WO2024072995A1 - Formulation for in vivo tissue regeneration and rejuvenation - Google Patents

Abstract

Provided herein is a composition for regenerating and rejuvenating a mammalian tissue in vivo. In some embodiments, the composition may comprise L-methionine, threonine, glycine, putrescine, cysteine and S-adenosylmethionine (SAM) in amounts effective for regenerating the tissue in vivo. The composition is formulated for oral administration. Also provided is a method for regenerating a mammalian tissue in vivo, comprising orally administering the composition to a mammalian subject in need thereof. Also provided is a method for rejuvenating a mammalian tissue in vivo, comprising orally administering the composition to a mammalian subject in need thereof.

Description

FORMULATION FOR 72V VIVO TISSUE REGENERATION AND REJUVENATION

CROSS-REFERENCING

This application claims the benefit of U.S. provisional application serial no. 63/412,123, filed on September 30, 2022, which application is incorporated by reference herein.

BACKGROUND

Cellular reprogramming can potentially be used to regenerate tissues that have been lost or damaged due to a degenerative disease, injury or aging. While it is possible to reprogram somatic cells in vitro (e.g., using a transcription factors (Takahasi, et al Cell 2006 126: 663-76)) or using cell fusions (Sanges Cell Rep. 2013 4: 271-286), such methods are challenging to implement in vivo because they involve transplanting cells and/or administering recombinant DNA. Better methods for regenerating and rejuvenating tissues in vivo are therefore needed.

SUMMARY

Provided herein is a composition comprising L-methionine, threonine, glycine, putrescine, cysteine and S -adenosylmethionine (SAM), wherein the composition is formulated for oral administration and wherein the L-methionine, threonine, glycine, putrescine, cysteine and S -adenosylmethionine (SAM) are present in amounts effective for in vivo tissue regeneration.

Also provided is a method for regenerating a mammalian tissue in vivo, wherein the method comprises orally administering the composition to a mammalian subject in need thereof.

Also provided is a method for rejuvenating a mammalian tissue in vivo, wherein the method comprises orally administering the composition to a mammalian subject in need thereof.

Without wishing to he bound to any specific theory, it is thought that the composition partially reprograms cell fates in the mammalian subject in vivo which, in turn, facilitates tissue regeneration and rejuvenation. Other mechanisms are possible. BRIEF DESCRIPTION OF THE FIGURES

The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

Figs. 1A-1D show that in vivo 1C-MIM intervention accelerates motor recovery from CTX injury.

Fig. 1A: Experimental design. The drinking intervention is maintained during all time, including three months before the CTX injection, and 8 days of recovery after CTX. The day of CTX-injection is marked in the center as 'O’, followed by 8 days in recovery. The green bar before CTX, marks the period of behavioral preconditioning of mice to the open field test (OFT), the orange bar after CTX marks the tracking of recovery by OFT.

Fig. IB: Representative tracks of behavioral motor test performed in control and 1 C-MIM supplemented mice 24 hours after CTX injury.

Fig. 1C: Kinetics of resting, evaluated by OFT in CTX-injured mice. The resting time was recorded at 1-day, 5-days, and 8-days after cardiotoxin injury, during a 60-minutes session. Pre-conditioned was performed in all animals one week before injury and considered as net 100% for subsequent analyses.

Fig. ID: Kinetics of jumps, velocity, and ambulatory time, evaluated by OFT in CTX-injured mice. Recorded 1-day, 4-days, 5-days, and 7-days after cardiotoxin injury, during a 60-minutes session. Pre-conditioned was performed in all animals one week before the injury. Total intervention n=87. Pair comparisons at indicated groups in Fig. 4A.

Figs. 2A-2H show that muscle repair is accelerated after the intervention with IC-metabolites in vivo

Fig. 2A: Analysis of the number of centrally nucleated cells expressed as 'number of fibers/per field' in control and cardiotoxin-injected muscle at day-2 (2d) on control and 1C-MIM supplemented mice. Random fields were assessed per condition.

Fig. 2B: Representative H&E staining of tibialis anterior 7-days after cardiotoxin injection, on control and 1 C-MIM-treated mice. Scale bar= 50pm.

Fig. 2C: Representative immunostaining of Pax7 -positive cells in tibialis anterior, 7-days after cardiotoxin injection, on control and IC-MIM-treated mice. White arrows indicate double positive DAP1+Pax7+. Scale bar = 15pm.

Fig. 2D: Quantification of Pax7 positive cells per field in the indicated group comparisons.

Fig. 2E-2H: Distribution of the fibers according to their size, in tibialis anterior muscle (TA), 7-days after cardiotoxin injection. Comparison between control vs. IC-MIM-intervention in the indicated groups. Inserts on the right of each, represent averagesi SD. Note, as the curve displaces to the right, it indicates an advance in the muscle regeneration (because of the bigger size of myofibers as these recover). In (H) comparisons of the scramble supplementation of six metabolites unrelated to IC-metabolism; only glycine supplementation; the conditions control (plain water) and 1 C-MIM, all were supplemented for three months.

Total intervention n=87. Pair comparisons at indicated groups in Fig. 4A.

Figs. 3A-3H show that modifications in acetylation and modulations on cell cycle drive the acceleration of regeneration by 1 C-MIM in muscle in vivo.

Fig. 3A: Proliferation quantified by immunofluorescence and Ki67 detection.

Fig. 3B: Primary myofibers were placed in vitro and treated with or without 1C-MIM for precise evaluation of double markers in nuclei. In the right find the summary of nuclear markers in single-myofibers for control and IC-MIM-treated double-positive and the ratio of Pax7 -positive vs. MyoD-positive or MyoD— negative in myofibers exposed to the indicated conditions.

Figs. 3C-3F: Transcriptomic analysis of 1 C-MIM-quadriceps extracted from old mice after 3 -months of supplementation. In (C) PC A, in (0) heatmap with the total of differential expressed genes; in panel (E) the respective volcano plot showing in blue dots DEG, gray dots indicate not significant genes, and in red-dots highlight of genes of interest (p-Adj <0.01 ). In (F) the enrichment analysis of the DEG with p- Adj. <0.01

Fig. 3G: Orthogonal readouts on the histone modifications detected by MassSpec on control astrocytes and 1 C-MIM supplemented.

Fig. 3H: Suggested mechanism of 1C-MIM for eliciting acceleration of the cell transition on regeneration. Several metabolic sensors respond to incoming metabolites, creating modulation feedback in the niche. Particularly, an increase in deacetylation will evoke relaxed chromatin facilitating the expression of new genes associated with specific cell programs required for a cell in a given time (e.g., during regeneration). Thus, pre-loading the cells with specific metabolites may have them prepared for the rapid gene activation required for any identity transition.

Total intervention n=87. Pair comparisons at indicated groups in Fig. 4A. See also Fig. 5.

Figures 4A-4D show the experimental design and immediate parameters evaluated for the in vivo intervention with 1 C-MIM.

Fig. 4A: Distribution per round of the 87 mice used in the study. Each round represents the experimental intervention used for the study. Only two mice including control and 1 C-MIM treated exhibited tumors, and only in the group of aged animals (round 6), therefore no correlation of tumor emergence was associated with the intervention.

Fig. 4B: Drinking volumes. The drinking supplementation was replaced every other day with a standard volume, and measurements of consumption were taken during the intervention. The volumes were normalized according to the number of mice per cage.

Fig. 4C: Bodyweight measurements after 1C-MIM drinking supplementation per round. Each dot represents one mouse.

Fig. 4D: Movement impairment elicited after 5 hours of CTX injection evaluated in terms of jumps, velocity, and resting time. Measurements derived from the Open Field Test, recorded for a period of 60 minutes, graphs represent the distribution including control and 1 C-MIM supplemented mice.

Figs. 5A-5E show analyses on the 1 C-MIM intervention in muscle with and without CTX injury.

Fig. 5A: Representative histology images ~5 hours after CTX-injury showing similar damage in control and 1C-MIM supplemented mice. Tibialis anterior sections are stained by H&E (scale bar =250pm).

Fig. 5B: Myofiber size distribution of centrally nucleated fibers without CTX injury of control and 1C-MIM supplemented mice. Insert to the right represents the average.

Fig. 5C: DNA methylation clock using the Horvath Clock demonstrates rejuvenation after 1C-MIM supplementation in quadriceps non-injured. Left panel, schematic representation of how same samples were processed for transcriptomics and methylation, middle correlation analysis; and right panel, estimation of the methylation age using the Horvath clock for muscle. Tissues were recovered at 88- weeks old mice.

Figs. 5D-5E Gene set enrichment analyses from transcriptomics. See also Fig. 4A.

DETAILED DESCRIPTION

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entireties.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred 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. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “an agonist” includes a mixture of two or more such agonists, and the like.

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 may be different from the actual publication dates which may need to be independently confirmed.

As used herein, the terms "mcg" and "pg" refer to micrograms.

Formulations

As noted above, this disclosure provides a composition comprising L- methionine, threonine, glycine, putrescine, cysteine and S-adenosylmethionine (SAM), wherein the composition is formulated for oral administration and wherein the L-methionine, threonine, glycine, putrescine, cysteine and S-adenosylmethionine (SAM) are present in amounts effective for in vivo tissue regeneration. The composition may be formulated in a variety of different ways.

In some embodiments, the composition may comprise, for every 1 mg of L- methionine: 0.65 mg to 1.95 mg threonine, 0.98 mg to 2.93 mg glycine, 0.85 mg to 2.54 mg putrescene, 0.5 mg to 0.1.5 mg cysteine and 0.13 mg to 0.39 mg SAM. For example, the composition may comprise, for every 1 mg of L-methionine: 1.04 mg to 1.57 mg threonine, 1.56 mg to 2.03 mg glycine, 1.36 mg to 2.03 mg putrescene, 0.8 mg to 1.2 mg cysteine and 0.21 mg to 0.31 mg SAM.

In some embodiments, the composition may comprise, for every 1 mg of L- methionine: 0.66 mg to 2 mg threonine, 1 mg to 3 mg glycine, 0.02 mg to 0.07 mg putrescene, 0.5 mg to 1.5 mg cysteine and 0.13 mg to 0.4 mg SAM. For example, the composition may comprise, for every 1 mg of L-methionine: 1.06 mg to 1.6 mg threonine, 1.6 mg to 2.4 mg glycine, 0.03 mg to 0.05 mg putrescene, 0.8 mg to 1.2 mg cysteine and 0.21 mg to 0.32 mg SAM.

In some embodiments, the composition may comprise a daily dose of: 750 mg to 2250 mg methionine, 1000 mg to 3000 mg threonine, 1500 mg to 4500 mg glycine, 35 mg to 105 mg putrescene, 750 mg to 2250 mg cysteine and 200 mg to 600 mg SAM or a fraction of the daily dose (e.g., a half dose, quarter dose, fifth dose, etc). For example, in some embodiments the composition may comprises a daily dose of: 1200 mg to 1800 mg methionine, 1600 mg to 2400 mg threonine, 2400 mg to 3600 mg glycine, 56 mg to 84 mg putrescene, 1200 mg to mg 1800 cysteine and 320 mg to mg 480 SAM, or a fraction of the daily dose (e.g., a half dose, quarter dose, fifth dose, etc.).

In some embodiments, the composition comprises a daily dose of 59.84 mg to 179.51 mg L-methionine, 78.05 mg to 234.15 mg threonine, 117.07 mg to 351.22 mg glycine, 101.46 mg to 304.39 mg putrescene, 59.84 mg to 179.51 mg cysteine, and 15.61 mg to 46.83 mg SAM, or a fraction of the daily dose (e.g., a half dose, quarter dose, fifth dose, etc.). For example, the composition may comprise a daily dose of: 95.74 mg to 143.61 mg L-methionine, 124.88 mg to 187.32 mg threonine, 187.32 mg to 280.98 mg glycine, 162.34 mg to 243.51 mg putrescene, 95.74 mg to 143.61 mg cysteine and 24.98 mg to 37.46 mg SAM, or a fraction of the daily dose (e.g., a half dose, quarter dose, fifth dose, etc.).

As noted above, the composition may be formulated for oral administration. As such, in some embodiments, the composition may comprise an inert diluent or edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly with the food. In some embodiments, the composition may be incorporated with an excipient and used in the form of a powder, an ingestible tablet, a buccal tablet, a coated tablet, a troche, a capsule, an elixir, a dispersion, a suspension, a solution, a syrup, a wafer, or the like. Tablets, troches, pills, capsules and the like typically contain one or more of the following: a binder such as gum tragacanth, acacia, com starch or gelatin; an excipient, such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; a sweetening agent such as sucrose, lactose or saccharin; or a flavoring agent such as peppermint, oil of wintergreen or cherry flavoring, for example.

If the composition is formulated as a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coating, for instance, tablets, pills, or capsules may be coated with shellac, sugar or both. In some embodiments, the composition may be in a vapor- tight casing that dissolves in the stomach. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor, and/or one or more flavor masking compounds. In other embodiments, the composition may be formulated as an aqueous liquid.

The composition may contain additional components that may have a biological activity (e.g., growth factors, vitamins, anti-oxidants, anti-inflammatories, etc.). However, the composition is not a cell culture medium (i.e., not DMEM, MEM, BME, or RPMI, etc). In some embodiments, the composition is protein-free and, a such, does not contain serum or any cell survival factors that are commonly found in cell culture media.

Methods

A method for regenerating and/or rejuvenating a mammalian tissue in vivo is provided, where the term “regenerating” is intended to include, e.g., increasing muscle mass in a subject that has a degenerative disease or is aging as well as accelerating the recovery of muscle that has been damaged (e.g., by physical trauma).

In some embodiments, the method may comprise orally administering to a mammalian subject the composition as described above. The mammalian subject may be a human or a non-human mammal, e.g., a pet such as a cat or dog, a farm animal such as a horse, cow, sheep or pig, or a test animal such as a mouse, rat, or non- human primate, for example.

In some embodiments, the mammalian subject may have a degenerative disease (which may affect balance, movement, talking, breathing, and heart function), an injury (e.g., a traumatic injury or injury caused by surgery) or is aging (e.g., may be a human that is over 65, 70 or 80 years old). In some embodiments, the mammalian subject may have a muscle condition, e.g., age-related muscle loss or a muscle injury. In other embodiments, the mammalian subject may be a human athlete that is in training and would like to build muscle and/or avoid injury. In other embodiments, the mammalian subject may have a wound. The tissue targeted by the method may be muscle (cardiac, skeletal or smooth), neural, bone, cartilage or skin, etc.

In some embodiments, the composition may be administered to the mammalian subject at least once daily over a course of at least 60 days, e.g., at least 90 days, at least 6 months, at least 1 year, or at least 2 years. In some cases, the mammalian subject may receive a fraction of the daily dose multiple times per day.

In some embodiments, the method further comprises testing the mammalian subject for tissue regeneration and/or rejuvenation after administration of the composition and, optionally, before administration of the composition. For example, the mammalian subject may be tested using a variety of behavioral characteristics, e.g., movement, agility and/or muscle strength. Molecular markers could also be analyzed. For example, DNA methylation age may be tested using an epigenetic molecular clock, e.g,. the Horvath Muscle clock.

In any embodiment, the method may further comprising ceasing the administration of the composition after the tissue has been regenerated and/or rejuvenated. In these embodiments, the physical characteristics of the mammalian subject have returned to strength.

In some embodiments the mammalian subject may receive a daily dose per kg bodyweight of: 12.26 mg to 36.8 mg methionine, 16 mg to 48 mg threonine, 24 mg to 72 mg glycine, 20.8 mg to 62.4 mg putrescene, 12.26 mg to 36.8 mg cysteine and 3.2 mg to 9.6 mg SAM. For example, the mammalian subject may receive a daily dose per kg bodyweight of: 19.62 mg to 29.44 mg methionine, 25.6 mg to 38.4 mg threonine, 38.4 mg to 57.6 mg glycine, 33.28 mg to 49.92 mg putrescene, 19.62 mg to 29.44 mg cysteine and 5.12 mg to 7.68 mg SAM.

If the mammalian subject is a human, then the mammalian subject may receive a daily dose per kg bodyweight of: 12.09 mg to 36.29 mg methionine, 16.12 mg to 48.38 mg threonine, 24.19 mg to 72.58 mg glycine, 0.56 mg to 1.69 mg putrescene, 12.09 mg to 36.29 mg cysteine and 3.22 mg to 9.67 mg SAM, for example a daily dose per kg bodyweight of: 19.35 mg to 29.03 mg methionine, 25.80 mg to 38.70 mg threonine, 38.70 mg to 58.06 mg glycine, 0.90 mg to 1.35 mg putrescene, 19.35 mg to 29.03 mg cysteine; and 5.16 mg to 7.74 mg SAM.

In some embodiments, a human may receive a daily dose of: 59.84 mg to 179.51 mg L-methionine, 78.05 mg to 234.15 mg threonine, 117.07 mg to 351.22 mg glycine, 101.46 mg to 304.39 mg putrescene, 59.84 mg to 179.51 mg cysteine and 15.61 mg to 46.83 mg SAM, e.g., a daily dose of 95.74 mg to 143.61 mg L- methionine, 124.88 mg to 187.32 mg threonine, 187.32 mg to 280.98 mg glycine, 162.34 mg to 243.51 mg putrescene, 95.74 mg to 143.61 mg cysteine and 24.98 mg to 37.46 mg SAM. EXAMPLES

Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

MATERIALS AND METHODS

1C-MIM formulation-. The following metabolites were dissolved in 150 ml of water to produce a formulation referred to as " 1C-MIM" below.

23 mg of L-methionine

30 mg threonine;

45 mg glycine;

39 mg putrescene;

23 mg cysteine; and

6 mg SAM

The mice to which the 1C-MIM formulation was administrated consumed approximately 4 mis 1C-MIM per day, yielding a daily dose of:

0.61 mg methionine:

0.8 mg threonine;

1.2 mg glycine;

1.04 mg putrescene;

0.61 mg cysteine; and

0.16 mg SAM.

Animals: ICR mice were purchased from Envigo and the colony was maintained and expanded at Salk Institute animal facility. C57BL mice were purchased from The Jackson Laboratory and the colony was maintained and expanded at both Salk Institute and Altos Labs animal facilities. Mice were utilized for in vivo interventions and primary culture assays. All animal handling was approved by the IACUC committee to conform to regulatory standards.

RNA isolation and gene expression analysis by RT-PCR: Total RNA was isolated at the needed time points, using the RNeasy Plus Mini kit QIAGEN (Cat. 74106), according to the manufacturer's protocol, including a DNA-removal step with DNAsel. Amount and purity of RNA were assessed using a NanoDrop spectrophotometer (Nanodrop Technologies); at least 500ng of total RNA was used to synthesize cDNA by reverse transcription, using MAXIMA™ H Minus cDNA Synthesis Master Mix (Thermo Fisher Scientific, Cat. M1662). 2.5-10ng of cDNA was used in the following qPCR performed on a CFX384 thermal cycler (Bio-Rad) using the SSOADVANCED™ Universal SYBR® Green Supermix (BIORAD, Cat. 1725274). Results were normalized to at least one reference genes

Actin, RPL38, GAPDH, Gus, CTCF, and Natl , specified per figure), selected for their highest stability among a pool of common housekeeping genes. Primers sequences in SI6. Statistical analysis of the results was performed using the 2ACt method. Results were expressed relative to the expression values of the experimental control.

In vivo intervention: C57BL or ICR mice were allocated into groups according to their age and strain, where possible siblings were split into control and treatment to reduce differences. All mice used in the experiment were wild-type, with no evident health problems. Mice between 3-4-months-old were used for experiments for the young phenotype, and mice between 17-20-months-old for experiments for the old phenotype. All animals were weighed at the beginning and end of the intervention. The drinking solution was prepared from the stock of water provided by the animal facility, for both controls and supplemented animals. A volume of 150mL of water was provided every other day for each cage containing 5 mice maximum. The remaining volumes of control and supplemented water were measured every time before discarding the remaining volumes and the volume consumed was correlated to the number of mice present in each cage, to determine the drinking volume. The treated water supplementation was delivered in sterile red bottles for a period of one to three months. After these interventions, mice were habituated to walking around an open field test every other day for a week before the muscle injury.

Muscle injury by cardiotoxin: A solution of cardiotoxin (Latoxan S.A.S.Cat. L8102) was prepared at a concentration of lOuM. For old-phenotype, a volume of 50uL was injected into both back legs in the tibialis anterior (TA) and in gastrocnemius (GAS) muscles, using a 29-gauge insulin syringe. For the young phenotype the injection was in TA, GAS, and quadriceps. To provide the injection mice were anesthetized using an isoflurane chamber, afterwards, legs were shaved for facilitating the visualization of muscle at the injection time. Mice were allowed to recover over a warm bed set at 38°C for 30 minutes and then returned to their usual housing when they were awake.

Open Field Tests: For pre-conditioning and for evaluation after muscle injury, animals were set in the behavioral room for 1 hour in advance to habituate them. Next, each animal was placed in its respective chamber to monitor its physical activity. The chamber for recording was connected to the Activity Monitor Software to quantify distance and movements including distance, velocity, resting time, and jumps. Representative snapshots in real-time were taken for a visual representation of the distance traveled, and the raw data was further analyzed using the Activity Monitor and Excel Software.

Immunostaining and HE-staining: After euthanasia muscles from the injury site tibialis anterior (TA) and gastrocnemius (GAS) were collected and frozen in OCT for further sectioning. Transversal sections from both control and treated were obtained using a Leica cryostat set at lOum thickness. Three slices of muscle were arranged per glass slide for downstream experiments. Briefly, muscle slides were fixed with 4% PFA solution, washed with PBS, and cleared with incubation of lOOmM Glycine (Sigma, Cat. G5417) for 15 min. Then, samples were blocked and permeabilized (5% goat serum + 0.02%triton + 0.01% NaN3 + 2% BSA, in PBS) for Ih at room temperature followed by an overnight incubation with the needed primary antibody at 4°C. The next day, samples were washed with PBS-tween20 at least three times, afterward, were incubated with the respective secondary antibodies and DAPI for Ih at room temperature. All secondary antibodies were diluted at a ratio of 1:500. Fluorescent images were captured using a confocal scanning microscope. Quantification using the ImageJ software. For HE staining, slides were incubated in hematoxylin for 5-min and eosin for 3-min, with clearance with ammonium hydroxide solution (1:500), followed by standard washes. Images were captured using bright field microscopy.

Myofibers area measurement: Muscles processed by cryostat as described above were utilized to capture images in Axioscan. For analysis of area of individual myofibers the software ImageJ was used and area of at least 50 center-nucleated myofibers per slice were measured. Then an automatic subdivision per rages was performed by the Prisma software and using those ranges the averages were estimated per condition. Single myofiber isolation: EDL muscles were extracted and carefully digested with 2mg/mL collagenase type I in DMEM (Worthington Biochem) for 45min at 37 DC. Digestion was stopped with a medium containing horse serum. Myofibers were released by gently flushing with a large bore glass pipette. Released single myofibers were treated with 1C-MIM or control medium and 3 days later fixed with 4%PFA for subsequent immunostaining.

DNA-methylation clock from quadriceps: After euthanasia muscles quadriceps (i.e., from non-injury-site) were extracted and flash-frozen and minced in liquid nitrogen, then samples from 25-50 mg of ground frozen tissue were separated for genomic DNA extraction. DNA was processed by the Clock Foundation (https://clockfoundation.org/data-tools/), which retrieved the averaged results of three-biological replicates per condition.

RNA-sequencing from quadriceps: After euthanasia muscles quadriceps (i.e., from non-injury-site) were extracted and flash-frozen, and minced in liquid nitrogen, then samples from 50-100 mg of ground frozen tissue were separated for RNA extraction. The processing from total-RNA extraction, library prep, sequencing, and analysis was performed at the company Active Motif (https://www.activemotif.com) according to their standard procedures.

Quantification and statistical analysis: Statistical analysis applied, exact n values, and precision measures are indicated in each figure legend. All readouts derived from metabolomics are derived from n=5 biological replicates. For other experiments, refer to each figure legend. No statistical methods were used to predetermine the sample size Randomization or stratification were not applied. In general, data is shown as averages ± S.D. or S.E.M, as indicated in each figure legend. Statistics were performed using GraphPad Prism Software. Comparisons between two groups were analyzed using t-Test Two-tailed, or one-way AN0VA followed by either Bonferroni or Turkey's post hoc test, as appropriate. Frequency distribution plots were obtained by analysis tool in Graph Pad Prism (version 9). The statistics from metabolome analyses were obtained under the Metabolon Portal Software as provided for the company (www.portal.metabolon.com/en). For transcriptomic analyses, R Software (R version 3.5.1) was used for statistics, other details about these analyses in their respective methods' section. For all experiments, values of P<0.05 were considered statistically significant. RESULTS

The following results shows that the 1C-MIM formulation rejuvenates muscle in old mice and accelerates muscle recovery after injury in young and old mice in vivo

The model of muscle degeneration by cardiotoxin (CTX) was chosen, which injures muscular fibers with subsequent activation of the repair mechanism involving sequential transformative cell events (Ramadasan-Nair et al, J Biol Chem 2014 289: 485-509). An in vivo intervention was performed using 87 wild-type mice (see allocated groups in Fig. 4A). Because this intervention consisted of a drinking supplementation, the drinking volumes were measured, observing just a slight trend of an increase in the drinking of IC-MIM-water compared to control standard water (Fig. 4B). The drinking supplementation did not induce weight changes (Fig. 4C). In addition, a scramble drinking control composed of six metabolites not directly related to IC-metabolism and an intervention of 3-month glycine supplementation was included (glycine, as an amino acid part of 1C and with reported effects in muscle building (see Lin et al Molecular Therapy 2020 28: 1339-1358)). Neither scramble nor glycine altered the volume of drinking or weight (Fig. 4B-C).

As part of the in vivo validation, the motor capacity in the intervened young and old mice were compared. As the first step, the mice were preconditioned to the open field test (by placing the mice in the area for one hour, and 2-3 times at least one week before inducing the injury) being the last habituation the day before CTX- injection (Fig. 1A). Next, that CTX injury impaired the mice's movement was confirmed, as observed by a reduction in motor responses in terms of velocity and jumps, together with an increase in resting time, measured 5 hours after recovering from anesthesia (Fig. 4D). Interestingly, only after 24 hours after injury, it was observed an increase in ambulatory distance and consequently, a decrease in the resting time in those mice supplemented with 1C-MIM (Figs. 1B-C). Only a trend in the improvement of other parameters like the number of jumps, and ambulatory time was detected: 59% and 61% of the variation of the relative number of jumps and ambulatory time accounted for the regeneration progression. While the velocity variation is not accounted for that recovery (Fig. ID). Of note, the resting time was the parameter that showed the most relevant difference between groups after CTX injury (i.e., mice rest more time); it also was compared to a glycine intervention and a scramble intervention of metabolites to test specificity. In summary, by comparing with the other control supplementations: a faster motor recovery was detected in mice supplemented with 1C-MIM, in second place was the group supplemented with glycine; and control-untreated and scramble-supplemented were behind with no difference between them (Fig. 1C). Overall, mice reduce the resting time (likewise increase ambulatory distance) as muscle regeneration (recovery) progresses.

Next, observations were made at the tissue level. The observations corroborated the improvement seen in IC-MIM-supplemented mice. After an injury, the recovery of muscle architecture is concurrent with the appearance of centrally nucleated regenerating fibers; thus, whether 1C-MIM benefits the appearance rate of central-nucleated fibers was investigated. A 2.4-fold increase in central-nucleated fibers on day 2 in IC-MIM-supplemented mice was observed (i.e., meeting a number of center-nucleated fibers usually observed in controls after day 7), suggesting that the repair occurs faster in 1C-MIM animals (Figs. 2A-B). This effect occurs despite having started from a comparable degree of damage with CTX injection (Fig. 5A).

Previous studies have shown that activated satellite cells (SCs) are prominent on day 7 -after CTX injury- and reduced beyond 11-days (Ramadasan- air et al, J Biol Chem 2014 289: 485-509). It was observed that, indeed, 1C-MIM enhances this activation, as indicated by a significant increase in the levels of Pax7+ cells. These results were consistently obtained in independent treated groups, including young - males or females- and aged mice (Figs. 2C-D). Moreover, during the regeneration process, the myofibers from IC-MIM-supplemented mice exhibited increased cross- sectional areas compared to their untreated counterparts, indicating that this intervention accelerates recovery (Figs. 2E-G). The 1C-MIM intervention did not impact the myofiber size in control animals without injury (Fig. 5B). Of note, the scramble 3-months intervention did not impact the myofiber size (Fig. 2H). Whereas the 3-months glycine supplementation had an effect increasing the size as reported by others (Lin et al Molecular Therapy 2020 28: 1339-1358), but the effect of 1C-MIM supplementation was still superior (Fig. 2H).

Finally, to get mechanistic insights of 1C-MIM in muscle tissue, DNA methylation age in mice quadriceps was measured (without injury). It was found that IC-MIM-muscle exhibited a discrete but significant reduction in age according to Horvath’s Muscle-Clock (Fig. 5C). This suggests that rejuvenation by DNA- methylation plays a minor role. Because in the earlier in vitro models analyzed (both myofibers and astrocytes), modulations in the cell cycle occurred after the addition of 1C-MIM, the Ki67+ cells in mice muscle over the different supplementations were measured, and only 1C-MIM -but not scramble, glycine, nor control- had a significant increase in Ki67+ cells (Fig. 3A). To distinguish whether 1C-MIM influences proliferating MBs or quiescent SCs, coimmunostaining was performed on isolated single myofibers using MyoD and Pax7. It was found that 1C-MIM increased the number of proliferating SCs (Pax7+MyoD+) and the number of quiescent SCs (Pax7+MyoD-) by 1.96 and 1.7-folds, respectively (Fig. 3B). Next, RNA- transcriptomics was performed, PCA showed a clear separation of the control muscle from the IC-MIM-supplemented mice (Fig. 3C). DEG analysis (cut-off value of fold changeLog2 -0.5, +0.5 and P-adj <0.01) showed 789 genes, from which 67% were downregulated and were associated to immune response and collagen formation (Fig. 3D-F). Particularly, it was observed significant down-regulation of the pro- tumorigenic gene Saa3, the pro dystrophy gene Timpl, the glycoprotein involved in inflammation and cell death Cite, and the downregulation of methallothioneins Mtl and Mt2, which abrogation has been associated to increases in myotube size and muscle strength ¹⁶. No significant changes were found in senescent markers (Fig. 5D). Conversely, supplemented 1C-MIM muscle, particularly showed up-regulation of genes such as Lep (a gene known for modulating the expression of metabolic and myokine genes), Gdf5 (an inhibitor of muscle atrophy), several cadherins, Efcab6 (related to the recruitment of the histone deacetylase complex), and Hdac3 (histone deacetylase that regulates skeletal muscle fuel metabolism). Correspondingly, GSEA revealed that 1C-MIM intervention was positively correlated with epigenetic regulation of gene expression, chromatin modifying enzymes, and histone acetylation (Fig. 5E). The up-regulated DEG enriched for regulation of transcriptional activity by acetylation, suggesting this as the via for modulating cell cycle (Fig. 3F). Orthogonal readouts obtained in astrocytes treated with 1C-MIM corroborate this regulation, as changes in the relative abundance of histone modifications induced by 1C-MIM (including acetylation, methylation, and unmarked histones) were observed. No relevant changes in methylation marks were observed, but 1C-MIM increased global acetylation marks, an event supporting the acquisition of chromatin plasticity (Fig.

5E, Fig. 3G).

Overall, the foregoing results show that 1C-MIM administration in vivo accelerates the regeneration in mouse muscle emulating the intermediate flexibility observed in the transitional state of differentiating myoblasts, therefore without impacting the lineage identity. Moreover, this intervention favors reprogramming transitions that enhance the myogenic potential after an injury event, thereby accelerating the recovery of a healthy state in damaged muscle. While some embodiments have been illustrated and described above, it will be appreciated that various changes can be made therein without departing from the spirit and scope of this disclosure.

Claims

What is claimed is:

1. A composition comprising L- methionine, threonine, glycine, putrescine, cysteine and S-adenosylmethionine (SAM), wherein the composition is formulated for oral administration and wherein the L-methionine, threonine, glycine, putrescine, cysteine and S-adenosylmethionine (SAM) are present in amounts effective for in vivo tissue regeneration.

2. The composition of claim 1, wherein the composition comprises, for every 1 mg of L-methionine:

0.65 mg to 1.95 mg threonine;

0.98 mg to 2.93 mg glycine;

0.85 mg to 2.54 mg putrescene;

0.5 mg to 1.5 mg cysteine; and

0.13 mg to 0.39 mg SAM.

3. The composition of claim 1 or 2, wherein the composition comprises, for every 1 mg of L-methionine:

1.04 mg to 1.57 mg threonine;

1.57 mg to 2.03 mg glycine;

1.36 mg to 2.03 mg putrescene;

0.8 mg to 1.2 mg cysteine; and

0.21 mg to 0.31 mg SAM.

4. The composition of any prior claim, wherein the comprises, for every 1 mg of L-methionine:

0.66 mg to 2 mg threonine;

1 mg to 3 mg glycine;

0.02 mg to 0.07 mg putrescene;

0.5 mg to 1.5 mg cysteine; and

0.13 mg to 0.4 mg SAM.

5. The composition of any prior claim, wherein the composition comprises, for every 1 mg of L-methionine:

1.06 mg to 1.6 mg threonine;

1.6 mg to 2.4 mg glycine;

0.03 mg to 0.05 mg putrescene;

0.8 mg to 1.2 mg cysteine; and

0.21 mg to 0.32 mg SAM.

6. The composition of any prior claim, wherein the composition comprises a daily dose of:

750 mg to 2250 mg methionine;

1000 mg to 3000 mg threonine;

1500 mg to 4500 mg glycine;

35 mg to 105 mg putrescene;

750 mg to 2250 mg cysteine; and

200 mg to 600 mg SAM or a fraction of the daily dose.

7. The composition of any prior claim, wherein the composition comprises a daily dose of:

1200 mg to 1800 mg methionine;

1600 mg to 2400 mg threonine;

2400 mg to 3600 mg glycine;

56 mg to 84 mg putrescene;

1200 mg to 1800 mg cysteine; and

320 mg to 480 mg SAM; or a fraction of the daily dose.

8. The composition of any of claims 1-7, wherein the composition is a tablet or capsule.

9. The composition of any of claims 1-8, wherein the composition is a vapor- tight capsule that dissolves in the stomach.

10. The composition of any of claims 1-7, wherein the composition is a liquid.

11. A method for regenerating a mammalian tissue in vivo, comprising orally administering to a mammalian subject a composition of any of claims 1-10.

12. The method of claim 11, wherein the mammalian subject has a degenerative disease, an injury or is aging.

13. The method of claim 11 or 12, wherein the subject has age-related muscle loss or a muscle injury.

14. The method of any of claims 11-13, wherein the composition is administered to the mammalian subject at least once daily over a course of at least 60 days.

15. The method of any of claims 11-14, wherein the method further comprises testing the subject for tissue regeneration after administration of the composition and, optionally, before administration of the composition.

16. The method of claim 15, wherein the marker is movement, agility and/or muscle strength.

17. The method of any of claims 11-16, further comprising ceasing the administration of the composition after the tissue has been regenerated.

18. The method of any of claims 11-17, wherein the mammalian subject receives a daily dose per kg bodyweight of:

12.26 mg to 36.8 mg methionine;

16 mg to 48 mg threonine;

24 mg to 72 mg glycine;

20.8 mg to 62.4 mg putrescene;

12.26 mg to 36.8 mg cysteine; and

3.2 mg to 9.6 mg SAM.

19. The method of any of claims 11-18, wherein the mammalian subject receives a daily dose per kg bodyweight of:

19.62 mg to 29.44 mg methionine;

25.6 mg to 38.4 mg threonine;

38.4 mg to 57.6 mg glycine;

33.28 mg to 49.92 mg putrescene;

19.62 mg to 29.44 mg cysteine; and

5.12 mg to 7.68 mg SAM.

20. The method of any of claims 11-17, wherein the mammalian subject is a human and the human receives a daily dose per kg bodyweight of:

12.09 mg to 36.29 mg methionine;

16.12 mg to 48.38 mg threonine;

24.19 mg to 72.58 mg glycine;

0.56 mg to 1.69 mg putrescene;

12.09 mg to 36.29 mg cysteine; and

3.22 mg to 9.67 mg SAM.

21. The method of any of claims 11-17, wherein the mammalian subject is a human and the human receives a daily dose per kg bodyweight of:

19.35 mg to 29.03 mg methionine;

25.80 mg to 38.70 mg threonine;

38.70 mg to 58.06 mg glycine;

0.90 mg to 1.35 mg putrescene;

19.35 mg to 29.03 mg cysteine; and

5.16 mg to 7.74 mg SAM.