capacity. As an illustrative example, an enhancement of muscle tissue may be shown by an increase in strength.

In some embodiments, enhancing tissue promotes the growth or gain of the tissue mass by at least about 2.5%, at least about 3%, at least about 3.5%, at least about 4%, at least about 4.5%, at least about 5%, at least about 5.5%, at least about 6%, at least about 6.5%, or at least about 7%. In some embodiments, enhancing tissue promotes the growth or gain of the tissue mass by up to about 2.6%, up to about 2.7%, up to about 2.8%, up to about 2.9%, up to about 3%, up to about 3.5%, up to about 4%, up to about 4.5%, up to about 5%, up to about 5.5% up to about 6%, up to about 6.5%, up to about 7%, up to about 7.5%, up to about 8%, up to about 8.5%, up to about 9%, up to about 9.5%, or up to about 10%. In some embodiments, the enhancing tissue promotes the growth or gain of the tissue mass by about between 2.5% to about 25%, between about 3% to about 25%, between about 4% to about 30%, between about 5% to about 25%, between about 5% to about 20%, between about 5% to about 15%, or between about 10% to about 20%. In some embodiments, enhancing tissue promotes the growth or gain of the tissue mass by about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 10%, about 10.5%, about 11%, about 11.5%, about 12%, about 12.5%, about 13%, about 13.5%, about 14%, about 14.5%, about 15%, about 15.5%, about 16%, about 16.5%, about 17%, about 17.5%, about 18%, about 18.5%, about 19%, about 19.5%, or about 20%.

In some embodiments, the desired therapeutic effect of enhancing tissue mass is measured by comparing before administration of the composition and after a scheduled administration plan. In some embodiments, the scheduled administration plan is one administration of the composition. In some embodiments, the scheduled administration plan is more than one administration of the composition. In some embodiments, the composition is more than two administrations of the composition. In some embodiments, the composition is administered daily. In some embodiments, the composition is administered more than once daily. In some embodiments, the composition is administered every other day. In some embodiments, the composition is administered weekly. In some embodiments, the composition is administered monthly. The scheduled administration plan may be proscribed by a person having skill in the art. The scheduled administration plan is adjusted based on the subject and environment.

In some embodiments, the enhancement of the tissue results in an increase in anabolic effects. In some embodiments, the level of the proteins or metabolites indicative of promoting tissue growth increase compared to the protein or metabolite levels prior to the method. In some embodiments, the increase in protein and/ or metabolites indicative of promoting tissue growth is significant.

In some embodiments, the enhancement of the tissue is measured by a functional test. In an illustrative embodiment, the musculoskeletal tissue is enhanced when the subject's strength increases as a result of the method.

In some embodiments, the desired therapeutic effect from administration of the composition in a therapeutic effective amount is to maintain tissue mass. In some embodiments, the maintenance of tissue mass is measured by comparing before administration of the composition and after a scheduled administration plan. In some embodiments, the scheduled administration plan is one administration of the composition. In some embodiments, the scheduled administration plan is more than one administration of the composition. In some embodiments, the administration plan is more than two administrations of the composition. In some embodiments, the composition is administered daily. In some embodiments, the composition is administered more than once daily. In some embodiments, the composition is administered every other day. In some embodiments, the composition is administered weekly. In some embodiments, the composition is administered monthly. The scheduled administration plan may be proscribed by a person having skill in the art. Illustrative examples of persons having skill in the art include, doctors, nurse practitioners, physical therapists, dieticians, or physical trainers. The scheduled administration plan is adjusted based on the subject and environment. In some embodiments, the subject is exposed to an environment that results in the effect of mechanical unloading on the subject's tissue. For example, the subject may be sedentary. In some embodiments, the subject may be immobile. In some embodiments, the subject may be partially immobile. In some embodiments, the subject relies on a device to move around. In some embodiments, the subject is living in microgravity as observed in space flight.

In some embodiments, the method further comprises applying a mechanical load to the tissue. In some embodiments, the mechanical load is a suboptimal mechanical load. In some embodiments, the suboptimal mechanical load is applied as passive activity. In some embodiments, the suboptimal load is applied by active activity. In some embodiments, the suboptimal load is applied by exercise.

In some embodiments, the mechanical load is an optimal mechanical load. In some embodiments, the optimal mechanical loading is applied by active activity. In some embodiments, the optimal mechanical load is applied by exercise. In some embodiments, L-BAIBA enhances or improves the anabolic effects of mechanical loading on tissue. In some embodiments, the optimal mechanical loading may be applied passively.

In some embodiments, the method applies suboptimal skeletal loading conditions, such as occurs with space flight, immobilization, and aging, while also improving the force output of skeletal muscles. In some embodiments, the subject may be at a risk for deterioration of the musculoskeletal system due to aging or space flight. In some embodiments, the subject is or will be sedentary. In some embodiments, the subject is bed-ridden, suffers from paralysis, is partially or fully immobilized, or is engaged in space flight.

In some embodiments, the composition may be administered by any suitable administration route, including oral, enteral, percutaneous, intradermal, mucosal, submucosal, subcutaneous, interstitial, intrafat, peritumoral, or intramuscular injection administration. In some embodiments, the L-p-aminoisobutyric acid is administered orally. In some embodiments, the L-p-aminoisobutyric acid may be administered with a pharmaceutically acceptable carrier. In some embodiments, the composition is administered by parenteral administration. In some embodiments, the composition is administered prior to the application of a mechanical load. In some embodiments, the composition is administered during the application of a mechanical load. In some embodiments, the composition is administered subsequent to the application of a mechanical load.

The timing and amount of L-BAIBA administration could have different effects on anabolic loading. L-BAIBA may also have effects on other types of loading and exercise such as running, walking, weight lifting, etc. Astronauts now spend three to five hours per day maintaining bone and muscle; the disclosed method could significantly shorten this duration, permitting more time for required tasks and duties. Aging individuals are frequently limited by the amount of exercise they are capable of performing, and may not be able to lift heavy weights or to run due to illness, devices, etc. Inclusion of L-BAIBA with walking or with lighter weights may desirably have an equivalent effect to running or lifting heavier loads. Immobilized patients frequently cannot move or their movement may be limited. L-BAIBA may assist with patients that are manually manipulated, those that require assistance through prosthetic devices, those requiring a synthetic exoskeleton for movement, or those undergoing physical therapy. These and other individuals may use L-BAIBA to maintain tissue or potentially enhance tissue.

The following Examples provide illustrative embodiments of methods of enhancing or maintaining tissue with the administration of L-BAIBA.

EXAMPLE 1

L-BAIBA was synthesized as follows. All reagents and solvents were purchased from commercially available sources and used without further purification. NMR spectra were obtained on a Bruker 300 MHz NMR instrument. The chemical shifts are reported as ppm (d) relative to the residual solvent peak. 1H NMR coupling constants (J) are reported in Hertz (Hz), and the multiplicities are indicated as follows: s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet.

(S)-3-amino-2-methylpropanoic acid (L-BAIBA) was synthesized according to the literature (Beddow, James E. Chemical Communication, 2004, 2778-2779) as White solid. Melting point: 182-184 °C, 1H NMR (300 MHz, d4-CD₃0D): d 2.96-2.93 (m, 2H), 2.47 (m, 1H), 1.20 (d, J = 7.5, 3H). 13C NMR (75 MHz, d4-CD₃OD): d 179.9,

42.5, 38.6, 14.9. Purity >95% (l 254 nm).

EXAMPLE 2

Animals. Mice were obtained from the National Institute of Aging (NIA, Bethesda, MD, USA) and housed at the Indiana University Animal Care Facility. C57BL/ 6 male and female mice of 5 months of age were selected because it is past the period of rapid bone accrual that occurs with growth, and near the age of 'peak bone mass' based on measures of total bone BMD, size and strength. Mice were housed 4-5 per cage under standard conditions for 1-2 weeks before they were single-caged and used in the experiment. Three days prior to the beginning of the experiment mice were separated in groups; L-BAIBA (100 mg/ kg/ day, AdipoGen Life Sciences) was provided in drinking water ad libitum to the treated group while control groups were provided with regular water. Referring to Figs 1 and 3, amounts of consumed water/ day were similar in both control (male, 4.82 ± 0.34 mL; female, 4.75 ± 0.68 mL) and treated mice (male, 4.95 ± 0.34 mL; female, 4.80 ± 1.31 mL). Mice were fed regular chow (2018 Teklad Global 18% Protein Extruded Rodent Diet, ENVIGO), water ad libitum and housed communally in a room maintained at a constant temperature (25°C) on a 12 hour light/ dark schedule. Referring to Figs. 2 and 4, water intake, weight, and overall health of the animal was been monitored and documented during the total duration of the experiment.

EXAMPLE 3

In vivo loading. Mice were anesthetized (2-3% isofluorane) and their right tibiae subjected to axial compression for 220 cycles/ day, loading was repeated every other day, 3 times a week for two weeks total, using the EnduraTEC ELF 3200 (Bose, Mass, USA) system and the Wintest (Bose) software. We selected age-specific peak forces in order to generate peak periosteal strains between -1500 and -2200 me at the cortical mid-shaft which resulted in a magnitude of 7N and 8.25N applied for the male mice, while females were loaded at 8. IN (for both groups n=8). After each loading session were returned to their cages where they were observed to resume unrestricted activity. The left tibia served as a contralateral control.

EXAMPLE 4

Dynamic histomorphometry. Two fluorochromes were used to label and track bone formation using dynamic histomorphometry of the bones; Calcein green (10 mg/ kg; Sigma, Saint Louis, MO, USA) was injected intraperitoneally the first day of loading and Alizarin complexone (30 mg/ kg; Sigma) was injected the last day of tibial loading. Mice were sacrificed 4 days after the last tibial compression. Both loaded and unloaded tibiae were collected and fixed in 4% Paraformaldehyde. After 48 hours, fixative was removed and substituted with 70% Etoh. Tibiae were then embedded in Methyl-Metacrylate for further analysis. To measure bone formation, dynamic histomorphometric analysis was performed on 20-40 pm thick sections taken at the mid-diaphysis located 1mm distal from the tibial-fibular junction using a Lionheart FX, Automated Microscope (BioTek Instruments, Inc., Winooski, Vermont, USA) and the free software ImageJ (NIH, https:/ / imagej.nih.gov/ij/). Measures include: mineralizing surface (MS/BS), mineral apposition rate (MAR), and bone-formation rate (BFR/BS). For each section, we analyzed the entire endocortical (Ec) and periosteal (Ps) surfaces separately. Values obtained from the right tibia were then normalized to the left corresponding tibia of the same animal. This was done to further reduce the biological variability present among non-littermate animals.

Anabolic Loading: A total of 325-month-old C57BL6 male mice and 16 female mice of the same strain were used to induce anabolic bone formation through mechanical loading using suboptimal anabolic loading, 7N and 8.25N for males and 8.1N for females. Compression was applied to the right tibiae of the animals for 220 cycles at 2Hz every other day for 2 weeks. One subgroup of animals received L-BAIBA in water (100mg/Kg/ day). Fluorochrome compounds (Calcein and Alizarin Red) were injected intraperitoneally at the beginning and at the end of the experiment in order to measure the dynamic growth of the bone (Displayed as Mineralizing surface-MS/ BS, Mineral apposition rate-MAR and Bone formation rate-BFR). Values obtained from the right tibia were then normalized to the left corresponding tibia of the same animal (relative measurements: rMS/BS, rMAR and rBFR). Water intake and weight of the animals were measured daily throughout the experiment.

Isobutyric acid measurement in serum: Approximately 1ml of blood per animal was collected by cardiac puncture upon sacrifice. Blood was then centrifuged at 10000RPM for 10 minutes in order to separate serum from the blood cells. Serum obtained was then analyzed to identify the presence of Butyric Acids. LC-MS/MS analysis was performed on a Shimadzu LCMS-8050 triple quadrupole mass spectrometer. The instrument was operated and optimized with positive electrospray ionization in multiple reaction mode (MRM). Six aminobutyric acid isomers were separated and analyzed on a chiral LC column (AZYP LLC-Separation & Analytics, Arlington, TX).

EXAMPLE 5

Referring to Figs 5, 6, and 7, grip strength. The day before the sacrifice, absolute grip strength was tested in all animals. Five measurements were registered per animal, and the top three measurements were included in the analysis using a grip strength meter (Columbus Instruments, Columbus, OH, USA). Mean of the values obtained was calculated in order to avoid habituation of the animals to the repetition of the test. Results were then normalized per body weight in order to reduce the biological variability among mice and analyzed for statistical significance.

RNAseq: Whole genome mRNA Sequencing was performed using an ultra- low mRNAseq chip for purified RNA extracted from the tibiae of 165-months-old C57BL6 male mice that received 8.25N axial loading on the right tibiae for 220 cycles at 2Hz for 2 weeks. Briefly, tibiae were collected, the epiphysis cut and the bone marrow flushed. Tibiae were then cut in pieces and digested twice with collagenase and once with EDTA. Osteocyte enriched bone chips remaining from the digestion were collected and snap-frozen to later be pulverized for RNA extraction. Reactome software analysis was used to analyze the data.

EXAMPLE 6

Blood Serum. Blood was collected by heath puncture the day of the sacrifice. Around 1ml of blood per animal was collected in Blood Collection Tubes (Becton, Dickinson and Company, NJ, USA). Blood was then centrifuged 10000RPM for 10 minutes in order to separate serum from the blood cells. Serum obtained was then analyzed to identify the presence of Butyric Acids.

EXAMPLE 7

Statistical Analysis. One-way ANOVA (Turkey's multiple comparisons test) was used to investigate the significant differences in bone growth in all groups for both males and females for all the parameters considered. T-test was also used to analyze the results in the grip strength and the serum levels collected from different mice groups. Data analysis was performed using Graphpad Prism 7 for Windows (GraphPad Software, La Jolla California USA). A p < 0.05 was considered significant.

EXAMPLE 8

Referring to Figs. 10 to 15, L-BAIBA potential to enhance bone formation by enhancement of the anabolic effects of mechanical loading on bone formation was evaluated. Specifically, five month old C57bl/ 6 male (n=8) and female mice (n=5) were subjected to tibial cyclic compressive loading at 7.0 N and 8.25 N (males) and 8.1 N (females) 220 cycles (-1500 me), 2 Hz, three times per week for two weeks total. Throughout the two week duration, the drinking water for the mice either lacked or contained L-BAIBA to provide approximately 100 mg/ kg/ day. At sacrifice, serum was collected for quantitation of butyric acids.

In mice that did not have access to L-BAIBA, there was no detectable level of L-BAIBA in the serum. In mice that did have access to L-BAIBA, the serum concentration of L-BAIBA averaged 7.12 mM ± 7.09 mM, with no significant effects on the other related butyric acids, L- and a-aminobutyric acid (D-AABA) and gamma-amino butyric acid (GABA). L-BAIBA alone had no effect on bone formation.

L-BAIBA also reduced loss of both bone and muscle due to unloading of the hindlimb of mice, a method to examine lack of loading on bone and analogous to the immobilization and lack of loading as occurs in space. This was achieved through prevention of osteocyte cell death. L-BAIBA synergizes with suboptimal anabolic load to induce bone formation, enhances the effects of voluntary wheel running on bone and muscle, and increases grip strength within two weeks.

EXAMPLE 9

Referring to Figs. 8 and 9, the effect of providing a load on the muscle, i.e., loading, significantly increased bone formation. This anabolic response was further increased in male mice (n=8) by adding L-BAIBA. In male mice, the relative values of loaded plus L-BAIBA, compared to loaded alone, were:

MS/BS 0.232 ± 0.136 loaded + L-BAIBA 0.049 ± 0.141 loaded without L-BAIBA MAR 1.081 ± 0.358 loaded + L-BAIBA 0.252 ± 0.381 loaded without L-BAIBA BFR 0.268 ± 0.209 loaded + L-BAIBA 0.031 ± 0.072 loaded without L-BAIBA

All differences were statistically significant at p < .05. Values in female mice trended towards significance.

Grip strength of the mice given L-BAIBA was significantly increased in both males and females. Force normalized to weight L-BAIBA vs. controls were as follows:

Males 7.66 ± 1.12 L-BAIBA 6.25 ± 0.56 controls

Females 9.08 ± 0.91 L-BAIBA 8.24 ± 0.55 controls

Relative strength g/ gr (n=8). All differences were statistically significant at p< .05. These results demonstrated that the muscle metabolite, L-BAIBA, enhanced the anabolic effects of loading on bone formation while increasing muscle strength. L- BAIBA synergizes with a sub-optimal mechanical load to induce bone formation and will enhance the effects of voluntary wheel running on bone and muscle. Importantly, specific to bone tissue, L-BAIBA alone did not enhance bone tissue.

EXAMPLE 10

To assess L-BAIBA function in muscle, C2C12 cells were separately treated with either D-BAIBA or L-BAIBA. L-BAIBA had no effect on the C2C12 cells. D- BAIBA had modest but significant inhibitory effects on myotube formation, inhibited mitochondrial function, and significantly inhibited adenosine monophosphate-activated protein kinase (AMPK) signaling. Administration of L- BAIBA and D-BAIBA to mice resulted in positive effects for L-BAIBA and negative effects for D-BAIBA. Both L-BAIBA and D-BAIBA were provided in water to mice for 30 days. L-BAIBA increased gastrocnemius and quadriceps muscle weight, increased the oxidative capacity of the gastrocnemius, increased peroxisome proliferator-activated receptor gamma coactivator-l-alpha (PGCla), cytochrome C (Cyt C), optic atrophy 1 (OPA1), and pAMPK) in the gastrocnemius muscle. D- BAIBA inhibited gastrocnemius fiber cross sectional area and oxidative capacity.

EXAMPLE 11

The impact of L-BAIBA on protein expression of PDK4, OPA1, and PGCla, markers of mitochondrial homeostasis and respiration, in C2C12 myotubes treated with 10 mM, 20 mM, or 30 mM L-BAIBA or D-BAIBA for 48 hours was tested. There was no significant effect of either L-BAIBA or D-BAIBA on any of these proteins.

Lig 16 shows protein expression of pAMPK, an indicator of muscle anabolism, in C2C12 myotubes treated with 10 mM, 20 mM, or 30 mM L-BAIBA or D-BAIBA for 48 hours. The results showed that D-BAIBA completely inhibited pAMK, as noted by the arrow. These results suggested that D-BAIBA induced muscle catabolism or breakdown.

Other results showed that L-BAIBA treatment, alone, retained soleus muscle function in spite of hindlimb unloading, but did not retain extensor digitorus longus (EDL) muscle function with hind limb unloading. Fig 17 shows size of three types of skeletal muscle, gastrocnemius (GSN), quadriceps, and tibialis anterior, in control mice and in mice receiving either L- BAIBA or D-BAIBA for four weeks. L-BAIBA significantly increased muscle size of the gastrocnemius muscle and the quadriceps muscle but not the tibialis anterior muscle. D-BAIBA had no effect. C57BL/ 6 male mice were used; control mice (n=5), mice receiving L-BAIBA (n=5), mice receiving D-BAIBA (n=5). Weights were normalized to the initial body weight (IBW) and expressed as weight/ 100 mg IBW. Data were expressed as means ± SD. Significance of the differences: *p<0.05 vs. control. This demonstrated that L-BAIBA had a positive effect on muscle.

Muscle morphology by hematoxylin and eosin stained sections, and quantification of the cross-sectional muscle fiber area (CSA) in the gastrocnemius muscle of control mice and mice treated with L-BAIBA or D-BAIBA for four weeks was studied (data not shown). Although the muscle size was not decreased, the fiber size in animals treated with D-BAIBA was decreased. This suggested that D- BAIBA had a detrimental effect on muscle.

Fig. 18 shows succinate dehydrogenase (SDH) staining and Integrity density (IntDen) quantification were performed in the gastrocnemius muscle of control mice and mice treated with either L-BAIBA or D-BAIBA. Data are expressed as means ± SD. Significance of the differences: 0.05 vs. control. The data demonstrate that L- BAIBA increased the number of oxidative fibers in muscle, suggesting that L-BAIBA increased functional capacity. No effects were observed with D-BAIBA.

Fig. 19 shows representative Western blotting and quantification for peroxisome proliferator-activated receptor gamma coactivator-l-alpha (PGCla) and cytochrome C (Cyt C), markers of mitochondrial homeostatsis and respiration, in whole muscle protein extracts from mice receiving either L-BAIBA or D-BAIBA. Tubulin was used as a loading control. The graphs show quantitation of each of PGCla as a master regulator of mitochondrial biogenesis, and cytochrome C protein that is associated with the mitochondrial inner membrane. The results demonstrated that L-BAIBA significantly increased both PGC1 a and cytochrome C, suggesting that L-BAIBA enhanced mitochondrial function in muscle. Data are expressed as arbitrary units (A.U.) vs. control and reported as means ± SD. Significance of the differences: *p<0.05 vs. control.

Fig. 20 shows representative Western blotting and quantification for optic atrophy 1 (OPA1), mitofusin, and pyruvate dehydrogenase kinase 4 (PDK4) in whole muscle protein extracts from mice receiving L-BAIBA or D-BAIBA. OPA1 protein helps regulate the shape of mitochondria by playing a key role in the mitochondrial fusion process. Mitofusin represents a key player in mitochondrial fusion, trafficking, and turnover. PDK4 is a glucose metabolism regulator. The results demonstrated that L-BAIBA significantly increased OPA1 protein expression, but had no effect on mitofusin and PDK4. D-BAIBA had no significant effect on any of these mitochondrial proteins.

Fig. 21 shows Western blotting analysis performed on whole muscle protein extracted from mice receiving L-BAIBA or D-BAIBA for four weeks. Representative Western blotting and quantification are shown for pAMPK/ AMPK and pAKT / AKT in whole muscle protein extracts from mice receiving L-BAIBA or D- BAIBA. Tubulin was used as loading control. The data suggested that L-BAIBA had an anabolic effect on muscle. Data are expressed as arbitrary units (A.U.) vs. control and reported as means ± SD. Significance of the differences: *p<0.05 vs. control.

Both L-BAIBA and D-BAIBA were evaluated for effects on C2C12 myotube formation. Effects of L-BAIBA are different from its enantiomer, D-BAIBA. L- BAIBA had no effect, but D-BAIBA exhibited myotube size effects and completely blocked pAMPK. In vivo in mice, specifically, D-BAIBA administered for four weeks had negative effects on muscle by decreasing fiber size, while L BAIBA administered for four weeks had positive effects on muscle by increasing size of the gastrocnemius and quadriceps muscles, increasing energy capacity, increasing mitochondrial respiration and homeostasis, and increasing anabolism.

These data demonstrate that the D-enantiomer of BAIBA could have negative effects on muscle formation and function, and thus that administering a racemic mixture of BAIBA would be undesirable.

Claims

Claims:

1. A method comprising: administering an effective amount of a composition to a subject to enhance tissue mass, wherein the composition is L-BAIBA.

2. The method of claim 1, wherein the composition comprises at least about

90% L-BAIBA.

3. The method of claim 1, wherein the tissue is selected from the group consisting of bone, adipose, brain, or muscle.

4. The method of claim 1, wherein the tissue mass is enhanced by at least about 3%.

5. The method of claim 1, wherein the step of administering occurs through oral administration.

6. The method of claim 1, wherein the effective amount enhances the tissue mass.

7. The method of claim 1, wherein the subject is not exposed to a mechanical load

8. A method comprising: administering an effective amount of a composition to a subject to maintain or enhance tissue mass, wherein the composition in L-BAIBA.

9. The method of claim 8 further comprising applying a mechanical load to the tissue.

10. The method of claim 9, wherein the L-BAIBA is administered prior to the application of force.

11. The method of claim 9, wherein the L-BAIBA is administered during the application of the mechanical load.

12. The method of claim 9, wherein the L-BAIBA is administered subsequent to the application of the mechanical load.

13. The method of claim 8, wherein the composition is at least about 90% L- BAIBA.

14. The method of claim 8, wherein the tissue is selected from the group consisting of bone, liver, adipose, brain, or muscle.

15. The method of claim 8, wherein the step of administering occurs through oral administration.

16. The method of claim 8, wherein the effective amount enhances the tissue mass.

17. The method of claim 9, wherein the mechanical load is suboptimal.

18. The method of claim 9, wherein the mechanical load is optimal.

19. The method of claim 8, wherein the subject is exposed to an environment of mechanical unloading.

20. The method of claim 9, wherein the tissue mass is maintained.