Combination of metformin and rapamycin for treatment of neurological, muscular and proliferative diseases

Abstract

The present disclosure relates to methods of treating diseases such as neurological, muscular and proliferative diseases using a combination of metformin and rapamycin.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 460,487, filed April 19, 2023, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present disclosure relates to methods of treating diseases such as neurological, muscular, and proliferative diseases using a combination of metformin and rapamycin. BACKGROUND

[0004] The underlying pathophysiology of many neurological, muscular, and proliferative diseases (e.g., myotonic dystrophy type 1 (DM1), Duchenne muscular dystrophy (DMD), and spinocerebellar ataxia type 3 (SCA-3)) is associated with impaired activation of active adenosine monophosphate-activated protein kinase (AMPK), mammalian target of rapamycin complex 1 (mTORCl) dysfunction, and / or autophagic flux disorder. There are currently no known methods of treating neurological, muscular, and proliferative diseases such as DM1, DMD, and SCA-3, and the existing methods of treatment are rarely effective in treating these diseases with adverse side effects, and many of the treatment methods are limited to improving symptoms to improve the quality of life of patients. Treatments for DM1 include, for example, mexiletine, beta-blockers, and angiotensin-converting enzyme (ACE) inhibitors. Treatment methods for DMD include, for example, prednisone and deflazacort. New therapies for neuromuscular diseases, including gene therapy, small molecule therapy, and RNA-targeted therapy, are still in the experimental stage and need to be further tested before they can be used in clinical practice.

[0005] Accordingly, there is a need for a method of treating and / or preventing neurological, muscular, and proliferative diseases associated with impaired activation of active adenosine monophosphate-activated protein kinase (AMPK), mammalian target of rapamycin complex 1 (mTORCl) dysfunction, and / or autophagic flux disorder, rather than managing or improving the symptoms of such diseases; and wherein the method has lighter side effects and is conveniently administered as a single dosage form. SUMMARY

[0006] Described herein are methods of treating or preventing a neurological, muscular, or proliferative disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof; wherein the neurological, muscular, or proliferative disease is selected from the group consisting of: myotonic dystrophy type 1 (DM1), Duchenne muscular dystrophy (DMD), spinocerebellar ataxia type 3 (SCA-3), Alzheimer's disease, Parkinson's disease, vascular dementia, Lewy body dementia (DLB), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Lafora disease, glioblastoma, and diffuse intrinsic pontine glioma (DIPG).

[0007] In some embodiments, the neurological, muscular, or proliferative disease is myotonic dystrophy type 1 (DM1). In some embodiments, a reduction in reactive oxygen species (ROS) is measured in the subject after administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0008] In some embodiments, the neurological, muscular, or proliferative disease is Duchenne muscular dystrophy (DMD).

[0009] In some embodiments, the neurological, muscular, or proliferative disease is spinocerebellar ataxia type 3 (SCA-3).

[0010] In some embodiments, the neurological, muscular, or proliferative disease is Alzheimer's disease.

[0011] In some embodiments, the neurological, muscular, or proliferative disease is Parkinson's disease.

[0012] In some embodiments, the neurological, muscular, or proliferative disease is vascular dementia.

[0013] In some embodiments, the neurological, muscular, or proliferative disease is Lewy body dementia (DLB).

[0014] In some embodiments, the neurological, muscular, or proliferative disease is Huntington's disease (HD).

[0015] In some embodiments, the neurological, muscular, or proliferative disease is amyotrophic lateral sclerosis (ALS).

[0016] In some embodiments, the neurological, muscular, or proliferative disease is Lafora disease.

[0017] In some embodiments, the method comprises preventing the disease.

[0018] In some embodiments, the neurological, muscular, or proliferative disease is glioblastoma.

[0019] In some embodiments, the neurological, muscular, or proliferative disease is diffuse intrinsic pontine glioma (DIPG). In some embodiments, Akt is activated in the subject after administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0020] Some embodiments provide a method of treating or preventing a neurological cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof; wherein the cancer is a glioblastoma or diffuse intrinsic pontine glioma (DIPG).

[0021] In some embodiments, the neurological cancer is a glioblastoma.

[0022] In some embodiments, the neurological cancer is diffuse intrinsic pontine glioma (DIPG). In some embodiments, Akt is activated in the subject after administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0023] Some embodiments provide a method of increasing muscle strength in a subject identified or diagnosed with muscle weakness, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0024] Some embodiments provide a method of preventing or reversing muscle weakness in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, an increase in muscle mass is measured in the subject using magnetic resonance imaging (MRI) after administration of a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to the subject.

[0025] In some embodiments, the method comprises preventing cell senescence in the subject.

[0026] Some embodiments provide a method of increasing motor ability in a subject, comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0027] In some embodiments, the method comprises increasing the number of voluntary muscle contractions per minute in the subject relative to before administration of a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to the subject.

[0028] In some embodiments, the method comprises increasing the duration of voluntary muscle contractions in the subject prior to administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0029] In some embodiments, the method comprises determining an increase in the distance walked by the subject during a 10-meter walk test after administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0030] In some embodiments, the method comprises determining a decrease in the time required for the subject to complete a 100-meter walk test after administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0031] In some embodiments, an increase in AMPK activation, inhibition of mTORCl, inhibition of S6 kinase, or any combination thereof, is measured in the subject after administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0032] In some embodiments, metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof are administered orally. In some embodiments, metformin or a pharmaceutically acceptable salt thereof is administered daily.

[0033] In some embodiments, the dose of metformin or a pharmaceutically acceptable salt thereof is about 500 mg to about 3000 mg, based on the free base of metformin. In some embodiments, the dose of metformin or a pharmaceutically acceptable salt thereof is about 500 to about 1750 mg, based on the free base of metformin.

[0034] In some embodiments, rapamycin or a pharmaceutically acceptable salt thereof is administered daily.

[0035] In some embodiments, the dose of rapamycin or a pharmaceutically acceptable salt thereof is about 0.1 mg to about 2 mg, based on the free base of rapamycin. In some embodiments, the dose of rapamycin or a pharmaceutically acceptable salt thereof is about 0.5 mg to about 1 mg, based on the free base of rapamycin. In some embodiments, the dose of rapamycin or a pharmaceutically acceptable salt thereof is about 0.7 mg, based on the free base of rapamycin.

[0036] In some embodiments, metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof are administered simultaneously. In some embodiments, metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof are administered simultaneously as a fixed-dose form.

[0037] In some embodiments, the rapamycin or pharmaceutically acceptable salt thereof is administered once a week.

[0038] In some embodiments, the dose of rapamycin or pharmaceutically acceptable salt thereof is about 1 mg to about 10 mg, based on the free base of rapamycin. In some embodiments, the dose of rapamycin or pharmaceutically acceptable salt thereof is about 2 mg to about 10 mg, based on the free base of rapamycin. In some embodiments, the dose of rapamycin or pharmaceutically acceptable salt thereof is about 5 mg, based on the free base of rapamycin.

[0039] In some embodiments, the subject has not been previously administered cyclosporine, tacrolimus, and mycophenolate mofetil within 1 month of administration of metformin or pharmaceutically acceptable salt thereof and rapamycin or pharmaceutically acceptable salt thereof. In some embodiments, the subject has not been identified or diagnosed with a disease associated with the kidney. In some embodiments, the subject has not been identified or diagnosed with a disease associated with the liver. In some embodiments, the subject has not been identified or diagnosed with a disease associated with the heart. In some embodiments, the subject has not been identified or diagnosed with diabetes. In some embodiments, the subject has not been identified or diagnosed with abnormal endocrine function. In some embodiments, the subject has not been previously administered a therapeutic agent that modulates the insulin transduction pathway within 1 year of administration of metformin or pharmaceutically acceptable salt thereof and rapamycin or pharmaceutically acceptable salt thereof.

[0040] The details of one or more embodiments of the application are set forth in the description below. Other features and advantages of the application will be apparent from the description, the drawings, and the claims. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 Figure 1 is a diagram depicting the design and timeline of the study of the effects of rapamycin, metformin, and combinations thereof on C. elegans lifespan and healthspan.

[0042] Figure 2 Figure 3 is a series of survival curves for N2 wild-type worms treated with rapamycin, metformin, Combination A, Combination B, Combination C, and Combination D.

[0043] Figure 3 Figure 4 is a series of survival curves for N2 wild-type worms treated with rapamycin, metformin, Combination A, Combination B, Combination C, and Combination D to day 17.5 (corresponding to about 50% survival for the negative control DMSO 1%).

[0044] Figure 4The following are a series of survival curves of N2 wild-type nematodes treated with rapamycin, metformin, combination A, combination B, combination C and combination D up to day 20 (corresponding to approximately 25% survival of the negative control DMSO 1%).

[0045] Figure 5 A-5D is the effect observed in wild-type N2 nematodes treated with rapamycin, metformin, combination A, combination B, combination C, and combination D, from injection of L4 larvae to the production of the first ( Figure 5 A) and the last one ( Figure 5 B) The time of egg laying (in hours), and the span of egg laying time ( Figure 5 C) and the average number of eggs laid per nematode during the oviposition period ( Figure 5 The bar chart for D).

[0046] Figure 6 This is a bar chart (area under the curve) of N2 wild-type nematodes treated with rapamycin, metformin, combination A, combination B, combination C and combination D.

[0047] Figures 7A-7D This is a graph showing the motility of N2 wild-type nematodes treated with rapamycin, metformin, combination A, combination B, combination C, and combination D. Figure 7A Head amplitude; Figure 7B , medium amplitude; Figure 7C Tail amplitude; Figure 7D Bending frequency; Figure 7E ,speed).

[0048] Figures 8A-8C Using rapamycin and metformin ( Figure 8A Combinations A and B ( Figure 8B ); and combinations C and D ( Figure 8C Radar graphs of nematode locomotion (head amplitude, midbody amplitude, tail amplitude, velocity, and bending frequency) at different stages (1: D0 to D5; 2: D6 to D10; 3: D11 to D15; 4: D16-D20) of wild-type N2 nematodes treated with nematodes. Detailed Implementation

[0049] Described herein are (i) methods of treating and / or preventing a neurological, muscular, or proliferative disease, disorder, or condition, and (ii) methods of reducing and / or preventing processes associated with aging, such as cellular senescence, comprising administering a combination of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. The AMP-activated protein kinase (AMPK) and the mammalian target of rapamycin (mTOR) targets are two signaling pathways that play a role in cellular metabolism, growth, and proliferation. For example, AMPK activation and mTOR inhibition can (1) protect neurons from injury and improve cognitive function, thus becoming a potential target for treating neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease; (2) enhance muscle protein synthesis, thus being a potential therapy for treating muscle diseases such as muscular dystrophy and myotonic dystrophy; and (3) inhibit tumor growth, induce cancer cell death, thus being a potential strategy for cancer treatment. Surprisingly, the inventors found that metformin (e.g., activating AMPK) and rapamycin (e.g., inhibiting mTORCl) synergistically treat the above-mentioned diseases, thus reducing the required dosage and, therefore, the adverse effects of targeting unwanted metabolic pathways. Other benefits include, for example, reducing the concentration of insulin and insulin growth factor 1 (IGF-1), inducing energy stress, restoring cerebral blood flow (CBF) and cerebral vascular density, reducing cerebral amyloid angiopathy and microhemorrhages, reducing inflammatory cytokines (such as IL-6), reducing proinflammatory T helper 1 (Thl) and T helper 17 (Thl7) cells. Moreover, the safety and efficacy of metformin and rapamycin have been extensively studied. Without wishing to be bound by theory, the two drugs are believed to have complementary side effects. For example, it is believed that metformin can reduce the risk of hyperglycemia and hyperlipidemia induced by rapamycin, while rapamycin can reduce the gastrointestinal side effects of metformin. Moreover, the combination of metformin and rapamycin can have a synergistic effect on health and longevity.

[0050] Definitions

[0051] As used herein, the terms “about” and “approximately” can be used interchangeably, when used in connection with a numerical value, to include an imprecise numerical range of 0-10% of that numerical value.

[0052] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0053] The terms "treat" or "treatment" as used herein refer to a therapeutic or palliative measure. Beneficial or desired clinical results include, but are not limited to, detectable or undetectable, complete or partial remission of one or more symptoms associated with a disease or disorder or condition, lessening of disease extent, stabilization (i.e., not worsening) of disease state, delay or slowing of disease progression, amelioration or palliation of the disease state (e.g., one or more symptoms of the disease), and remission (partial or total). "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0054] The term "prevent" or "prevention" as used herein means to completely or partially stop the onset, recurrence or spread of a disease or condition as described herein or a symptom thereof. The term "preventive measure" refers to an action taken to prevent the occurrence, recurrence or spread of a disease or condition described herein in whole or in part.

[0055] As used herein, the terms "subject," "individual," and "patient" are used interchangeably and refer to any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine; cattle, sheep, horses, primates, and humans. In some embodiments, the subject is a human. In some embodiments, the subject has experienced and / or displays at least one symptom of a disease or disorder to be treated and / or prevented.

[0056] As used herein, the term "adverse reaction" refers to an undesired reaction resulting from a normal physiological change in a subject.

[0057] As used herein, the term "pharmaceutical composition" is intended to encompass a product comprising one or more active ingredients, and one or more inert ingredients that constitute carriers, as well as any product which results from the combination, complexation or aggregation of any two or more ingredients, or from the degradation or

[0058] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" means a carrier or adjuvant that can be administered to a patient, together with a compound of the disclosure, or a pharmaceutically acceptable salt thereof, and that does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound. In some embodiments, each component is "pharmaceutically acceptable" in the sense of being compatible with pharmaceutical formulations other ingredients of a drug formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenecity, or other problems or complications commensurate with a reasonable benefit / risk ratio. For example, see Remington: The Science and Practice of Pharmacy, 21stEdition; LWW Publishing Company (Lippincott Williams & Wilkins): Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6thEdition; Rowe et al. Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rdEdition; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2ndEdition; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009.

[0059] As used herein, the phrase "fixed dosage form" means the simultaneous administration of two or more therapeutic agents in a single dosage form (e.g., in a single oral dosage form, such as a pill, tablet, or capsule). In this context, when metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof are administered in a fixed dosage form, the two drugs are administered together in a single dosage form (e.g., an oral dosage form, such as a pill, tablet, or capsule) that contains both metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof at the same time. In some embodiments, the fixed dosage form can include metformin, rapamycin, and one or more additional therapeutic agents.

[0060] For example, the use of the word "abnormal" in the terms "abnormally high," "abnormally elevated," or "abnormally low," refers to a deviation from the range of parameters found in healthy subjects that a medical professional (such as a physician, nurse practitioner, medical laboratory scientist, nurse practitioner, physical therapist, or physician's assistant) would recognize as indicative or predictive of a dysfunction or pathological state. In addition, in some embodiments, "abnormal" can refer to a physiological response that persists beyond the time at which a normal, healthy subject recovers from the response; or a physiological response that is exaggerated in degree and / or duration relative to that which occurs in a normal, healthy subject.

[0061] As used herein, "autophagy" refers to the natural, conservative degradation of cells that degrades, removes, and recycles unnecessary or dysfunctional components in cells. In some embodiments, promoting autophagy in a subject includes increasing the frequency of the number of cells and / or cellular components in a subject that undergo autophagy and / or the autophagy process.

[0062] The term "therapeutically effective amount" as used herein refers to the amount of one or more active chemical entities or pharmaceutical agents (e.g., metformin and rapamycin) administered which will elicit the biological or medical response of a tissue, system, animal, individual or human sought to be treated. In some embodiments, the response includes reduction and / or alleviation of signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate "therapeutically effective amount" can be ascertained by any suitable technique, including, for example, a dose escalation study, in any individual case.

[0063] The phrase "pharmaceutically acceptable" means within the scope of sound medical judgment these compounds, materials, compositions, and / or dosage forms are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable salt" refers to salts formed by the addition of a pharmaceutically acceptable acid or base to a compound disclosed herein. In some cases, a pharmaceutically acceptable salt is obtained by reacting a compound described herein with an acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. The term "pharmaceutically acceptable salt" can refer to a pharmaceutically acceptable addition salt that is formed by reacting a compound having an acidic group with a base, or that is formed by other methods previously determined, such as an ammonium salt, an alkali metal salt, such as a sodium or potassium salt, an alkaline earth metal salt, such as a calcium or magnesium salt, a salt with an organic base, such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl) methylamine, and a salt with an amino acid, such as arginine, lysine, and the like. There is no specific limitation on the pharmacologically acceptable salt as long as it can be used for a drug. Examples of the salt of the compound described herein with a base include the following: a salt with an inorganic base such as sodium, potassium, magnesium, calcium, and aluminum; a salt with an organic base such as methylamine, ethylamine, and ethanolamine; a salt with a basic amino acid such as lysine and ornithine; and an ammonium salt. The salt can be an acid addition salt, and specific examples thereof are addition salts with an acid, such as a mineral acid, for example, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; an organic acid, for example, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, and ethanesulfonic acid; and an acidic amino acid, for example, aspartic acid and glutamic acid.

[0064] For clarity, unless otherwise specified herein, when a variable of a subject (e.g., a condition, a characteristic, a status, a parameter, a score, an assessment, a test, or a statistic) is increased, decreased, or improved, unless otherwise specified herein, the increase, decrease, or improvement is measured, assessed, or obtained, e.g., relative to the same variable measured, assessed, or obtained prior to the start of treatment (e.g., prior to administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof). The variable can be a single measurement, assessment, or score; an average of multiple measurements, assessments, or scores; or a daily average of multiple measurements, scores, or assessments. Unless otherwise specified herein, the measurement, assessment, or score is typically made within 1 month (e.g., within 3 weeks, 2 weeks, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 18 hours, 12 hours, 6 hours, 3 hours, 2 hours, 1 hour, 30 minutes, or 15 minutes) of administration of metformin and / or rapamycin (e.g., metformin and rapamycin). For example, a subject can have a reduced frequency of symptom onset when the number or average number of symptom onset episodes perceived by the subject within a period of time after administration of metformin and / or rapamycin (e.g., metformin and rapamycin) is less than the number or average number of symptom onset episodes perceived by the subject within the same period of time prior to administration of metformin and / or rapamycin.

[0065] The present disclosure provides a method of treating or preventing a neurological, muscular, or proliferative disease, disorder, or condition in a subject in need thereof. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the neurological, muscular, or proliferative disease is selected from the group consisting of: myotonic dystrophy type 1 (DM1), Duchenne muscular dystrophy (DMD), spinocerebellar ataxia type 3 (SCA-3, also known as Machado Joseph disease), Alzheimer’s disease, Parkinson’s disease, vascular dementia, Lewy body dementia (DLB), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), Lafora disease, glioblastoma, and diffuse intrinsic pontine glioma (DIPG).

[0066] In some embodiments, the method comprises determining whether the disease is associated with impaired activation of active adenosine monophosphate-activated protein kinase (AMPK). In some embodiments, the method comprises determining whether the disease is associated with dysfunction of mammalian target of rapamycin complex 1 (mTORCl). In some embodiments, the method comprises determining whether the disease is associated with autophagic flux disorder. In some embodiments, if the disease is determined to be associated with impaired activation of active adenosine monophosphate-activated protein kinase (AMPK), overactivation of mammalian target of rapamycin complex 1 (mTORCl), and / or autophagic flux disorder, the method comprises administering to the subject metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0067] Some embodiments provide a method of treating or preventing a neurological, muscular, or proliferative disease, disorder, or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof; wherein the neurological, muscular, or proliferative disease is selected from the group consisting of: myotonic dystrophy type 1 (DM1), Duchenne muscular dystrophy (DMD), spinocerebellar ataxia type 3 (SCA-3, i.e., Machado Joseph disease), Alzheimer's disease, Parkinson's disease, vascular dementia, Lewy body dementia (DLB), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Lafora disease, glioblastoma, and diffuse intrinsic pontine glioma (DIPG).

[0068] Some embodiments provide a method of treating a neurological, muscular, or proliferative disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof; wherein the neurological, muscular, or proliferative disease is selected from the group consisting of: myotonic dystrophy type 1 (DM1), Duchenne muscular dystrophy (DMD), spinocerebellar ataxia type 3 (SCA-3), Alzheimer's disease, Parkinson's disease, vascular dementia, Lewy body dementia (DLB), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Lafora disease, glioblastoma, and diffuse intrinsic pontine glioma (DIPG).

[0069] Some embodiments provide a method of preventing a neurological, muscular, or proliferative disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof; wherein the neurological, muscular, or proliferative disease is selected from the group consisting of: myotonic dystrophy type 1 (DM1), Duchenne muscular dystrophy (DMD), spinocerebellar ataxia type 3 (SCA-3), Alzheimer's disease, Parkinson's disease, vascular dementia, dementia with Lewy bodies (DLB), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Lafora disease, glioblastoma, and diffuse intrinsic pontine glioma (DIPG).

[0070] Some embodiments provide a method of treating or preventing a neurological, muscular, or proliferative disease in a subject in need thereof, the method comprising (a) determining whether the disease is associated with impaired activation of active adenosine monophosphate-activated protein kinase (AMPK), dysfunction of mammalian target of rapamycin complex 1 (mTORCl), and / or autophagic flux disorder; and (b) administering to the subject metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof if the disease is determined to be associated with impaired activation of active adenosine monophosphate-activated protein kinase (AMPK), hyperactivation of mammalian target of rapamycin complex 1 (mTORCl), and / or autophagic flux disorder; wherein the neurological, muscular, or proliferative disease is selected from the group consisting of: myotonic dystrophy type 1 (DM1), Duchenne muscular dystrophy (DMD), spinocerebellar ataxia type 3 (SCA-3), Alzheimer's disease, Parkinson's disease, vascular dementia, dementia with Lewy bodies (DLB), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Lafora disease, glioblastoma, and diffuse intrinsic pontine glioma (DIPG).

[0071] In some embodiments, the dysfunction of mammalian target of rapamycin complex 1 (mTORCl) comprises hyperactivation of mammalian target of rapamycin complex 1 (mTORCl).

[0072] In some embodiments, the subject has a neurological, muscular, or proliferative disease selected from the group consisting of: myotonic dystrophy type 1 (DM1), Duchenne muscular dystrophy (DMD), spinocerebellar ataxia type 3 (SCA-3), Alzheimer's disease, Parkinson's disease, vascular dementia, dementia with Lewy bodies (DLB), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Lafora disease, glioblastoma, and diffuse intrinsic pontine glioma (DIPG).

[0073] Some embodiments provide a method of treating or preventing a neurological, muscular or proliferative disease associated with impaired activation of active AMP-activated protein kinase (AMPK), mammalian target of rapamycin complex 1 (mTORCl) dysfunction, and / or autophagy flux disruption in a subject, the method comprising:

[0074] determining whether a neurological, muscular or proliferative disease is associated with impaired activation of active AMP-activated protein kinase (AMPK), mammalian target of rapamycin complex 1 (mTORCl) dysfunction, and / or autophagy flux disruption; and

[0075] administering to a subject determined to have a neurological, muscular or proliferative disease associated with impaired activation of active AMP-activated protein kinase (AMPK), mammalian target of rapamycin complex 1 (mTORCl) dysfunction, and / or autophagy flux disruption, a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof; wherein the neurological, muscular or proliferative disease is selected from the group consisting of: myotonic dystrophy type 1 (DM1), Duchenne muscular dystrophy (DMD), spinocerebellar ataxia type 3 (SCA-3), Alzheimer's disease, Parkinson's disease, vascular dementia, dementia with Lewy bodies (DLB), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Lafora disease, glioblastoma and diffuse intrinsic pontine glioma (DIPG).

[0076] Some embodiments provide a method of treating or preventing a neurological, muscular or proliferative disease associated with impaired activation of active AMP-activated protein kinase (AMPK), mammalian target of rapamycin complex 1 (mTORCl) dysfunction, and / or autophagy flux disruption in a subject, the method comprising administering to a subject identified or diagnosed as having a neurological, muscular or proliferative disease associated with impaired activation of active AMP-activated protein kinase (AMPK), mammalian target of rapamycin complex 1 (mTORCl) target overactivation, and / or autophagy flux disruption, metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof; wherein the neurological, muscular or proliferative disease is selected from the group consisting of: myotonic dystrophy type 1 (DM1), Duchenne muscular dystrophy (DMD), spinocerebellar ataxia type 3 (SCA-3), Alzheimer's disease, Parkinson's disease, vascular dementia, dementia with Lewy bodies (DLB), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Lafora disease, glioblastoma and diffuse intrinsic pontine glioma (DIPG).

[0077] Some embodiments provide a method of selecting a treatment or prevention measure for a subject having a neurological, muscular or proliferative disease, the method comprising:

[0078] whether the determined subject has a neurological, muscular, or proliferative disease associated with impaired activation of active adenosine monophosphate-activated protein kinase (AMPK), mammalian target of rapamycin complex 1 (mTORCl) dysfunction, and / or autophagy flux disruption; and selecting the subject for a treatment or prophylactic measure comprising administering a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof; wherein the neurological, muscular, or proliferative disease is selected from the group consisting of: myotonic dystrophy type 1 (DM1), Duchenne muscular dystrophy (DMD), spinocerebellar ataxia type 3 (SCA-3), Alzheimer's disease, Parkinson's disease, vascular dementia, Lewy body dementia (DLB), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Lafora disease, glioblastoma, and diffuse intrinsic pontine glioma (DIPG).

[0079] Some embodiments provide a method of selecting a subject for a treatment or prophylactic comprising administering a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof, the method comprising:

[0080] identifying a subject having a neurological, muscular, or proliferative disease associated with impaired activation of active adenosine monophosphate-activated protein kinase (AMPK), mammalian target of rapamycin complex 1 (mTORCl) dysfunction, and / or autophagy flux disruption; and

[0081] selecting the subject for a treatment or prophylactic comprising administering a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof; wherein the neurological, muscular, or proliferative disease is selected from the group consisting of: myotonic dystrophy type 1 (DM1), Duchenne muscular dystrophy (DMD), spinocerebellar ataxia type 3 (SCA-3), Alzheimer's disease, Parkinson's disease, vascular dementia, Lewy body dementia (DLB), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Lafora disease, glioblastoma, and diffuse intrinsic pontine glioma (DIPG).

[0082] Some embodiments provide a method of selecting a treatment or prophylactic measure for a subject, the method comprising selecting a treatment or prophylactic measure comprising administering a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof for a subject identified or diagnosed as having a neurological, muscular or proliferative disease associated with impaired activation of active adenosine monophosphate-activated protein kinase (AMPK), mammalian target of rapamycin complex 1 (mTORCl) target dysfunction, and / or autophagy flux disruption, wherein the neurological, muscular or proliferative disease is selected from the group consisting of: myotonic dystrophy type 1 (DM1), Duchenne muscular dystrophy (DMD), spinocerebellar ataxia type 3 (SCA-3), Alzheimer's disease, Parkinson's disease, vascular dementia, dementia with Lewy bodies (DLB), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Lafora disease, glioblastoma, and diffuse intrinsic pontine glioma (DIPG).

[0083] Some embodiments provide a method of selecting a subject having a neurological, muscular or proliferative disease for a treatment comprising administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof, the method comprising:

[0084] determining whether the disease in the subject is a neurological, muscular or proliferative disease associated with impaired activation of active adenosine monophosphate-activated protein kinase (AMPK) and / or mTORCl hyperactivation; and

[0085] selecting a subject determined to have a neurological, muscular or proliferative disease associated with impaired activation of active adenosine monophosphate-activated protein kinase (AMPK), mammalian target of rapamycin complex 1 target (mTORCl) dysfunction, and / or autophagy flux disruption for a treatment comprising administering a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof; wherein the neurological, muscular or proliferative disease is selected from the group consisting of: myotonic dystrophy type 1 (DM1), Duchenne muscular dystrophy (DMD), spinocerebellar ataxia type 3 (SCA-3), Alzheimer's disease, Parkinson's disease, vascular dementia, dementia with Lewy bodies (DLB), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Lafora disease, glioblastoma, and diffuse intrinsic pontine glioma (DIPG).

[0086] In some embodiments, the method comprises selecting a treatment for the subject. In some embodiments, the method comprises selecting a prophylactic measure for the subject.

[0087] In some embodiments, the neurological, muscular or proliferative disease is selected from the group consisting of: myotonic dystrophy type 1 (DM1), Duchenne muscular dystrophy (DMD), and spinocerebellar ataxia type 3 (SCA-3).

[0088] In some embodiments, the neurological, muscular, or proliferative disease is selected from Alzheimer’s disease, Parkinson’s disease, vascular dementia, dementia with Lewy bodies (DLB), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), and Lafora disease.

[0089] In some embodiments, the neurological, muscular, or proliferative disease is selected from glioblastoma and diffuse intrinsic pontine glioma (DIPG).

[0090] In some embodiments, the method comprises treating the disease. In some embodiments, the method comprises preventing the disease.

[0091] In some embodiments, the method comprises treating one or more symptoms. In some embodiments, the method comprises preventing one or more symptoms.

[0092] In some embodiments, the neurological, muscular, or proliferative disease is myotonic dystrophy type 1 (DM1). In some embodiments, treating or preventing myotonic dystrophy type 1 (DM1) comprises treating or preventing one or more symptoms of myotonic dystrophy type 1. In some embodiments, the one or more symptoms is selected from the group of muscle weakness, muscle atrophy, muscle stiffness, muscle pain, ptosis, low blood oxygen saturation, intellectual disability, behavioral disorder, fatigue, cataracts, retinal damage, difficulty breathing, shortness of breath, diabetes (e.g., type I or type II diabetes), sleep apnea, pneumonia, low testosterone, pilomatrixoma, low gamma-globulinemia, erectile dysfunction, testicular failure, gonadal atrophy, arrhythmia, cardiomyopathy, difficulty swallowing, abdominal pain, irritable bowel syndrome (IBS), constipation, and diarrhea.

[0093] In some embodiments, the symptom is muscle weakness.

[0094] In some embodiments, the symptom is muscle atrophy.

[0095] In some embodiments, the symptom is muscle stiffness.

[0096] In some embodiments, the symptom is muscle pain.

[0097] In some embodiments, the symptom is ptosis.

[0098] In some embodiments, the symptom is low blood oxygen saturation. In some embodiments, low blood oxygen saturation refers to blood oxygen saturation below 95% (e.g., below 94%, below 93%, below 92%, below 91%, below 90%, below 89%, below 88%, below 87%, below 86%, or below 85%).

[0099] In some embodiments, the symptom is intellectual disability. In some embodiments, the intellectual disability comprises abnormally low duration of attention. In some embodiments, the intellectual disability comprises impairment in visual-spatial skills.

[0100] In some embodiments, the symptom is a behavioral disorder. In some embodiments, the behavioral disorder is an autism spectrum disorder.

[0101] In some embodiments, the symptom is fatigue. In some embodiments, the fatigue comprises excessive daytime sleepiness.

[0102] In some embodiments, the symptom is cataracts.

[0103] In some embodiments, the symptom is retinal damage.

[0104] In some embodiments, the symptom is difficulty breathing.

[0105] In some embodiments, the symptom is dyspnea.

[0106] In some embodiments, the symptom is diabetes (e.g., Type I or Type II diabetes).

[0107] In some embodiments, the symptom is sleep apnea.

[0108] In some embodiments, the symptom is pneumonia.

[0109] In some embodiments, the symptom is low testosterone. In some embodiments, low testosterone refers to total testosterone in ng / dL that is below the 50th percentile (e.g., below the 45th percentile, below the 40th percentile, below the 35th percentile, below the 30th percentile, below the 25th percentile, below the 20th percentile, below the 15th percentile, below the 10th percentile, or below the 5th percentile) in males of the same age or same age group.

[0110] In some embodiments, the symptom is pili matrical tumors.

[0111] In some embodiments, the symptom is hypogammaglobulinemia.

[0112] In some embodiments, the symptom is erectile dysfunction.

[0113] In some embodiments, the symptom is testicular failure.

[0114] In some embodiments, the symptom is gonadal atrophy.

[0115] In some embodiments, the symptom is cardiac arrhythmia.

[0116] In some embodiments, the symptom is cardiomyopathy.

[0117] In some embodiments, the symptom is dysphagia.

[0118] In some embodiments, the symptom is abdominal pain.

[0119] In some embodiments, the symptom is irritable bowel syndrome (IBS).

[0120] In some embodiments, the symptom is constipation.

[0121] In some embodiments, the symptom is diarrhea.

[0122] In some embodiments, a reduction in reactive oxygen species (ROS) is measured in the subject after administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0123] In some embodiments, a reduction in reactive oxygen species (ROS) is measured in a tissue sample of the subject. In some embodiments, a reduction in reactive oxygen species (ROS) is measured in the blood of the subject.

[0124] Various methods for measuring reactive oxygen species in a subject are known in the art, including but not limited to electron spin resonance (ESR) and spectrophotometry.

[0125] In some embodiments, the reduction in reactive oxygen species (ROS) is measured by electron spin resonance (ESR). In some embodiments, the reduction in reactive oxygen species (ROS) is measured by spectrophotometry.

[0126] In some embodiments, the neurological, muscular, or proliferative disease is Duchenne muscular dystrophy (DMD).

[0127] In some embodiments, treating or preventing Duchenne muscular dystrophy (DMD) comprises treating or preventing one or more symptoms of Duchenne muscular dystrophy (DMD). In some embodiments, the one or more symptoms are selected from the group consisting of muscle weakness, muscle atrophy, difficulty walking, impaired ability to perform routine tasks, falling, learning impairment, calf hypertrophy, fatigue, poor motor skills, difficulty breathing, and loss of lumbar lordosis.

[0128] In some embodiments, the symptom is muscle weakness.

[0129] In some embodiments, the symptom is muscle atrophy.

[0130] In some embodiments, the symptom is difficulty walking. In some embodiments, difficulty walking comprises ataxia, dizziness, poor balance, or lightheadedness.

[0131] In some embodiments, the symptom is impaired ability to perform routine tasks.

[0132] In some embodiments, the symptom is falling. In this case, falling is considered an abnormal frequency of falls that would not occur in otherwise healthy individuals (e.g., as determined by a medical professional such as a physician, a nurse practitioner, a nurse, or a medical technician).

[0133] In some embodiments, the symptom is a learning disorder. In some embodiments, the learning disorder is dyslexia. In some embodiments, the learning disorder is dyscalculia. In some embodiments, the learning disorder is dysgraphia.

[0134] In some embodiments, the symptom is calf hypertrophy.

[0135] In some embodiments, the symptom is fatigue. In some embodiments, the fatigue includes excessive daytime sleepiness.

[0136] In some embodiments, the symptom is poor motor skills.

[0137] In some embodiments, the symptom is difficulty breathing.

[0138] In some embodiments, the symptom is lumbar lordosis.

[0139] In some embodiments, the neurological, muscular, or proliferative disease is spinocerebellar ataxia type 3 (SCA-3).

[0140] In some embodiments, treating or preventing spinocerebellar ataxia type 3 (SCA-3) comprises treating or preventing one or more symptoms of spinocerebellar ataxia type 3 (SCA-3). In some embodiments, the one or more symptoms are selected from the group consisting of involuntary eye movements, poor hand-eye coordination, poor balance and coordination, slurred speech, learning disorder, impaired memory, and uncoordinated walking.

[0141] In some embodiments, the symptom is involuntary eye movements.

[0142] In some embodiments, the symptom is poor hand-eye coordination.

[0143] In some embodiments, the symptom is poor balance and coordination.

[0144] In some embodiments, the symptom is slurred speech.

[0145] In some embodiments, the symptom is a learning disorder.

[0146] In some embodiments, the symptom is impaired memory.

[0147] In some embodiments, the symptom is uncoordinated walking.

[0148] In some embodiments, the neurological, muscular, or proliferative disease is Alzheimer's disease.

[0149] In some embodiments, treating or preventing Alzheimer's disease comprises treating or preventing one or more symptoms of Alzheimer's disease. In some embodiments, the one or more symptoms is selected from the group of: decreased memory, lack of flexibility and hesitation in trying new things, confusion, disorientation, compulsive behavior, repetitive behavior, impulsive behavior, delusions, speech impairment, aphasia, sleep disturbances, frequent and unusual mood swings, depression, anxiety, despondency, difficulty performing spatial tasks, difficulty judging distances, agnosia, difficulty changing locations or walking around without assistance, weight loss, weight gain, and loss of speech abilities.

[0150] In some embodiments, the symptom is decreased memory. In some embodiments, the symptom is decreased short-term memory. In some embodiments, the symptom is decreased long-term memory. In some embodiments, decreased memory comprises misplacing items, forgetting the names of places and objects, repeating things in a regular pattern, and asking the same question multiple times.

[0151] In some embodiments, the symptom is lack of flexibility and hesitation in trying new things.

[0152] In some embodiments, the symptom is confusion.

[0153] In some embodiments, the symptom is disorientation.

[0154] In some embodiments, the symptom is compulsive behavior.

[0155] In some embodiments, the symptom is repetitive behavior.

[0156] In some embodiments, the symptom is impulsive behavior.

[0157] In some embodiments, the symptom is delusions.

[0158] In some embodiments, the symptom is speech impairment.

[0159] In some embodiments, the symptom is aphasia.

[0160] In some embodiments, the symptom is sleep disturbances.

[0161] In some embodiments, the symptom is frequent and unusual mood swings.

[0162] In some embodiments, the symptom is depression.

[0163] In some embodiments, the symptom is anxiety.

[0164] In some embodiments, the symptom is depression.

[0165] In some embodiments, the symptom is difficulty performing spatial tasks.

[0166] In some embodiments, the symptom is difficulty judging distance.

[0167] In some embodiments, the symptom is agnosia.

[0168] In some embodiments, the symptom is difficulty changing position or walking around. In some embodiments, the symptom is without assistance.

[0169] In some embodiments, the symptom is weight loss.

[0170] In some embodiments, the symptom is weight gain.

[0171] In some embodiments, the symptom is loss of speech.

[0172] In some embodiments, the method comprises determining that the subject has an abnormal amount of extracellular amyloid plaques and / or tau protein in the brain prior to administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the method comprises determining that the subject has an abnormal amount of extracellular amyloid plaques in the brain prior to administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the method comprises determining that the subject has an abnormal amount of tau protein in the brain prior to administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the method comprises determining that the extracellular amyloid plaques and / or tau protein in the brain is reduced after administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0173] In some embodiments, the method comprises determining that the subject has an abnormal amount of intracellular neurofibrillary tangles in the brain prior to administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the method comprises determining that the intracellular neurofibrillary tangles in the brain is reduced after administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0174] In some embodiments, determining the amount of extracellular amyloid plaques, tau protein, and / or intracellular neurofibrillary tangles in the brain comprises performing an imaging technique on the brain of the subject. In some embodiments, the imaging technique is magnetic resonance imaging (MRI). In some embodiments, the imaging technique is positron emission tomography (PET).

[0175] In some embodiments, the neurological, muscular, or proliferative disease is Parkinson's disease.

[0176] In some embodiments, treating or preventing Parkinson's disease comprises treating or preventing one or more symptoms of Parkinson's disease. In some embodiments, the one or more symptoms is selected from the group consisting of tremor, arm stiffness, leg stiffness, trunk stiffness, slow movement, poor balance and coordination, and difficulty speaking.

[0177] In some embodiments, the symptom is tremor.

[0178] In some embodiments, the symptom is arm stiffness.

[0179] In some embodiments, the symptom is leg stiffness.

[0180] In some embodiments, the symptom is trunk stiffness.

[0181] In some embodiments, the symptom is slow movement.

[0182] In some embodiments, the symptom is poor balance and coordination.

[0183] In some embodiments, the symptom is difficulty speaking.

[0184] In some embodiments, the neurological, muscular, or proliferative disease is vascular dementia. In some embodiments, treating or preventing vascular dementia comprises treating or preventing one or more symptoms of vascular dementia. In some embodiments, the one or more symptoms is selected from the group consisting of short-term memory loss, getting lost in known environments, decreased ability to pay attention and plan, difficulty managing finances, difficulty following instructions, inability to control bladder, inability to control bowel, delusions, and hallucinations.

[0185] In some embodiments, the symptom is short-term memory loss.

[0186] In some embodiments, the symptom is getting lost in known environments.

[0187] In some embodiments, the symptom is decreased ability to pay attention and plan.

[0188] In some embodiments, the symptom is difficulty managing finances.

[0189] In some embodiments, the symptom is difficulty following instructions.

[0190] In some embodiments, the symptom is an inability to control the bladder.

[0191] In some embodiments, the symptom is an inability to control the bowels.

[0192] In some embodiments, the symptom is delusion.

[0193] In some embodiments, the symptom is hallucination.

[0194] In some embodiments, the neurological, muscular, or proliferative disease is dementia with Lewy bodies (DLB).

[0195] In some embodiments, the neurological, muscular, or proliferative disease is dementia with Lewy bodies (DLB). In some embodiments, treating or preventing dementia with Lewy bodies (DLB) comprises treating or preventing one or more symptoms of dementia with Lewy bodies (DLB). In some embodiments, the one or more symptoms are selected from the group consisting of visual hallucinations, muscle stiffness, slowness of movement, dragging of the feet or trembling of the legs, tremor, inability to balance the body, abnormally high or abnormally low blood pressure, sweating, digestion, confusion, difficulty concentrating, memory loss, and difficulty sleeping.

[0196] In some embodiments, the symptom is visual hallucination.

[0197] In some embodiments, the symptom is muscle stiffness.

[0198] In some embodiments, the symptom is slowness of movement (i.e., slowing of movement).

[0199] In some embodiments, the symptom is dragging of the feet or trembling of the legs.

[0200] In some embodiments, the symptom is tremor.

[0201] In some embodiments, the symptom is inability to balance the body.

[0202] In some embodiments, the symptom is abnormally high or abnormally low blood pressure.

[0203] In some embodiments, the symptom is sweating.

[0204] In some embodiments, the symptom is digestion.

[0205] In some embodiments, the symptom is confusion.

[0206] In some embodiments, the symptom is difficulty concentrating.

[0207] In some embodiments, the symptom is memory loss.

[0208] In some embodiments, the symptom is sleep difficulty.

[0209] In some embodiments, the neurological, muscular, or proliferative disease is Huntington's Disease (HD). In some embodiments, treating or preventing Huntington's Disease (HD) comprises treating or preventing one or more symptoms of Huntington's Disease (HD). In some embodiments, the one or more symptoms is selected from chorea, tremor, muscle rigidity, slow or abnormal eye movements, impaired gait, impaired posture, impaired balance, difficulty swallowing, difficulty speaking, poor organizational skills, repetitive thinking, repetitive behavior, repetitive movements, lack of impulse control, lack of social or behavioral awareness, inability to process thoughts, difficulty learning, difficulty selecting speech, difficulty writing, depression, mania, irritability, social withdrawal, insomnia, fatigue, suicidal thoughts or tendencies, and apathy.

[0210] In some embodiments, the symptom is chorea.

[0211] In some embodiments, the symptom is tremor.

[0212] In some embodiments, the symptom is muscle rigidity.

[0213] In some embodiments, the symptom is slow or abnormal eye movements.

[0214] In some embodiments, the symptom is impaired gait.

[0215] In some embodiments, the symptom is impaired posture.

[0216] In some embodiments, the symptom is impaired balance.

[0217] In some embodiments, the symptom is difficulty swallowing.

[0218] In some embodiments, the symptom is difficulty speaking.

[0219] In some embodiments, the symptom is poor organizational skills.

[0220] In some embodiments, the symptom is repetitive thinking.

[0221] In some embodiments, the symptom is repetitive behavior.

[0222] In some embodiments, the symptom is repetitive movements.

[0223] In some embodiments, the symptom is lack of impulse control.

[0224] In some embodiments, the symptom is lack of social or behavioral awareness.

[0225] In some embodiments, the symptom is inability to process thoughts.

[0226] In some embodiments, the symptom is learning difficulties.

[0227] In some embodiments, the symptom is difficulty selecting speech.

[0228] In some embodiments, the symptom is writing difficulties.

[0229] In some embodiments, the symptom is depression.

[0230] In some embodiments, the symptom is mania.

[0231] In some embodiments, the symptom is irritability.

[0232] In some embodiments, the symptom is social withdrawal.

[0233] In some embodiments, the symptom is insomnia.

[0234] In some embodiments, the symptom is fatigue.

[0235] In some embodiments, the symptom is suicidal thoughts or tendencies.

[0236] In some embodiments, the symptom is apathy.

[0237] In some embodiments, the neurological, muscular, or proliferative disease is amyotrophic lateral sclerosis (ALS). In some embodiments, treating or preventing amyotrophic lateral sclerosis (ALS) comprises treating or preventing one or more symptoms of amyotrophic lateral sclerosis (ALS). In some embodiments, the one or more symptoms are selected from the group of muscle weakness, difficulty walking, difficulty performing normal daily activities, clumsiness, slurred speech, difficulty swallowing, muscle spasms, twitching, poor posture, and falling.

[0238] In some embodiments, the symptom is muscle weakness.

[0239] In some embodiments, the symptom is difficulty walking.

[0240] In some embodiments, the symptom is difficulty performing normal daily activities.

[0241] In some embodiments, the symptom is clumsiness.

[0242] In some embodiments, the symptom is slurred speech.

[0243] In some embodiments, the symptom is difficulty swallowing.

[0244] In some embodiments, the symptom is muscle spasms.

[0245] In some embodiments, the symptom is twitching.

[0246] In some embodiments, the symptom is poor posture.

[0247] In some embodiments, the symptom is falling.

[0248] In some embodiments, the neurological, muscular, or proliferative disease is Lafora disease. In some embodiments, treating or preventing Lafora disease comprises treating or preventing one or more symptoms of Lafora disease. In some embodiments, the one or more symptoms are selected from the group consisting of seizures, myoclonus, dementia, headache, and hallucinations (e.g., visual hallucinations).

[0249] In some embodiments, the symptom is seizures.

[0250] In some embodiments, the symptom is myoclonus.

[0251] In some embodiments, the symptom is dementia.

[0252] In some embodiments, the symptom is headache.

[0253] In some embodiments, the symptom is hallucinations (e.g., visual hallucinations).

[0254] In some embodiments, the neurological, muscular, or proliferative disease is glioblastoma. In some embodiments, treating or preventing glioblastoma comprises treating or preventing one or more symptoms of glioblastoma. In some embodiments, the one or more symptoms are selected from the group consisting of headache, nausea, vomiting, blurred vision, double vision, and seizures.

[0255] In some embodiments, the symptom is headache.

[0256] In some embodiments, the symptom is nausea.

[0257] In some embodiments, the symptom is vomiting.

[0258] In some embodiments, the symptom is blurred vision.

[0259] In some embodiments, the symptom is double vision.

[0260] In some embodiments, the symptom is seizures.

[0261] In some embodiments, the neurological, muscular, or proliferative disease is diffuse intrinsic pontine glioma (DIPG). In some embodiments, treating or preventing diffuse intrinsic pontine glioma (DIPG) comprises treating or preventing one or more symptoms of diffuse intrinsic pontine glioma. In some embodiments, the one or more symptoms are selected from the group consisting of blurred vision, double vision, abnormal eye movements, and headache.

[0262] In some embodiments, the symptom is blurred vision.

[0263] In some embodiments, the symptom is double vision.

[0264] In some embodiments, the symptom is abnormal eye movement.

[0265] In some embodiments, the symptom is headache.

[0266] Some embodiments provide a method of increasing muscle strength in a subject identified or diagnosed as having muscle weakness, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0267] Some embodiments provide a method of preventing or reversing muscle weakness in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0268] Some embodiments provide a method of preventing, slowing or reversing sarcopenia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0269] Some embodiments provide a method of treating or preventing myotonia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0270] In some embodiments, the method comprises determining an improvement in a stiffness visual analog scale (VAS). In some embodiments, the method comprises determining an improvement of at least 1 point (e.g., 1 point, 2 points, 3 points, or 4 points) in a stiffness visual analog scale (VAS). For more information on stiffness VAS, see Hammarén, Elisabet & Kjellby-Wendt, Gunilla & Lindberg, Christopher. (2005). Physiotherapists' quantification of activity impairment and self-assessment of stiffness in patients with myotonic dystrophy. Neuromuscul Disord, 15. 5-6. 353-359, the entire contents of which are incorporated herein by reference.

[0271] In some embodiments, the method comprises determining a reduction in electrical activity in a muscle of the subject. In some embodiments, the method comprises determining a reduction in electrical activity in a muscle of the subject using electromyography (EMG) (e.g., a reduction of at least 1%, a reduction of at least 2%, a reduction of at least 5%, a reduction of at least 7%, a reduction of at least 10%, a reduction of at least 15%, a reduction of at least 20%, a reduction of at least 30%, or a reduction of at least 40%).

[0272] Disclosed herein is a method of preventing muscle cell necrosis in a subject, comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0273] Disclosed herein is a method of improving balance and / or coordination in a subject, comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0274] In some embodiments, the method comprises determining an improvement in the Short Physical Performance Battery (SPPB) after administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the method comprises determining an improvement of at least 1 point (e.g., 2 points, 3 points, 4 points, 5 points, 6 points, 7 points, 8 points, 9 points, 10 points, or 11 points) in the Short Physical Performance Battery (SPPB) after administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, determining an improvement in the Short Physical Performance Battery (SPPB) comprises determining an improvement in the three-stand balance test. In some embodiments, determining an improvement in the Short Physical Performance Battery (SPPB) comprises determining an improvement in the gait speed test. In some embodiments, determining an improvement in the Short Physical Performance Battery (SPPB) comprises determining an improvement in the chair stand test. For more information on SPPB, see Cassidy B, Arena S. The Short Physical Performance Battery as a Predictor of Functional Decline. Home Healthcare Now. 2022 May 1;40(3):168-9, which is incorporated by reference herein in its entirety.

[0275] In some embodiments, the method comprises determining an increase in grip strength by a dynamometer after administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. More information can be found in, for example, Journal of Bodywork and Movement Therapies, 2020, 24(1), 235-243, which is incorporated by reference herein in its entirety.

[0276] In some embodiments, the method comprises increasing handgrip strength by at least 5% (e.g., by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, or by at least 95%) as measured by a dynamometer after administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0277] Some embodiments provide a method of increasing motor ability in a subject, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, increasing motor ability in the subject comprises increasing the number of voluntary muscle contractions per unit of time (e.g., per minute, per 6 minutes, per 10 minutes, per 15 minutes, per 30 minutes, per 45 minutes, per hour, per 3 hours, per 12 hours, per 24 hours, per 2 days, per week, or per month) in the subject relative to before administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, increasing motor ability in the subject comprises increasing the number of voluntary muscle contractions per minute in the subject. In some embodiments, increasing motor ability in the subject comprises increasing the duration of voluntary muscle contractions in the subject relative to before administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, increasing motor ability in the subject comprises decreasing the length of time required for the subject to perform a task or activity requiring movement and / or muscle contractions relative to before administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the method comprises determining improvement in the Activity and Social Participation DM1 Activity Scale after administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. More information about the Activity and Social Participation DM1 Activity Scale can be found, for example, in PLoS One 2015, 10: e0139944, which is incorporated by reference herein in its entirety.

[0278] In some embodiments, the method comprises determining an improvement in the Health-Related Quality of Life InQoL questionnaire after administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, determining an improvement in the Health-Related Quality of Life InQoL questionnaire comprises determining an improvement in the Fatigue subscale. In some embodiments, determining an improvement in the Health-Related Quality of Life InQoL questionnaire comprises determining an improvement in the Activity subscale. In some embodiments, determining an improvement in the Health-Related Quality of Life InQoL questionnaire comprises determining an improvement in the Independence subscale. For more information on the Health-Related Quality of Life InQoL questionnaire, see Taylor VR. Measuring healthy days; population assessment of health-related quality of life. Atlanta: Centers for Disease Control and Prevention; 2000, the entire contents of which are incorporated herein by reference.

[0279] In some embodiments, the method comprises determining an improvement in at least one clinical parameter selected from the group consisting of: myotonia index, muscle function and strength parameters, quality of life parameters, gait parameters, and total mechanical power of gait after administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. For more information on myotonia index, muscle function and strength parameters, quality of life parameters, gait parameters, and total mechanical power of gait, see Brain 2018: 141; 2855-2865, the entire contents of which are incorporated herein by reference.

[0280] In some embodiments, the method comprises determining an increase in the distance walked during a 6-minute walk test (6MWT) in the subject after administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the method comprises determining an increase of at least 5% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%) in the distance walked during a 6-minute walk test (6MWT) in the subject after administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. For more information on the 6-minute walk test (6MWT) can be found in, for example, Endocrinology 2005; 146: 1328-1337, the entire contents of which are incorporated herein by reference.

[0281] In some embodiments, the method comprises determining a reduction in the time required for the subject to complete a 10-meter walk test after administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the method comprises determining a reduction of at least 5% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%) in the time required for the subject to complete a 10-meter walk test after administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. More information on the 10-meter walk test can be found, for example, in Journal of Neurologic Physical Therapy, 2018; 42(2): 174-220, the entire contents of which are incorporated herein by reference. In some embodiments, the method comprises determining an improvement in at least one of muscle stiffness index, muscle function and strength, quality of life, and total mechanical force after administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the method comprises determining a reduction of at least 5% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%) in the time required for the subject to complete a 10-meter walk test after administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. More information on the 10-meter walk test can be found, for example, in Journal of Neurologic Physical Therapy, 2018; 42(2): 174-220, the entire contents of which are incorporated herein by reference.

[0282] In some embodiments, the method comprises determining a reduction in the time required for the subject to complete a 100-meter walk test after administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the method comprises determining a reduction of at least 5% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%) in the time required for the subject to complete a 100-meter walk test after administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. For more information on the 100-meter walk test, see Alfano, Lindsay & Miller, Natalie & Berry, Katherine & Yin, Han & Rolf, Kimberly & Flanigan, Kevin & Mendell, Jerry & Lowes, Linda. (2017). The 100-meter timed test: Normative data in healthy males and comparative pilot outcome data for use in Duchenne muscular dystrophy clinical trials. Neuromuscular Disorders. 27. 10.1016 / j.nmd.2017.02.007, which is incorporated by reference herein in its entirety.

[0283] In some embodiments, the method comprises determining an increase in muscle mass of the subject using magnetic resonance imaging (MRI) after administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the method comprises determining an increase of at least 1% (e.g., an increase of at least 2%, an increase of at least 3%, an increase of at least 4%, an increase of at least 5%, an increase of at least 7%, an increase of at least 9%, an increase of at least 11%, an increase of at least 13%, an increase of at least 15%, an increase of at least 20%, an increase of at least 25%, an increase of at least 30%, an increase of at least 35%, or an increase of at least 40%) in muscle mass of the subject using magnetic resonance imaging (MRI) after administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the muscle mass is skeletal muscle mass. In some embodiments, the increase in muscle mass is an increase in whole body muscle mass. More information can be found, for example, in Eur. J. Neurol. 2022 Mar; 29(3):843-854 or Radiography, 2015, 21(1), e35-e39, each of which is incorporated by reference in its entirety.

[0284] In some embodiments, the method comprises determining an improvement in the Muscle Impairment Rating Scale (MIRS) after administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the improvement in the MIRS comprises a scale improvement of at least 1 point (e.g., 1 point, 2 points, 3 points, or 4 points). For more information on the MIRS, see, for example, Neurology, 2001 Feb 13; 56(3):336-40, which is incorporated by reference in its entirety.

[0285] In some embodiments, the method comprises determining a reduction in the time measured by a Timed Up and Go (TUG) test performed by the subject after administration of the metformin or pharmaceutically acceptable salt thereof and the rapamycin or pharmaceutically acceptable salt thereof. In some embodiments, the method comprises determining at least a 2% reduction (e.g., at least a 3% reduction, at least a 4% reduction, at least a 5% reduction, at least a 7% reduction, at least a 9% reduction, at least an 11% reduction, at least a 13% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 50% reduction, at least a 60% reduction, or at least a 70% reduction) in the time measured by a Timed Up and Go (TUG) test performed by the subject after administration of the metformin or pharmaceutically acceptable salt thereof and the rapamycin or pharmaceutically acceptable salt thereof. Further information regarding the Timed Up and Go (TUG) test can be found, for example, in Osteosarcopenia, 2022, pp. 181-204, the entire contents of which are incorporated herein by reference. Other tests that can be used to assess the subject’s mobility include, but are not limited to: the lift a weight test (see, e.g., Reuben DB, Siu AL. “Objective measures of physical performance of ambulatory older patients.” J Am Geriatr Soc. 1990; 38(11): 1113- 1115), the 6-minute walk test (see, e.g., ATS Statement: Guidelines for the Six-Minute Walk Test. Am J Respir Crit Care Med. 2002; 166(3): 111-117), the 4-stair climb test (see, e.g., Jones CJ, Raisz LG. “Assessment of muscle strength as a functional outcome measure in sarcopenia.” J Gerontol A Biol Sci Med Sci. 1999; 54(10): M761-765), and the chair stand test (see, e.g., Jones CJ, Raisz LG. “Assessment of muscle strength as a functional outcome measure in sarcopenia.” J Gerontol A Biol Sci Med Sci. 1999; 54(10): M761-765).An objective measure of physical function of elderly outpatients. The Physical Performance Test”) J Am Geriatr Soc, 1990, vol. 38 (pg. 1105-12), incorporated by reference in its entirety, the Half Turn Test (see, e.g., Berg K, Wood-Dauphinee S, Williams JI, et al. Measuring balance in the elderly: preliminary development of an instrument. Physiother Can, 1989, vol. 41 (pg. 304-11), incorporated by reference in its entirety), the Alternate Step Test (see, e.g., Anne Tiedemann, Hiroyuki Shimada, Catherine Sherrington, Susan Murray, Stephen Lord, Age and Ageing, vol. 37, no. 4, July 2008, pp. 430-435, incorporated by reference in its entirety), and the Stair Up and Down Test (see, e.g., Anne Tiedemann, Hiroyuki Shimada, Catherine Sherrington, Susan Murray, Stephen Lord, Age and Ageing, vol. 37, no. 4, July 2008, pp. 430-435, incorporated by reference in its entirety).

[0286] Some embodiments provide a method of reducing or preventing genomic instability, telomere attrition, epigenetic alterations (e.g., DNA methylation, histone modification, or regulation of gene activity by RNA and / or proteins), protein homeostasis dysregulation or collapse, nutrient-sensing dysregulation, mitochondrial dysfunction, cellular senescence, stem cell depletion, and / or altered intercellular communication in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0287] Some embodiments provide a method of preventing genomic instability, telomere attrition, epigenetic alterations (e.g., DNA methylation, histone modification, or regulation of gene activity by RNA and / or proteins), dysregulation or deregulation of protein homeostasis, dysregulation of nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell depletion, and / or altered intercellular communication in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0288] In some embodiments, the method comprises reducing or preventing cellular senescence. In some embodiments, the method comprises preventing cellular senescence.

[0289] In some embodiments, the method comprises reducing or preventing genomic instability.

[0290] In some embodiments, the method comprises reducing or preventing telomere attrition.

[0291] In some embodiments, the method comprises reducing or preventing epigenetic alterations (e.g., DNA methylation, histone modification, or regulation of gene activity by RNA and / or proteins).

[0292] In some embodiments, the method comprises reducing or preventing dysregulation or deregulation of protein homeostasis.

[0293] In some embodiments, the method comprises reducing or preventing dysregulation of nutrient sensing.

[0294] In some embodiments, the method comprises reducing or preventing mitochondrial dysfunction.

[0295] In some embodiments, the method comprises reducing or preventing stem cell depletion.

[0296] In some embodiments, the method comprises reducing or preventing altered intercellular communication.

[0297] Some embodiments provide a method of promoting autophagy in a subject, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0298] Some embodiments provide a method of darkening hair color in a subject, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof, wherein the subject has white or gray hair prior to administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0299] In some embodiments, darkening comprises changing the color of the hair to a brown color. In some embodiments, darkening comprises changing the color of the hair to a black color.

[0300] Some embodiments provide a method of improving memory in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the improvement in memory of the subject is determined by a number span test, a number-letter sequencing test, a California Verbal Learning Test, a Rey Auditory Verbal Learning Test, or a Wechsler Memory Scale. For more information on the number span test, the number-letter sequencing test, the California Verbal Learning Test, the Rey Auditory Verbal Learning Test, and the Wechsler Memory Scale, see Am Fam Physician. 2019, 99(2), 101-108.

[0301] In some embodiments, the method comprises measuring an improvement in a number span test in the subject after administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0302] In some embodiments, the method comprises measuring an improvement in a number-letter sequencing test in the subject after administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0303] In some embodiments, the method comprises measuring an improvement in a California Verbal Learning Test in the subject after administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0304] In some embodiments, the method comprises measuring an improvement in a Rey Auditory Verbal Learning Test in the subject after administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0305] In some embodiments, the method comprises measuring an improvement in a Wechsler Memory Scale in the subject after administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0306] Some embodiments provide a method of preventing a decline in memory in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0307] Some embodiments provide a method of treating or preventing a neurologic cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof; wherein the cancer is glioblastoma or diffuse intrinsic pontine glioma (DIPG).

[0308] In some embodiments, the neurologic cancer is glioblastoma. In some embodiments, the neurologic cancer is diffuse intrinsic pontine glioma (DIPG).

[0309] Some embodiments provide a method of inhibiting cell proliferation, the method comprising administering to a subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a brain cell or a spinal cord cell. In some embodiments, the brain cell or spinal cord cell is a glial cell, an astrocyte, an oligodendrocyte progenitor cell, a neuron, or a neural stem cell. In some embodiments, the inhibition is performed in vivo. In some embodiments, the inhibition is performed in vitro.

[0310] Some embodiments provide a method of treating a neurologic cancer and / or inhibiting metastasis associated with a neurologic cancer, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof; wherein the cancer is glioblastoma or diffuse intrinsic pontine glioma (DIPG).

[0311] Some embodiments provide a method of providing supportive care to a cancer patient, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof; wherein the cancer is glioblastoma or diffuse intrinsic pontine glioma (DIPG).

[0312] Some embodiments provide a method for reversing or preventing acquired resistance to an anti-cancer drug in a subject having a neurologic cancer, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof; wherein the cancer is glioblastoma or diffuse intrinsic pontine glioma (DIPG).

[0313] Some embodiments provide a method of treating a subject having a neurologic cancer and an increased likelihood of developing resistance to an anti-cancer drug, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof; wherein the cancer is glioblastoma or diffuse intrinsic pontine glioma (DIPG).

[0314] In some embodiments, the subject has been identified or diagnosed as having a neurologic cancer selected from the group consisting of glioblastoma and diffuse intrinsic pontine glioma (DIPG).

[0315] In the field of medical oncology, it is common practice to treat each patient with cancer using a combination of different forms of therapy. In medical oncology, the other component of such combination therapy or regimen, in addition to metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof, can be, for example, surgery, radiotherapy and chemotherapeutic drugs such as other kinase inhibitors, signal transduction inhibitors and / or monoclonal antibodies. For example, the surgery can be open surgery or minimally invasive surgery. The combination of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof can also be used as an adjuvant to cancer treatment, i.e. they can be used in conjunction with one or more other therapies or therapeutic agents, for example chemotherapeutic agents that act via the same or different mechanism of action.

[0316] In some embodiments of any of the methods described herein, metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof are administered in combination with a therapeutically effective amount of at least one additional therapeutic agent selected from one or more additional therapies or therapeutic agents (e.g., chemotherapeutic agents). In some embodiments, the at least one additional therapeutic agent is selected from the group of alkylating agents (e.g., altretamine, busulfan, carboplatin, carmustine, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, melphalan, temozolomide, trabectedin), antimetabolites (e.g., 5-fluorouracil, 6-mercaptopurine, azacitidine, capecitabine, cladribine, cytarabine, floxuridine, fludarabine, gemcitabine, methotrexate, pemetrexed, pentostatin, pralatrexate, or a combination of trifluridine and tipiracil), alkaloids (e.g., vincristine, vinblastine, vinorelbine, paclitaxel, docetaxel, etoposide, teniposide, irinotecan, topotecan), and antitumor antibiotics (e.g., daunorubicin, doxorubicin, doxorubicin liposomal, epirubicin, idarubicin, valrubicin). Some therapeutic agents that can be used to treat glioblastoma include, but are not limited to, cyclophosphamide, dinutuximab (Unituxin®), daratumumab, doxorubicin, naxitamumab, Unituxin (dinutuximab), vincristine sulfate, busulfan, and melphalan. Some therapies that can be used to treat diffuse intrinsic pontic glioma (DIPG) include, but are not limited to, the following: fractionated radiation therapy with concurrent administration of anti-inflammatory steroids (e.g., dexamethasone), anti-GD2 CAR T cells, and anti-EGFR drugs (e.g., nimotuzumab, gefitinib, and erlotinib). In some embodiments, the at least one additional therapeutic agent is selected from the group of alkylating agents (e.g., altretamine, busulfan, carboplatin, carmustine, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, melphalan, temozolomide, trabectedin), antimetabolites (e.g., 5-fluorouracil, 6-mercaptopurine, azacitidine, capecitabine, cladribine, cytarabine, floxuridine, fludarabine, gemcitabine, methotrexate, pemetrexed, pentostatin, pralatrexate, or a combination of trifluridine and tipiracil), alkaloids (e.g., vincristine, vinblastine, vinorelbine, paclitaxel, docetaxel, etoposide, teniposide, irinotecan, topotecan), and antitumor antibiotics (e.g., daunorubicin, doxorubicin, doxorubicin liposomal, epirubicin, idarubicin, valrubicin), cyclophosphamide, dinutuximab (Unituxin®), daratumumab, doxorubicin, naxitamumab, Unituxin (dinutuximab), vincristine sulfate, busulfan, melphalan, anti-GD2 CAR T cells, and anti-EGFR drugs (e.g., nimotuzumab, gefitinib, and erlotinib).

[0317] Accordingly, also provided herein is a method of treating a neurologic cancer, comprising administering to a patient in need thereof a pharmaceutical combination for treating a cancer, the pharmaceutical combination comprising (a) metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof, (b) an additional therapeutic agent, and (c) optionally at least one pharmaceutically acceptable carrier, for simultaneous, separate or sequential use in treating a cancer, wherein the amounts of the compounds of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof and the additional therapeutic agent together are effective in treating the cancer; wherein the cancer is a glioblastoma or a diffuse intrinsic pontine glioma (DIPG).

[0318] Some embodiments provide a method for inhibiting, preventing, aiding in the prevention of, or reducing the metastatic symptoms of a neurologic cancer in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof; wherein the cancer is a glioblastoma or a diffuse intrinsic pontine glioma (DIPG). The term "metastasis" is a term known in the art and refers to the formation of additional tumors (e.g., solid tumors) at sites remote from the primary tumor of the subject or patient, wherein the additional tumors comprise cancer cells that are the same or similar to the cancer cells of the primary tumor.

[0319] In some embodiments, an increase in AMPK activation, inhibition of mTORCl, inhibition of S6 kinase, or any combination thereof, is measured in the subject after administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, an increase in AMPK activation, inhibition of mTORCl, and inhibition of S6 kinase are measured in the subject after administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0320] In some embodiments, p70 S6 kinase is inhibited or eliminated in the subject after administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0321] In some embodiments, Akt is activated in the subject after administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0322] In some embodiments, the metformin or a pharmaceutically acceptable salt thereof is metformin hydrochloride.

[0323] In some embodiments, the rapamycin or a pharmaceutically acceptable salt thereof is rapamycin.

[0324] In some embodiments, the subject has not been previously administered cyclosporine, tacrolimus, and mycophenolate mofetil within 1 month of administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0325] In some embodiments, the subject has not been identified or diagnosed as having a disease associated with the kidney.

[0326] In some embodiments, the subject has not been identified or diagnosed as having a disease associated with the liver.

[0327] In some embodiments, the subject has not been identified or diagnosed as having a disease associated with the heart.

[0328] In some embodiments, the subject has not been identified or diagnosed as having diabetes.

[0329] In some embodiments, the subject has not been identified or diagnosed as having abnormal endocrine function.

[0330] In some embodiments, the subject has not been previously administered a therapeutic agent that modulates the insulin transduction pathway within 1 year of administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0331] The amount administered depends on the formulation of the compound, the route of administration, the disease, the subject, and the result sought. It is generally determined empirically and can vary from subject to subject. Generally, the amount of active compound in unit dosage formulations will range from 0.01 mg to 1000 mg, preferably from 0.01 mg to 100 mg, and more preferably from 0.01 mg to 10 mg, according to the particular application.

[0332] In some embodiments, metformin or a pharmaceutically acceptable salt thereof is administered monthly, every 3 weeks, every 2 weeks, every 10 days, every 9 days, every 8 days, every 7 days, every 6 days, every 5 days, every 4 days, every 3 days, every 2 days, every 1 day (i.e., daily), 3 times a week, 2 times a week, twice a day, or three times a day. In some embodiments, metformin or a pharmaceutically acceptable salt thereof is administered daily. In some embodiments, an initial dose of metformin (i.e., a “loading dose”) is administered to the subject as the first dose of treatment. In some embodiments, the initial dose of metformin is higher than the dose subsequently administered to the subject.

[0333] In some embodiments, the dosage of metformin, or a pharmaceutically acceptable salt thereof, is about 100 mg to about 5000 mg (e.g., about 100 mg to about 500 mg, about 100 mg to about 800 mg, about 100 mg to about 1000 mg, about 500 mg to about 1000 ng, about 1000 mg to about 1500 mg, about 1500 mg to about 2250 mg, about 1500 mg to about 2000 mg, about 2000 mg to about 2500 mg, about 2250 mg to about 3000 mg, about 2500 mg to about 3000 mg, about 3000 mg to about 3500 mg, about 3500 mg to about 4000 mg, about 4000 mg to about 5000 mg, about 500 mg to about 3000 mg, about 500 to about 1750 mg, about 500 mg to about 1125 mg, about 1125 mg to about 2250 mg, about 1500 mg to about 3000 mg, about 500 mg, about 650 mg, about 750 mg, about 850 mg, about 1000 mg, about 1750 mg, about 2000 mg, or about 3000 mg) based on the free base of metformin. In some embodiments, the dosage of metformin, or a pharmaceutically acceptable salt thereof, is about 500 to about 3000 mg based on the free base of metformin. In some embodiments, the dosage of metformin, or a pharmaceutically acceptable salt thereof, is about 1500 to about 3000 mg based on the free base of metformin. In some embodiments, the dosage of metformin, or a pharmaceutically acceptable salt thereof, is about 500 to about 1750 mg based on the free base of metformin.

[0334] In some embodiments, the method comprises treating or preventing type 1 myotonic dystrophy (DM1); and the dosage of metformin, or a pharmaceutically acceptable salt thereof, is about 1500 mg to about 3000 mg per day based on the free base of metformin.

[0335] In some embodiments, the method comprises treating or preventing Duchenne muscular dystrophy; and the dosage of metformin, or a pharmaceutically acceptable salt thereof, is about 1500 mg to about 3000 mg per day based on the free base of metformin.

[0336] In some embodiments, the method comprises treating or preventing type 3 spinocerebellar ataxia (SCA-3); and the dosage of metformin, or a pharmaceutically acceptable salt thereof, is about 1500 mg to about 3000 mg per day based on the free base of metformin.

[0337] In some embodiments, the method comprises treating or preventing Alzheimer’s disease; and the dosage of metformin, or a pharmaceutically acceptable salt thereof, is about 500 mg to about 1750 mg per day based on the free base of metformin.

[0338] In some embodiments, the method comprises promoting autophagy; and the free base of metformin, metformin, or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 500 mg to about 1750 mg.

[0339] In some embodiments, the method comprises reducing or preventing genomic instability, telomere attrition, epigenetic alterations (e.g., DNA methylation, histone modification, or regulation of gene activity by RNA and / or proteins), dysregulation or deregulation of protein homeostasis, dysregulation of nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and / or alterations in intercellular communication; and the free base of metformin, metformin, or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 500 mg to about 1750 mg.

[0340] In some embodiments, the rapamycin or pharmaceutically acceptable salt thereof is administered monthly, every 3 weeks, every 2 weeks, every 10 days, every 9 days, every 8 days, every 7 days, every 6 days, every 5 days, every 4 days, every 3 days, every 2 days, every 1 day (i.e., daily), 3 times a week, 2 times a week, twice a day, or three times a day. In some embodiments, the rapamycin or pharmaceutically acceptable salt thereof is administered daily. In some embodiments, the rapamycin or pharmaceutically acceptable salt thereof is administered once a week. In some embodiments, the rapamycin or pharmaceutically acceptable salt thereof is administered daily in addition to another dose administered weekly. In some embodiments, an initial dose of rapamycin (i.e., a“loading dose”) is administered to the subject as the first dose of treatment. In some embodiments, the initial dose of rapamycin is higher than the doses subsequently administered to the subject. In some embodiments, the dose of rapamycin or pharmaceutically acceptable salt thereof is, based on the free base of rapamycin, about 0.1 mg to about 25 mg (e.g., about 0.1 mg to about 20 mg, about 0.1 mg to about 18 mg, about 0.1 mg to about 15 mg, about 0.1 mg to about 13 mg, about 0.1 mg to about 10 mg, about 0.1 mg to about 7 mg, about 0.1 mg to about 5 mg, about 0.1 mg to about 3 mg, about 0.1 mg to about 2 mg, about 0.1 mg to about 1 mg, about 0.5 mg to about 20 mg, about 0.5 mg to about 18 mg, about 0.5 mg to about 15 mg, about 0.5 mg to about 13 mg, about 0.5 mg to about 10 mg, about 0.5 mg to about 7 mg, about 0.5 mg to about 5 mg, about 0.5 mg to about 3 mg, about 0.5 mg to about 2 mg, about 0.5 mg to about 1 mg, about 1 mg to about 20 mg, about 1 mg to about 18 mg, about 1 mg to about 15 mg, about 1 mg to about 13 mg, about 1 mg to about 10 mg, about 1 mg to about 7 mg, about 1 mg to about 5 mg, about 1 mg to about 3 mg, about 1 mg to about 2 mg, about 2 mg to about 12 mg, about 4 mg to about 10 mg, about 4 mg to about 8 mg, about 10 mg to about 30 mg, about 13 mg to about 17 mg, about 2 mg to about 4 mg, about 2 mg to about 10 mg, about 1 mg to about 3 mg, about 3 mg to about 7 mg, about 4 mg to about 6 mg, about 15 mg to about 25 mg, about 18 mg to about 22 mg, about 10 mg to about 14 mg, about 0.5 mg, about 1 mg, about 2 mg, about 2.5 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, or about 15 mg).In some embodiments, the dosage of rapamycin, or a pharmaceutically acceptable salt thereof, is about 0.5 mg to about 1 mg, on a free base basis of rapamycin. In some embodiments, the dosage is about 50% of the dosage given to a subject who is 12 years of age or older. In some embodiments, the dosage of rapamycin, or a pharmaceutically acceptable salt thereof, is about 0.7 mg, on a free base basis of rapamycin. In some embodiments, the dosage of rapamycin, or a pharmaceutically acceptable salt thereof, is about 1 mg to about 10 mg, on a free base basis of rapamycin. In some embodiments, the dosage of rapamycin, or a pharmaceutically acceptable salt thereof, is about 2 mg to about 10 mg, on a free base basis of rapamycin. In some embodiments, the dosage of rapamycin, or a pharmaceutically acceptable salt thereof, is about 5 mg, on a free base basis of rapamycin.

[0341] In some embodiments, the rapamycin, or a pharmaceutically acceptable salt thereof, is administered in a daily dosage of about 0.1 mg to about 2 mg, on a free base basis of rapamycin; and an additional weekly dosage of about 1 mg to about 10 mg, on a free base basis of rapamycin.

[0342] In some embodiments, the rapamycin, or a pharmaceutically acceptable salt thereof, is administered in a daily dosage of about 0.5 mg to about 1 mg, on a free base basis of rapamycin; and an additional weekly dosage of about 2 mg to about 10 mg, on a free base basis of rapamycin.

[0343] In some embodiments, the rapamycin, or a pharmaceutically acceptable salt thereof, is administered in a daily dosage of about 0.7 mg, on a free base basis of rapamycin; and an additional weekly dosage of about 5 mg, on a free base basis of rapamycin.

[0344] In some embodiments, the method comprises treating or preventing myotonic dystrophy type 1 (DM1); and the rapamycin, or a pharmaceutically acceptable salt thereof, is administered in a daily dosage of about 0.5 mg to about 1 mg, on a free base basis of rapamycin; and an additional weekly dosage of about 2 mg to about 10 mg, on a free base basis of rapamycin.

[0345] In some embodiments, the method comprises treating or preventing myotonic dystrophy type 1 (DM1); and the rapamycin, or a pharmaceutically acceptable salt thereof, is administered in a daily dosage of about 0.5 mg to about 1 mg, on a free base basis of rapamycin.

[0346] In some embodiments, the method comprises treating or preventing myotonic dystrophy type 1 (DM1); and the rapamycin, or a pharmaceutically acceptable salt thereof, is administered in a daily dosage of about 2 mg to about 10 mg, on a free base basis of rapamycin.

[0347] In some embodiments, the method comprises treating or preventing Duchenne Muscular Dystrophy; and rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin, is administered at a daily dose of about 0.5 mg to about 1 mg; and an additional weekly dose of about 2 mg to about 10 mg based on the free base of rapamycin.

[0348] In some embodiments, the method comprises treating or preventing Duchenne Muscular Dystrophy; and rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin, is administered at a daily dose of about 0.5 mg to about 1 mg.

[0349] In some embodiments, the method comprises treating or preventing Duchenne Muscular Dystrophy; and rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin, is administered at a weekly dose of about 2 mg to about 10 mg.

[0350] In some embodiments, the method comprises treating or preventing Spinocerebellar ataxia type 3 (SCA-3); and rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin, is administered at a daily dose of about 0.5 mg to about 1 mg; and an additional weekly dose of about 2 mg to about 10 mg based on the free base of rapamycin.

[0351] In some embodiments, the method comprises treating or preventing Spinocerebellar ataxia type 3 (SCA-3); and rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin, is administered at a daily dose of about 0.5 mg to about 1 mg.

[0352] In some embodiments, the method comprises treating or preventing Spinocerebellar ataxia type 3 (SCA-3); and rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin, is administered at a weekly dose of about 2 mg to about 10 mg.

[0353] In some embodiments, the method comprises treating or preventing Alzheimer's Disease; and rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin, is administered at a daily dose of about 0.5 mg to about 1 mg; and an additional weekly dose of about 1 mg to about 10 mg based on the free base of rapamycin.

[0354] In some embodiments, the method comprises treating or preventing Alzheimer's Disease; and rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin, is administered at a daily dose of about 0.5 mg to about 1 mg.

[0355] In some embodiments, the method comprises treating or preventing Alzheimer's disease; and the rapamycin-based free base, rapamycin, or pharmaceutically acceptable salt thereof is administered at a weekly dose of about 1 mg to about 10 mg.

[0356] In some embodiments, the method comprises promoting autophagy; and the rapamycin or pharmaceutically acceptable salt thereof, rapamycin-based free base, is administered at a daily dose of about 0.5 mg to about 1 mg; and an additional weekly dose of about 1 mg to about 10 mg of the rapamycin-based free base.

[0357] In some embodiments, the method comprises promoting autophagy; and the rapamycin or pharmaceutically acceptable salt thereof, rapamycin-based free base, is administered at a daily dose of about 0.5 mg to about 1 mg.

[0358] In some embodiments, the method comprises promoting autophagy; and the rapamycin or pharmaceutically acceptable salt thereof, rapamycin-based free base, is administered at a weekly dose of about 0.5 mg to about 1 mg.

[0359] In some embodiments, the method comprises reducing or preventing genomic instability, telomere attrition, epigenetic alterations (e.g., DNA methylation, histone modification, or regulation of gene activity by RNA and / or proteins), dysregulation or deregulation of protein homeostasis, dysregulation of nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell depletion, and / or alterations in intercellular communication; the rapamycin or pharmaceutically acceptable salt thereof, rapamycin-based free base, is administered at a daily dose of about 0.5 mg to about 1 mg; and an additional weekly dose of about 1 mg to about 10 mg of the rapamycin-based free base.

[0360] In some embodiments, the method comprises reducing or preventing genomic instability, telomere attrition, epigenetic alterations (e.g., DNA methylation, histone modification, or regulation of gene activity by RNA and / or proteins), dysregulation or deregulation of protein homeostasis, dysregulation of nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell depletion, and / or alterations in intercellular communication; and the rapamycin or pharmaceutically acceptable salt thereof, rapamycin-based free base, is administered at a daily dose of about 0.5 mg to about 1 mg.

[0361] In some embodiments, the method comprises reducing or preventing genomic instability, telomere attrition, epigenetic alterations (e.g., DNA methylation, histone modification, or regulation of gene activity by RNA and / or proteins), dysregulation or deregulation of protein homeostasis, dysregulation of nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell depletion, and / or alterations in intercellular communication; and the rapamycin-based free base, rapamycin, or a pharmaceutically acceptable salt thereof is administered at a weekly dose of about 1 mg to about 10 mg.

[0362] In some embodiments, the metformin or a pharmaceutically acceptable salt thereof is administered at a daily dose of about 1500 mg to about 3000 mg; and the rapamycin-based free base, rapamycin, or a pharmaceutically acceptable salt thereof is administered at a daily dose of about 0.5 mg to about 1 mg.

[0363] In some embodiments, the metformin or a pharmaceutically acceptable salt thereof is administered at a daily dose of about 1500 mg to about 3000 mg; and the rapamycin-based free base, rapamycin, or a pharmaceutically acceptable salt thereof is administered at a weekly dose of about 2 mg to about 10 mg.

[0364] In some embodiments, the metformin or a pharmaceutically acceptable salt thereof is administered at a daily dose of about 1500 mg to about 3000 mg; and the rapamycin or a pharmaceutically acceptable salt thereof, based on the rapamycin-based free base, is administered at a daily dose of about 0.5 mg to about 1 mg; and an additional weekly dose of about 2 mg to about 10 mg, based on the rapamycin-based free base.

[0365] In some embodiments, the metformin or a pharmaceutically acceptable salt thereof is administered at a daily dose of about 1500 mg to about 3000 mg; and the rapamycin or a pharmaceutically acceptable salt thereof, based on the rapamycin-based free base, is administered at a daily dose of about 0.5 mg to about 1 mg; and an additional weekly dose of about 2 mg to about 10 mg, based on the rapamycin-based free base; wherein the daily dose of metformin or a pharmaceutically acceptable salt thereof and the daily dose of rapamycin or a pharmaceutically acceptable salt thereof are administered simultaneously (e.g., as a fixed dosage form).

[0366] In some embodiments, the metformin or a pharmaceutically acceptable salt thereof is administered at a daily dose of about 500 mg to about 1750 mg; and the rapamycin or a pharmaceutically acceptable salt thereof, based on the rapamycin-based free base, is administered at a daily dose of about 0.5 mg to about 1 mg; and an additional weekly dose of about 1 mg to about 10 mg, based on the rapamycin-based free base.

[0367] In some embodiments, metformin or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 500 mg to about 1750 mg; and rapamycin or a pharmaceutically acceptable salt thereof, on a free base basis, in a daily dose of about 0.5 mg to about 1 mg; and an additional weekly dose of about 1 mg to about 10 mg, on a free base basis of rapamycin; wherein the daily dose of metformin or a pharmaceutically acceptable salt thereof and the daily dose of rapamycin or a pharmaceutically acceptable salt thereof are administered concurrently (e.g., as a fixed dosage form).

[0368] In some embodiments, when metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof are administered on the same day, they are administered concurrently (e.g., as a fixed dosage form (e.g., an oral fixed dosage form)). In some embodiments, when metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof are administered on the same day, they are administered together as a fixed dosage form. In some embodiments, when metformin or a pharmaceutically acceptable salt thereof is administered daily and rapamycin or a pharmaceutically acceptable salt thereof is administered weekly, the weekly dose of rapamycin or a pharmaceutically acceptable salt thereof is administered in a fixed dosage form with the daily dose of metformin or a pharmaceutically acceptable salt thereof, the daily dose of metformin or a pharmaceutically acceptable salt thereof and the weekly dose of rapamycin or a pharmaceutically acceptable salt thereof are administered on the same day.

[0369] In some embodiments, the method comprises treating or preventing type 1 myotonic dystrophy (DM1); metformin or a pharmaceutically acceptable salt thereof, on a free base basis of metformin, is administered in a daily dose of about 1500 mg to about 3000 mg; and rapamycin or a pharmaceutically acceptable salt thereof, on a free base basis of rapamycin, in a daily dose of about 0.5 mg to about 1 mg (e.g., in a fixed dosage form with metformin or a pharmaceutically acceptable salt thereof); and an additional weekly dose of about 2 mg to about 10 mg, on a free base basis of rapamycin.

[0370] In some embodiments, the method comprises treating or preventing type 1 myotonic dystrophy (DM1); metformin or a pharmaceutically acceptable salt thereof, on a free base basis of metformin, is administered in a daily dose of about 1500 mg to about 3000 mg; and rapamycin or a pharmaceutically acceptable salt thereof, on a free base basis of rapamycin, in a daily dose of about 0.5 mg to about 1 mg (e.g., in a fixed dosage form with metformin or a pharmaceutically acceptable salt thereof).

[0371] In some embodiments, the method comprises treating or preventing Duchenne Muscular Dystrophy; metformin or a pharmaceutically acceptable salt thereof, based on the free base of metformin, is administered at a daily dose of about 1500 mg to about 3000 mg; and rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin, is administered at a daily dose of about 0.5 mg to about 1 mg (e.g., in a fixed-dose formulation with metformin or a pharmaceutically acceptable salt thereof).

[0372] In some embodiments, the method comprises treating or preventing Duchenne Muscular Dystrophy; metformin or a pharmaceutically acceptable salt thereof, based on the free base of metformin, is administered at a daily dose of about 1500 mg to about 3000 mg; and rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin, is administered at a daily dose of about 0.5 mg to about 1 mg (e.g., in a fixed-dose formulation with metformin or a pharmaceutically acceptable salt thereof).

[0373] In some embodiments, the method comprises treating or preventing Duchenne Muscular Dystrophy; metformin or a pharmaceutically acceptable salt thereof, based on the free base of metformin, is administered at a daily dose of about 1500 mg to about 3000 mg; and rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin, is administered at a daily dose of about 0.5 mg to about 1 mg (e.g., in a fixed-dose formulation with metformin or a pharmaceutically acceptable salt thereof).

[0374] In some embodiments, the method comprises treating or preventing Duchenne Muscular Dystrophy; metformin or a pharmaceutically acceptable salt thereof, based on the free base of metformin, is administered at a daily dose of about 1500 mg to about 3000 mg; and rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin, is administered at a daily dose of about 0.5 mg to about 1 mg (e.g., in a fixed-dose formulation with metformin or a pharmaceutically acceptable salt thereof).

[0375] In some embodiments, the method comprises treating or preventing Spinocerebellar Ataxia Type 3 (SCA-3); metformin or a pharmaceutically acceptable salt thereof, based on the free base of metformin, is administered at a daily dose of about 1500 mg to about 3000 mg; and rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin, is administered at a daily dose of about 0.5 mg to about 1 mg (e.g., in a fixed-dose formulation with metformin or a pharmaceutically acceptable salt thereof); and an additional weekly dose of about 2 mg to about 10 mg, based on the free base of rapamycin.

[0376] In some embodiments, the method comprises treating or preventing Spinocerebellar ataxia type 3 (SCA-3); metformin or a pharmaceutically acceptable salt thereof, based on the free base of metformin, is administered at a daily dose of about 1500 mg to about 3000 mg; and rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin, is administered at a daily dose of about 0.5 mg to about 1 mg (e.g., in a fixed-dose form with metformin or a pharmaceutically acceptable salt thereof).

[0377] In some embodiments, the method comprises treating or preventing Spinocerebellar ataxia type 3 (SCA-3); metformin or a pharmaceutically acceptable salt thereof, based on the free base of metformin, is administered at a daily dose of about 1500 mg to about 3000 mg; and rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin, is administered at a weekly dose of about 2 mg to about 10 mg (e.g., in a fixed-dose form with metformin or a pharmaceutically acceptable salt thereof).

[0378] In some embodiments, the method comprises treating or preventing Alzheimer’s disease; metformin or a pharmaceutically acceptable salt thereof, based on the free base of metformin, is administered at a daily dose of about 500 mg to about 1750 mg; and rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin, is administered at a daily dose of about 0.5 mg to about 1 mg (e.g., in a fixed-dose form with metformin or a pharmaceutically acceptable salt thereof); and an additional weekly dose of rapamycin, based on the free base of rapamycin, of about 1 mg to about 10 mg.

[0379] In some embodiments, the method comprises treating or preventing Alzheimer’s disease; metformin or a pharmaceutically acceptable salt thereof, based on the free base of metformin, is administered at a daily dose of about 500 mg to about 1750 mg; and rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin, is administered at a daily dose of about 0.5 mg to about 1 mg (e.g., in a fixed-dose form with metformin or a pharmaceutically acceptable salt thereof).

[0380] In some embodiments, the method comprises treating or preventing Alzheimer’s disease; metformin or a pharmaceutically acceptable salt thereof, based on the free base of metformin, is administered at a daily dose of about 500 mg to about 1750 mg; and rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin, is administered at a weekly dose of about 1 mg to about 10 mg (e.g., in a fixed-dose form with metformin or a pharmaceutically acceptable salt thereof).

[0381] In some embodiments, the method comprises promoting autophagy; metformin or a pharmaceutically acceptable salt thereof, at a daily dose of about 500 mg to about 1750 mg, on a free base basis of metformin; and rapamycin or a pharmaceutically acceptable salt thereof, at a daily dose of about 0.5 mg to about 1 mg, on a free base basis of rapamycin (e.g., in a fixed-dose form with metformin or a pharmaceutically acceptable salt thereof); and an additional weekly dose of about 1 mg to about 10 mg, on a free base basis of rapamycin.

[0382] In some embodiments, the method comprises promoting autophagy; metformin or a pharmaceutically acceptable salt thereof, at a daily dose of about 500 mg to about 1750 mg, on a free base basis of metformin; and rapamycin or a pharmaceutically acceptable salt thereof, at a daily dose of about 0.5 mg to about 1 mg, on a free base basis of rapamycin (e.g., in a fixed-dose form with metformin or a pharmaceutically acceptable salt thereof).

[0383] In some embodiments, the method comprises promoting autophagy; metformin or a pharmaceutically acceptable salt thereof, at a daily dose of about 500 mg to about 1750 mg, on a free base basis of metformin; and rapamycin or a pharmaceutically acceptable salt thereof, at a weekly dose of about 1 mg to about 10 mg, on a free base basis of rapamycin (e.g., in a fixed-dose form with metformin or a pharmaceutically acceptable salt thereof).

[0384] In some embodiments, the method comprises reducing or preventing genomic instability, telomere attrition, epigenetic alterations (e.g., DNA methylation, histone modification, or regulation of gene activity by RNA and / or proteins), dysregulation or deregulation of protein homeostasis, dysregulation of nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell depletion, and / or alterations in intercellular communication; metformin or a pharmaceutically acceptable salt thereof, at a daily dose of about 500 mg to about 1750 mg, on a free base basis of metformin; and rapamycin or a pharmaceutically acceptable salt thereof, at a daily dose of about 0.5 mg to about 1 mg, on a free base basis of rapamycin (e.g., in a fixed-dose form with metformin or a pharmaceutically acceptable salt thereof); and an additional weekly dose of about 1 mg to about 10 mg, on a free base basis of rapamycin.

[0385] In some embodiments, the method comprises reducing or preventing genomic instability, telomere attrition, epigenetic alterations (e.g., DNA methylation, histone modification, or modulation of gene activity by RNA and / or proteins), dysregulation or deregulation of protein homeostasis, dysregulation of nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell depletion, and / or alterations in intercellular communication; metformin or a pharmaceutically acceptable salt thereof, based on the free base of metformin, is administered in a daily dose of about 500 mg to about 1750 mg; and rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin, is administered in a daily dose of about 0.5 mg to about 1 mg (e.g., with metformin or a pharmaceutically acceptable salt thereof in a fixed-dosage form).

[0386] In some embodiments, the method comprises reducing or preventing genomic instability, telomere attrition, epigenetic alterations (e.g., DNA methylation, histone modification, or modulation of gene activity by RNA and / or proteins), dysregulation or deregulation of protein homeostasis, dysregulation of nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell depletion, and / or alterations in intercellular communication; metformin or a pharmaceutically acceptable salt thereof, based on the free base of metformin, is administered in a daily dose of about 500 mg to about 1750 mg; and rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin, is administered in a weekly dose of about 1 mg to about 10 mg (e.g., with metformin or a pharmaceutically acceptable salt thereof in a fixed-dosage form).

[0387] In some embodiments, metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof are administered separately, sequentially, or simultaneously. In some embodiments, metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof are administered separately. In some embodiments, metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof are administered sequentially. In some embodiments, metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof are administered simultaneously. In some embodiments, metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof are administered simultaneously as a fixed-dosage form.

[0388] In some embodiments, the dosing period of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, about 5 days to about 14 days, about 1 day to about 1 month, about 1 day to about 2 weeks, at least about 1 month, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, at least about 1 year, at least about 2 years, at least about 5 years, at least about 10 years, at least about 15 years, at least about 20 years, or more. In some embodiments, the dosing period of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof is about 1 day to about 1 month. In some embodiments, the dosing period of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof is about 1 day to about 2 weeks. In some embodiments, the dosing period of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof is about 2 weeks. In some embodiments, the dosing period of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof is about 12 days. In some embodiments, the dosing period of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof is about 1 week. In some embodiments, the dosing period of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof is at least about one month. In some embodiments, the dosing period of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof is at least about 1 year. In some embodiments, the time of cessation of administration is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more than 12 months. In some embodiments, the time of cessation of administration is up to 1 day, up to 2 days, up to 3 days, up to 4 days, up to 5 days, up to 6 days, up to 7 days, up to 8 days, up to 9 days, up to 10 days, up to 11 days, up to 12 days, up to 13 days, up to 14 days, up to 3 weeks, up to 4 weeks, up to 5 weeks, up to 6 weeks, up to 7 weeks, up to 8 weeks, up to 9 weeks, up to 10 weeks, up to 11 weeks, up to 12 weeks, up to 4 months, up to 5 months, up to 6 months, up to 7 months, up to 8 months, up to 9 months, up to 10 months, up to 11 months, or up to 12 months. In one embodiment, the individual is administered metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof for one period of time followed by a separate period of time alone.In another embodiment, metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof are administered for a first period of time, followed by a second period of time during which administration is ceased, followed by a third period of time during which metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof are administered, followed by a fourth period of time after the third period of time during which administration is ceased. In one aspect of this embodiment, the administration periods of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof are repeated for a determined or undetermined period of time, followed by a period of time during which administration is ceased. In another embodiment, the administration period is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more. In another embodiment, the period of time during which administration is ceased is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more.

[0389] In some embodiments, the subject experiences gastrointestinal symptoms after administration (e.g., oral administration) of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof, which are improved by eating prior to administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the subject eats for up to about 6 hours prior to administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. For example, the subject eats for up to about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 30 minutes, about 20 minutes, about 15 minutes, about 10 minutes, about 5 minutes, about 1 minute, about 30 seconds, or about 5 seconds prior to administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. For example, the subject eats at the same time as administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0390] In some embodiments, the subject is less than 90 years old, e.g., less than 80 years old, less than 70 years old, less than 60 years old, less than 50 years old, less than 40 years old, less than 30 years old, less than 15 years old, less than 10 years old, less than 5 years old, from about 1 week to about 5 years old, from about 5 years old to about 12 years old, from about 12 years old to about 21 years old, from about 21 years old to about 34 years old, from about 34 years old to about 45 years old, from about 45 years old to about 55 years old, from about 55 years old to about 65 years old, from about 65 years old to about 75 years old, or from about 75 years old to about 90 years old. In some embodiments, the subject is less than 80 years old. In some embodiments, the subject is less than 70 years old. In some embodiments, the subject is less than 60 years old.

[0391] Solid dosage forms of the immediate drug composition for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one pharmaceutically-acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate and / or a) fillers or fillers such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrants such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, or sodium carbonate, e) solution retarders such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin or bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms can also contain buffering agents.

[0392] Solid pharmaceutical compositions of a similar type can also be employed as fillers in soft and hard filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols, and the like.

[0393] The solid dosage forms of the pharmaceutical compositions of the present application as tablets, dragees, capsules, pills, and granules can be prepared with coatings or shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They can optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric and waxy substances.

[0394] The active ingredients can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0395] Liquid forms in which the compositions of the present application for oral administration are included in pharmaceutically-acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, the liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, EtOAc, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0396] Suspensions of the compounds of the present application can also contain suspending agents as

[0397] Pharmaceutical compositions of the present application for parenteral injection comprise sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and 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), 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.

[0398] These pharmaceutical compositions can contain, in addition to inert diluents, adjuvants such as preserving, wetting, emulsifying, dispersing, sweetening, flavoring, and perfuming agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It can also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of injectable pharmaceutical forms can be brought about by the inclusion of agents which delay absorption, for example, aluminum monostearate and gelatin. These compounds can be incorporated into sustained release or targeted delivery systems such as polymer matrices, liposomes, and microspheres. Such formulations can provide for the control of the composition distribution.

[0399] Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders, these can be prepared by

[0400] Dosage forms for topical administration of a compound or pharmaceutical composition of this disclosure include powders, patches, sprays, ointments, and inhalers. Active compounds can be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants as can be required.

[0401] The compounds and compositions described herein can be administered orally, parenterally (e.g., subcutaneously, intradermally, intravenously, or intramuscularly), topically, rectally, nasally, buccally, or buccal, for example, in dosages, ranging from about 0.01 milligram per kilogram (mg / kg) to about 1000 mg / kg (e.g., from about 0.01 to about 100 mg / kg, from about 0.1 to about 100 mg / kg), or according to the requirements of the particular drug, dosage form and / or route of administration. Other routes of administration include enteral, intraarterial, intraperitoneal, and intrathecal. The interrelationship of dosages for animals and humans based upon mg per square meter of body surface is described by Freireich et al., Cancer Chemother. Rep. 50, 219-244 (1966). Body surface area can be estimated from weight and height of the patient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley, New York, 537 (1970). In certain embodiments, the compositions are administered orally or by injection. The methods herein contemplate administration of a therapeutically effective amount of a compound or compound composition to achieve the desired or stated effect. Generally, the pharmaceutical compositions of this disclosure will be administered from about 1 to about 6 times per day or alternatively, as a continuous infusion. Such administration can occur as a chronic or acute treatment.

[0402] Lower or higher doses than those recited above can be required. Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, disorder, or condition, the patient's disposition to the disease, and the judgment of the treating physician.

[0403] The present application provides a dosage form comprising metformin or a pharmaceutically acceptable salt thereof in an amount of about 100 mg to about 5000 mg (e.g., about 100 mg to about 500 mg, about 100 mg to about 800 mg, about 100 mg to about 1000 mg, about 500 mg to about 1000 mg, about 1000 mg to about 1500 mg, about 1500 mg to about 2250 mg, about 1500 mg to about 2000 mg, about 2000 mg to about 2500 mg, about 2250 mg to about 3000 mg, about 2500 mg to about 3000 mg, about 3000 mg to about 3500 mg, about 3500 mg to about 4000 mg, about 4000 mg to about 5000 mg, about 500 mg to about 3000 mg, about 500 to about 1750 mg, about 500 mg to about 1125 mg, about 1125 mg to about 2250 mg, about 1500 mg to about 3000 mg, about 500 mg, about 650 mg, about 750 mg, about 850 mg, about 1000 mg, about 1750 mg, about 2000 mg, or about 3000 mg) as the free base of metformin and rapamycin or a pharmaceutically acceptable salt thereof in an amount of about 0.1 mg to about 25 mg (e.g., about 0.1 mg to about 20 mg, about 0.1 mg to about 18 mg, about 0.1 mg to about 15 mg, about 0.1 mg to about 13 mg, about 0.1 mg to about 10 mg, about 0.1 mg to about 7 mg, about 0.1 mg to about 5 mg, about 0.1 mg to about 3 mg, about 0.1 mg to about 2 mg, about 0.1 mg to about 1 mg, about 0.5 mg to about 20 mg, about 0.5 mg to about 18 mg, about 0.5 mg to about 15 mg, about 0.5 mg to about 13 mg, about 0.5 mg to about 10 mg, about 0.5 mg to about 7 mg, about 0.5 mg to about 5 mg, about 0.5 mg to about 3 mg, about 0.5 mg to about 2 mg, about 0.1 mg, about 0.5 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, or about 20 mg) as the free base of rapamycin.5 mg to about 1 mg, about 1 mg to about 20 mg, about 1 mg to about 18 mg, about 1 mg to about 15 mg, about 1 mg to about 13 mg, about 1 mg to about 10 mg, about 1 mg to about 7 mg, about 1 mg to about 5 mg, about 1 mg to about 3 mg, about 1 mg to about 2 mg, about 2 mg to about 12 mg, about 4 mg to about 10 mg, about 4 mg to about 8 mg, about 10 mg to about 30 mg, about 13 mg to about 17 mg, about 2 mg to about 4 mg, about 2 mg to about 10 mg, about 1 mg to about 3 mg, about 3 mg to about 7 mg, about 4 mg to about 6 mg, about 15 mg to about 25 mg, about 18 mg to about 22 mg, about 10 mg to about 14 mg, about 0.5 mg, about 1 mg, about 2 mg, about 2.5 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, or about 15 mg). The dosage form can also include a pharmaceutically acceptable carrier and / or an additional therapeutic agent.

[0404] In some embodiments, the dosage form includes metformin, or a pharmaceutically acceptable salt thereof, in an amount of about 1500 mg to about 3000 mg, based on the free base equivalent of metformin, and rapamycin, or a pharmaceutically acceptable salt thereof, in an amount of about 0.5 mg to about 1 mg, based on the free base equivalent of rapamycin.

[0405] In some embodiments, the dosage form includes metformin, or a pharmaceutically acceptable salt thereof, in an amount of about 1500 mg to about 2250 mg, based on the free base equivalent of metformin, and rapamycin, or a pharmaceutically acceptable salt thereof, in an amount of about 0.5 mg to about 1 mg, based on the free base equivalent of rapamycin.

[0406] In some embodiments, the dosage form includes metformin, or a pharmaceutically acceptable salt thereof, in an amount of about 2250 mg to about 3000 mg, based on the free base equivalent of metformin, and rapamycin, or a pharmaceutically acceptable salt thereof, in an amount of about 0.5 mg to about 1 mg, based on the free base equivalent of rapamycin.

[0407] In some embodiments, the dosage form includes metformin, or a pharmaceutically acceptable salt thereof, in an amount of about 1500 mg, and rapamycin, or a pharmaceutically acceptable salt thereof, in an amount of about 0.5 mg, based on the free base equivalent of rapamycin.

[0408] In some embodiments, the dosage form comprises metformin, or a pharmaceutically acceptable salt thereof, in an amount of about 3000 mg as the free base equivalent of metformin, and rapamycin, or a pharmaceutically acceptable salt thereof, in an amount of about 0.5 mg as the free base equivalent of rapamycin.

[0409] In some embodiments, the dosage form comprises metformin, or a pharmaceutically acceptable salt thereof, in an amount of about 1500 mg as the free base equivalent of metformin, and rapamycin, or a pharmaceutically acceptable salt thereof, in an amount of about 1 mg as the free base equivalent of rapamycin.

[0410] In some embodiments, the dosage form comprises metformin, or a pharmaceutically acceptable salt thereof, in an amount of about 3000 mg as the free base equivalent of metformin, and rapamycin, or a pharmaceutically acceptable salt thereof, in an amount of about 1 mg as the free base equivalent of rapamycin.

[0411] In some embodiments, the dosage form comprises metformin, or a pharmaceutically acceptable salt thereof, in an amount of about 500 mg to about 1750 mg as the free base equivalent of metformin, and rapamycin, or a pharmaceutically acceptable salt thereof, in an amount of about 0.5 mg to about 1 mg as the free base equivalent of rapamycin.

[0412] In some embodiments, the dosage form comprises metformin, or a pharmaceutically acceptable salt thereof, in an amount of about 500 mg to about 1125 mg as the free base equivalent of metformin, and rapamycin, or a pharmaceutically acceptable salt thereof, in an amount of about 0.5 mg to about 1 mg as the free base equivalent of rapamycin.

[0413] In some embodiments, the dosage form comprises metformin, or a pharmaceutically acceptable salt thereof, in an amount of about 1125 mg to about 1750 mg as the free base equivalent of metformin, and rapamycin, or a pharmaceutically acceptable salt thereof, in an amount of about 0.5 mg to about 1 mg as the free base equivalent of rapamycin.

[0414] In some embodiments, the dosage form comprises metformin, or a pharmaceutically acceptable salt thereof, in an amount of about 500 mg as the free base equivalent of metformin, and rapamycin, or a pharmaceutically acceptable salt thereof, in an amount of about 0.5 mg as the free base equivalent of rapamycin.

[0415] In some embodiments, the dosage form comprises metformin, or a pharmaceutically acceptable salt thereof, in an amount of about 1750 mg as the free base equivalent of metformin, and rapamycin, or a pharmaceutically acceptable salt thereof, in an amount of about 0.5 mg as the free base equivalent of rapamycin.

[0416] In some embodiments, the dosage form comprises metformin or a pharmaceutically acceptable salt thereof, in an amount of about 500 mg, as the free base equivalent of metformin, and rapamycin or a pharmaceutically acceptable salt thereof, in an amount of about 1 mg, as the free base equivalent of rapamycin.

[0417] In some embodiments, the dosage form comprises metformin or a pharmaceutically acceptable salt thereof, in an amount of about 1750 mg, as the free base equivalent of metformin, and rapamycin or a pharmaceutically acceptable salt thereof, in an amount of about 1 mg, as the free base equivalent of rapamycin.

[0418] In some embodiments, the weight ratio of metformin to rapamycin is about 10000: 1 to about 100: 1 (e.g., about 10000: 1 to about 300: 1, about 5000: 1 to about 400: 1, about 4000: 1 to about 500: 1, about 3500: 1 to about 750: 1, about 3500: 1 to about 900: 1, about 35000: 1 to about 1000: 1, about 2500: 1 to about 1000: 1, about 2500: 1 to about 1700: 1, about 3000: 1 to about 2000: 1, about 2000: 1 to about 1500: 1, about 1300: 1 to about 700: 1, about 3500: 1, about 2500: 1, about 1750: 1, or about 1000: 1). In one embodiment, the weight ratio of metformin to rapamycin is about 3500: 1. In one embodiment, the weight ratio of metformin to rapamycin is about 2500: 1. In one embodiment, the weight ratio of metformin to rapamycin is about 1000: 1. In one embodiment, the weight ratio of metformin to rapamycin is about 1750: 1.

[0419] Suitable dosage levels can be determined by any appropriate method. Preferably, for topical administration, the active substances are administered with a frequency of between 1 and 4 times per day, or less frequently if a drug delivery system is used. However, the actual dose level and time course of administration of the active ingredients in the pharmaceutical compositions described herein can vary from patient to patient, from composition to composition, and from mode of administration to mode of administration, so as to obtain an amount of the active ingredients which is effective in obtaining the desired therapeutic response without causing persistent toxic effects for the patient. In some cases, the dosage can deviate from the stated amounts, especially depending on the age, sex, weight, diet, and general health of the patient, the route of administration, the individual response to the active ingredients, the nature of the formulation, and the time or interval at which the administration takes place. Thus, in some cases, it can be desirable to administer less than the minimum amounts stated above, while in other cases it can be desirable to exceed the stated upper limits. If the amount administered is large, it is recommended to divide it into several individual doses, spread over the day.

[0420] In some embodiments of the methods disclosed herein, metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof will be administered orally, parenterally, transdermally, intranasally, sublingually, neural axon, or ocularly. In some embodiments, metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof will be administered orally.

[0421] The disclosures of all articles and publications referenced herein are expressly incorporated herein by reference in their entirety for the purpose of disclosing and describing the compositions and methodologies used in connection with the disclosure and for the purpose of disclosing and describing various embodiments of the disclosure.

[0422] Examples

[0423] Example 1: Metformin and rapamycin combination therapy in mouse models of neuromuscular and proliferative diseases (such as myotonic dystrophy type 1 (DM1)) evaluation.

[0424] Materials and Methods:

[0425] DM1 mouse models can be used as described in Brockhoff et al., J Clin Invest, 2017, 127(2):549-563, which is incorporated herein by reference in its entirety. DM1 mouse models that can be used are transgenic mouse models that overexpress a mutant human DMPK gene containing CTG repeat expansions, which is characteristic of DM1. These mice exhibit skeletal muscle weakness and histological features of DM1, such as muscle fiber atrophy, nuclear clustering, and increased fibrosis. Mice can be bred to obtain DM1 mice that are either AMPKa2 wild type (AMPKa2WT) or skeletal muscle-specific deletion of AMPKa2 (AMPKa2MKO). Mice can be genotyped by PCR using tail DNA. Insulin resistance, hyperglycemia, glucose uptake, and HbAlc can be measured.

[0426] Myoblasts from the quadriceps muscle of control and DM1 mice can be studied in vitro. Myoblasts can be cultured in growth media, differentiated in differentiation media, and treated with AICAR or rapamycin. Western blot analysis can be performed to assess protein expression and phosphorylation levels of various markers of the AMPK and mTORCl pathways.

[0427] Dosing for the combination therapy can include treating DM1 mice with a combination of metformin and rapamycin to target the dysregulated AMPK / mTORCl pathway in muscle cells. Metformin can be used to activate AMPK, while rapamycin can be used to inhibit mTORCl. The effect of this combination therapy can be evaluated by measuring muscle function and gene expression in the treated mice. Doses can be determined based on previous studies, pharmacokinetic data, and toxicity profiles, and can be optimized using various techniques in the art, such as dose escalation studies. As one non-limiting example, metformin can be administered in drinking water at a dose of 300 mg / kg / day for 2 weeks, and rapamycin can be administered via intraperitoneal injection at a dose of 3 mg / kg / day for 2 weeks, for example.

[0428] Data analysis:

[0429] The efficacy of the combination of metformin and rapamycin on relevant disease parameters in the DM1 model, such as blood glucose levels, body weight, insulin sensitivity, or other relevant biomarkers, can be measured by comparing the effects of the combination to the effects of each drug used alone.

[0430] Blood glucose levels: Blood glucose levels can be analyzed by periodically collecting blood samples from the mice, measuring glucose concentration using a glucometer, or performing enzyme-linked immunosorbent assay (ELISA) on collected serum samples.

[0431] Body weight: Body weight can be measured periodically using a digital scale throughout the study. Statistical methods such as analysis of variance or linear regression can then be used to analyze the data to determine any significant differences between treatment groups.

[0432] Insulin sensitivity: Insulin sensitivity can be assessed by insulin tolerance tests (ITT) or glucose tolerance tests (GTT). These tests involve administering insulin or glucose to the mice and measuring the resulting changes in blood glucose levels. Statistical methods such as analysis of variance or linear regression can then be used to analyze the data to determine any significant differences between treatment groups.

[0433] Other relevant biomarkers: At the end of the study, other relevant biomarkers can be analyzed by collecting blood samples or tissue samples from the mice and performing various assays such as ELISA, Western blot, or PCR. Statistical methods such as analysis of variance or linear regression can then be used to analyze the data to determine any significant differences between treatment groups.

[0434] The degree of drug synergy can be assessed using quantitative methods such as the Combination Index (CI) or Bliss Independence Model. These models allow to accurately determine the degree of synergy of the drug combination of metformin and rapamycin in the treatment of type I myotonic dystrophy. To validate the results of the in vivo experiment, additional experiments can be performed such as dose response curves, time course studies or genetic manipulations.

[0435] Example 2: Microfluidic-based automated in vivo lifespan and healthspan assessment of rapamycin and metformin and their combination using Caenorhabditis elegans

[0436] SUMMARY

[0437] Objectives

[0438] The objective of this study was to investigate the effect of rapamycin (herein referred to as "Compound 1" or "Cpd 1"), metformin (herein referred to as "Compound 2" or "Cpd 2") and their combination on the lifespan and healthspan of Caenorhabditis elegans. To this end, a microfluidic-based technology was used.

[0439] Method summary

[0440] For the purpose of this study, the two compounds and their four combinations were tested on a microfluidic platform. Worms were injected at the L4 larval stage, confined in dedicated microfluidic chambers and continuously exposed to the presence of the two compounds alone or in combination from the L4 larval stage to the end of the experiment (corresponding to the day of death of the worms). During the trial, short video sequences of each microfluidic chamber containing worms were automatically performed every six (6) hours. Measurements of locomotor ability as well as worm size were performed on the acquired video sequences by using algorithms (3,4) to address compound toxicity (1) and reproductive assessment lifespan (2). Dead / alive worms were automatically identified according to locomotor ability parameters. Each condition was tested in four technical replicates on different microfluidic cartridges. The experiment was performed once.

[0441] Study Design and Methods

[0442] The proposed study is in accordance with Figure 1The design and timeline shown were followed. N2 wild-type C. elegans were grown to the adult stage on solid nematode growth medium (NGM) agar plates and harvested in complete S-medium. Eggs from adult populations were hatched overnight in S-medium and the resulting LI offspring were recovered using a dedicated filter. The LI larvae were then collected by filtration in S-medium and seeded on NGM agar plates and grown for 48 hours (h) at 20°C. After 48 hours, the nematode populations had reached the L4 larval stage. The L4 larvae were then collected by filtration in 5 mL of S-medium. In each condition, 20 to 30 L4 were injected into the microfluidic chip. The worms were then continuously fed on the chip by bacterial medium throughout the experiment. Videos of each microchamber after feeding were taken every 6 hours throughout the experiment.

[0443] Preparation of the test substances

[0444] Compounds 1 and 2 were suspended in DMSO at a stock concentration of 100 mM and 2.5 M, respectively (Table 1). The compounds were then mixed with the bacterial medium at the target concentration, alone or in combination (Table 2). For all test conditions, the final vehicle (DMSO) concentration was 1%. For the negative control condition, the worms were fed with bacterial medium containing only the vehicle solution (DMSO 1%). All stock solutions were stored at -20°C and freshly diluted on the day of the start of the experiment on the chip, then the compound / bacterial medium solution was changed every 72 hours.

[0445] Table 1. List of chemical reagents used in this study.

[0446]

[0447] Table 2. Conditions tested in this study.

[0448]

[0449] Bacterial preparation

[0450] The worms were continuously fed on the chip by bacterial medium (lyophilized E. coli OP50 in complete S-medium) at a concentration of 8.05E+9 cells per mL. Briefly, 500 mg of lyophilized OP50 were dissolved in 5 mL of complete S-medium and carefully homogenized using vortex. The bacterial concentration was monitored using a spectrophotometer and adjusted to 8.05E+9 if necessary (5).

[0451] Nematode preparation

[0452] Eggs of C. elegans (N2 wild type strain provided by the Caenorhabditis Genetics Center) were hatched on solid nematode growth medium (NGM) agar plates seeded with E. coli OP50 and grown at 20°C for 4 days. After 4 days, a mixed stage of N2 was present on the NGM agar plates, mainly comprising adult worms and eggs. The worm population was harvested and filtered in complete S-medium to separate the adults from the other mixed stage population. The separated adults were incubated overnight at room temperature. The hatched L1 larvae from the eggs produced by the adults were then collected by filtering in S-medium, seeded on NGM agar plates and grown for 48 hours at 20°C. After 48 hours, the worm population had reached the L4 larval stage. The L4 larvae were then collected by filtering in 5 mL h and the initial solution of bacteria and compound was replaced with a new solution containing bacteria. Every 60 minutes, 4 μL of fresh food / compound solution was injected into the microfluidic chip under each test condition. Videos were taken of each microchamber after feeding.

[0453] Phenotype analysis

[0454] Five time-resolved phenotype readouts were extracted during the test period, including:

[0455] - Nematode size (area): This parameter was measured to assess the potential effect of the tested compound on the growth retardation or larval stasis phenotype (7).

[0456] - Sexual maturity: This parameter was measured to assess the potential effect of the tested compound on the time required for the worms to reach sexual maturity, which was determined by observing the first embryo (i.e. egg) produced in each microfluidic chamber. This phenotype also indicates the general infertile phenotype (i.e. no egg production) (7).

[0457] - Nematode reproductive capacity: This parameter was measured to assess the potential effect of the tested compound on the average number of eggs produced by each worm during the reproductive period, which was calculated from the images obtained during the experiment (7).

[0458] - Reproductive period: This parameter was measured to assess the potential effect of the tested compound on the duration of the period during which the worms were fertile, which was determined by observing the last embryo produced in each microfluidic chamber.

[0459] - Nematode lifespan: This parameter was measured to assess the potential effect of the tested compound on the survival of the worms.

[0460] Nematode motility is an additional phenotype readout, and five parameters of motility were assessed from the videos collected every 6 hours (2 seconds long, 10 frames per second per video). At each time point, the values recorded for dead worms were excluded. The five motility parameters monitored include (3):

[0461] o Head movement amplitude

[0462] o Body movement amplitude in nematodes

[0463] o Tail movement amplitude

[0464] o Nematode bending frequency

[0465] o Nematode speed

[0466] These locomotor capacity parameters were analyzed by staging (area under the curve calculation followed by two-way ANOVA), defined as follows:

[0467] o Stage 1 : from day 1 to day 5

[0468] o Stage 2: from day 6 to day 10

[0469] o Stage 3: from day 11 to day 15

[0470] o Stage 4: from day 16 to day 20

[0471] Data statistics

[0472] Raw values were normalized and compared to their respective negative control (if indicated). Survival analysis was performed using the Kaplan-Meier method and the log-rank test was used to calculate the significance of the difference between survival curves. To compare the interaction between groups, a two-way ANOVA test was performed. When comparing more than two groups, the ANOVA was assessed using the Bonferroni multiple comparison test. GraphPad Prism 5 (GraphPad Software) was used for all other statistical analyses, including calculation of sd, standard error of the mean (sem), and area under the curve (AUC). All p values < 0.05 were considered significant. Figures 8A-8C The radar chart shown is: compared to the negative control (DMSO 1%), 2 Statistical data.

[0473] Summary and interpretation of the results of the longevity monitoring:

[0474] • Although all the compounds tested individually or in combination have a positive effect on survival, for example compared to the negative control, the combination D+14%, the tested conditions do not reach a significant effect on the overall survival of N2 nematodes under the current conditions. Figure 2 and Table 3).

[0475] · The combination D treatment has a significant effect on the survival of 50% of nematodes compared to the negative control (+56.0%; p<0.05) Figure 3 and Table 4). Cpd 1, combination A and combination B treatments show a modest improvement in the survival of 50% of nematodes, but not significant (0.05

[0476] Compared with the negative control, combination A treatment significantly affected 25% nematode survival (+77.8%; p<0.05). Figure 4 (and Table 5).

[0477] Reproduction and growth monitoring

[0478] observe:

[0479] Figure 5 A-5D was observed in wild-type N2 nematodes treated with the conditions listed in Table 2 from the injection of L4 larvae to the production of the first ( Figure 5 A) and the last one ( Figure 5 B) The time of egg laying (in hours), and the span of egg laying time ( Figure 5 C) and the average number of eggs laid per nematode during the oviposition period ( Figure 5 D) Bar chart. The bars represent the mean + / - sem. The p-values ​​were obtained by one-way ANOVA followed by a Bonferroni multiple comparison test (p<0.05) to compare the negative control (blue bar) with the treatment. *Statistical data with the negative control (DMSO 1%). Table 6-9 summarizes the statistics. Figure 5 Data from A-5D.

[0480] Table 6. Figure 5 Summary of Results A.

[0481]

[0482] n C This corresponds to the number of chambers where oviposition initiation was detected. Within the chambers, oviposition initiation is determined at the population level, not at the individual nematode level.

[0483] Table 7. Figure 5 Summary of Results B.

[0484]

[0485] n C This corresponds to the number of chambers where oviposition initiation was detected. Within each chamber, oviposition initiation is determined at the population level, not at the individual nematode level.

[0486] Table 8. Figure 5 Summary of C results.

[0487]

[0488] n C This corresponds to the number of chambers where oviposition initiation was detected. Within each chamber, oviposition initiation is determined at the population level, not at the individual nematode level.

[0489] Table 9. Figure 5 Summary of results for D.

[0490]

[0491] n C Number of chambers corresponding to the detection of the beginning of egg laying. Within the chamber, the beginning of egg laying was determined at the population level, not at the individual nematode level.

[0492] Figure 6 Bar chart of nematode growth (area under the curve) for N2 wild-type nematodes treated with the conditions summarized in Table 2. Negative control (blue line) was compared to treatments by two-way ANOVA to assess overall curve differences, followed by Bonferroni’s multiple comparison test to obtain p values. ** p < 0.01; *** p < 0.001; **** p < 0.0001. Table 10 summarizes the data in Figure 6

[0493] Table 10. Figure 6 Summary of results related to nematode size shown in

[0494]

[0495] Summary and interpretation of reproduction and growth monitoring results:

[0496] • All tested conditions (compounds tested alone or in combination) significantly reduced the number of eggs laid per nematode compared to the negative control (p < 0.05) Figure 5 A and Table 9).

[0497] • Combination A treatment had a slight impact on the time to the last egg laid compared to the negative control group (+9.9%, p < 0.05), thus extending the time span of total egg laying by 12.8% (p < 0.05) Figure 5 B-5C and Table 8).

[0498] • All tested conditions (compounds tested alone or in combination) significantly impacted nematode size compared to the negative control group (p < 0.05) Figure 6 and Table 10): Cpd 1, Combination A, Combination C and Combination D treatments showed a significant improvement in nematode size, while Cpd 2 and Combination B nematode size was significantly reduced compared to the negative control Figure 6 and Table 10).

[0499] Motor ability monitoring

[0500] Observations:

[0501] Figures 7A-7D ​The diagram shows the motility of wild-type N2 nematodes treated with the conditions shown in Table 2. Figure 7A Head amplitude; Figure 7B , medium amplitude; Figure 7C Tail amplitude; Figure 7D Bending frequency; Figure 7E (Speed). The difference in the overall curves was assessed by comparing the negative control (blue line) and the treatment using two-way ANOVA, followed by a Bonferroni multiple comparison test to obtain the p-value (p < 0.05). Error bars indicate: ↑: significantly increased phenotype compared to the negative control (blue curve) (two-way ANOVA curve comparison (p < 0.05)); ↓: significantly decreased phenotype compared to the negative control (blue curve) (two-way ANOVA curve comparison (p < 0.05)).

[0502] Figures 8A-8C It uses Cpd 1 (final concentration 100µM) and Cpd 2 (final concentration 25 mM) ( Figure 8A Combination A (Cpd 1100µM + Cpd 2 25 mM) and combination B (Cpd 1 33µM + Cpd 2 25 mM) Figure 8B Combinations C (Cpd 1 100µM + Cpd 2 8.5 mM) and D (Cpd 1 50µM + Cpd 2 12.5 mM) Figure 8C A series of radar plots showing the nematode locomotion (head amplitude, midbody amplitude, tail amplitude, velocity, and bending frequency) of N2 wild-type nematodes treated with the treatment at different stages (as shown clockwise around each radar plot, stage "1" corresponds to stages D0 to D5; stage "2" corresponds to stages D6 to D10; stage "3" corresponds to stages D11 to D15; stage "4" corresponds to stages D16-D20). The values ​​shown on the radar plots correspond to the ratio of the AUC calculated according to the curve shown in Figure 7 to the negative control (DMSO 1%). ↓: The analyzed phenotype was significantly reduced compared to the negative control (DMSO 1%, blue line) (two-way ANOVA curve comparison (p<0.05)); ↑: The analyzed phenotype was significantly increased compared to the negative control (DMSO 1%, blue line) (two-way ANOVA curve comparison (p<0.05)).

[0503] Summary and interpretation of motor ability and behavioral monitoring results under baseline conditions:

[0504] • The overall locomotor ability of nematodes treated with Cpd 1 (average improvement of 14% in all locomotor parameters), Cpd 2 (average improvement of 22% in all locomotor parameters), Combination A (average improvement of 19% in all locomotor parameters), Combination B (14% improvement in 1 out of 5 locomotor parameters) and Combination D (average improvement of 16% in 4 out of 5 locomotor parameters) was significantly increased compared to the negative control ( Figures 7A-7E ). No effect on locomotor ability was observed when nematodes were treated with Combination C ( Figures 7A-7E ).

[0505] • Only in Phase 1 (D0 to D5) and Phase 2 (D6 to D10) ( Figures 8A-8C ), almost all locomotor parameters were significantly increased, which explains the overall improvement of nematode locomotor ability in populations treated with Cpd 1, Cpd 2 and Combination A ( Figures 7A-7E ). No effect on locomotor ability was observed in Phase 3 (D11 to D15) and Phase 4 (D16 to D20).

[0506] • In Phase 1 (D0 to D5), Phase 2 (D6 to D10) and Phase 3 (D11 to D15) ( Figures 8A-8C ), most locomotor parameters were significantly increased, which explains the average improvement of 16% in nematode locomotor ability in populations treated with Combination D ( Figures 7A-7E ). No effect on locomotor ability was observed in Phase 4 (D16 to D20).

[0507] • Combination B treatment showed a significant improvement in all locomotor parameters in Phase 1 (D0 to D5) and Phase 2 (D6 to D10) ( Figures 8A-8C ). However, in later stages (Phase 3 and Phase 4), locomotor ability was clearly impaired, which explains the overall weaker effect of this particular condition on locomotor ability ( Figures 7A-7E ).

[0508] • Combination C treatment showed a significant improvement in all locomotor parameters only in Phase 1 (D0 to D5) ( Figures 8A-8C ). In Phase 3 (D11 to D15), locomotor ability was clearly impaired, which explains the overall lack of effect of this particular condition on locomotor ability ( Figures 7A-7E ).

[0509] CONCLUSIONS

[0510] Study summary

[0511] The biological effects of Cpd 1 and Cpd 2 and their combination on aging and related age-related phenotypes were analyzed and described. Overall, this study showed that the combination of Cpd 1 and Cpd 2 at high doses (100 µM and 25 mM, respectively, corresponding to Combination A) and half of the maximum dose (50 µM and 12.5 mM, respectively, corresponding to Combination D) was able to have a significant synergistic effect on the lifespan and the locomotor ability of C. elegans. Further conclusions are summarized below:

[0512] • Combination D (Cpd 1 50 µM + Cpd 2 12.5 mM) treatment had a significant effect on the survival of 50% of the worms compared to the negative control (+56.0%; p<0.05), while no effect was observed on the later lifespan (survival of 25% of the worms). In addition, Combination A (Cpd 1 100 µM + Cpd 2 25 mM) treatment showed a significant effect on the survival of 25% of the worms compared to the negative control (+77.8%; p<0.05), while a modest improvement was observed on the survival of 50% of the worms (p<0.1). Altogether, a synergistic effect of Combination A and Combination D on survival was observed since no significant effect was monitored for the 2 compounds tested alone. These results indicate that the combination of rapamycin and metformin synergistically extended the lifespan of C. elegans.

[0513] • Overall, while Combination A showed a trend to improve the overall lifespan of the worms (+14.4%; p=0.2), none of the tested conditions had a significant effect on the overall survival of the worms compared to the negative control.

[0514] • All the tested conditions significantly reduced the number of eggs laid per worm compared to the negative control. No synergistic effect was observed when the two compounds were tested in combination (i.e. the combination did not lead to a reduction of the number of eggs laid per worm). Without wishing to be bound by theory, it is believed that, across species, some interventions, in particular caloric restriction, promote beneficial effects on lifespan while reducing the ability to reproduce (2). Some compounds known to mimic the effects of caloric restriction also have this effect on reproduction.

[0515] • However, Combination A treatment had a slight effect on the time to the last egg laid compared to the negative control (+9.9%, p<0.05) and extended the total time span of egg laying by 12.8% (p<0.05), which can be interpreted as a beneficial effect on the reproductive system of C. elegans.

[0516] • Regarding nematode size, Cpd 1, Combination A, Combination C and Combination D treatments showed a significant improvement in nematode size compared to the negative control, while Cpd 2 and Combination B showed a significant decrease in nematode size. Based on these observations, high doses of Cpd 2 seemed to have a negative impact on nematode size (alone or in combination, as observed in Combination B): high doses (100 mM) of Cpd 1 were able to reverse this deleterious effect on size, as observed in Combination A conditions (an increase of 4.9% compared to 25 mM Cpd 2 alone).

[0517] • The overall locomotion capacity of nematodes treated with Cpd 1, Cpd 2, Combination A, Combination B and Combination D was significantly increased compared to the negative control. Interestingly, almost all locomotion capacity parameters were significantly increased only in Stage 1 (D0 to D5) and Stage 2 (D6 to D10), which explains the overall improvement in nematode locomotion capacity in populations treated with Cpd 1, Cpd 2 and Combination A. No effect on locomotion capacity was observed in Stage 3 (D11 to D15) and Stage 4 (D16 to D20) in these specific conditions. Moreover, most locomotion capacity parameters were significantly increased in Stage 1 (D0 to D5), Stage 2 (D6 to D10) and Stage 3 (D11 to D15), which explains the overall improvement in nematode locomotion capacity in populations treated with Combination D. This observation suggests that Combination D treatment is able to maintain and improve nematode health and locomotion capacity later in life.

[0518] • Moreover, while some deleterious effects on nematode locomotion capacity were observed in Combination B in the late stages of life (Stage 3 and Stage 4), these negative effects were reversed in Combination A and Combination D, with either a decrease in Cpd 2 concentration (Combination D) or an increase in Cpd 1 (Combination A).

[0519] Overall, this study shows that the combination of Cpd 1 and Cpd 2 at high doses (100 mM and 25 mM, respectively) and at half the maximum dose (50 mM and 12.5 mM, respectively) is able to provide a significant synergistic effect in terms of longevity and locomotion capacity improvement in C. elegans.

[0520] REFERENCES

[0521] 1) Hunt PR. The C. elegans model in intoxicity testing. J Appl Toxicol. 2017 Jan;37(1)

[0522] 2) Moatt JP, et al. The effect of dietary restriction on reproduction: a meta-analytic perspective. BMC Evol Biol. 2016 Oct 7; 16(1): 199

[0523] 3) Atakan HB, et al. Automated Platform for Long-Term Culture and High-Content Phenotyping of Single C. elegans Worms. Sci Rep. 2019 Oct 4; 9(1): 14340

[0524] 4) Atakan HB, et al. Automated high-content phenotyping from the first larval stage till the onset of adulthood of the nematode Caenorhabditis elegans. Lab Chip. 2018 Dec 18; 19(1): 120-135

[0525] 5) Gao AW, et al. High-content phenotypic analysis of a C. elegans recombinant inbred population identifies genetic and molecular regulators of lifespan. bioRxiv [Preprint]. 2024 Jan 16: 2024.01.15.575638

[0526] 6) Cornaglia et al., Automated longitudinal monitoring of in vivo protein aggregation in neurodegenerative disease C. elegans models. Mol Neurodegener. 2016 Feb 9;11:17.

[0527] 7) Mouchiroud L et al., Worm-on-Chip technology as a new alternative for early toxicity studies. Toxicology Letters. 2021 350, S118-S119.

[0528] Other implementation methods

[0529] It should be understood that although the invention has been described in conjunction with specific embodiments, the foregoing description is intended to illustrate, and not limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and improvements are within the scope of the claims.

Claims

1. A method for treating or preventing neurological, muscular, or proliferative disorders in a subject in need, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof; wherein the neurological, muscular, or proliferative disorder is selected from: myotonic dystrophy type 1 (DM1), Duchenne muscular dystrophy (DMD), spinocerebellar ataxia type 3 (SCA-3), Alzheimer's disease, Parkinson's disease, vascular dementia, Lewy body dementia (DLB), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Lafra disease, glioblastoma, and diffuse entropy-type pontine glioma (DIPG).

2. The method of claim 1, wherein the neurological, muscular, or proliferative disease is myotonic dystrophy type 1 (DM1).

3. The method of claim 2, wherein the reduction of reactive oxygen species (ROS) is measured in the subject after administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

4. The method of claim 1, wherein the neurological, muscular, or proliferative disease is Duchenne muscular dystrophy (DMD).

5. The method of claim 1, wherein the neurological, muscular, or proliferative disorder is spinocerebellar ataxia type 3 (SCA-3).

6. The method of claim 1, wherein the neurological, muscular, or proliferative disease is Alzheimer's disease.

7. The method of claim 1, wherein the neurological, muscular, or proliferative disease is Parkinson's disease.

8. The method of claim 1, wherein the neurological, muscular, or proliferative disease is vascular dementia.

9. The method of claim 1, wherein the neurological, muscular, or proliferative disorder is Lewy body dementia (DLB).

10. The method of claim 1, wherein the neurological, muscular, or proliferative disease is Huntington's disease (HD).

11. The method of claim 1, wherein the neurological, muscular, or proliferative disease is amyotrophic lateral sclerosis (ALS).

12. The method of claim 1, wherein the neurological, muscular, or proliferative disease is Lafra disease.

13. The method of any one of claims 6-12, wherein the method includes preventing the disease.

14. The method of claim 1, wherein the neurological, muscular, or proliferative disease is glioblastoma.

15. The method of claim 1, wherein the neurological, muscular, or proliferative disease is a diffuse engenerative pontine glioma (DIPG).

16. The method of any one of claims 14-15, wherein Akt in the subject is activated after administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

17. A method of treating or preventing neurocancer in a subject of need, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof; wherein the cancer is glioblastoma or diffuse endophytic pontine glioma (DIPG).

18. The method of claim 17, wherein the neurocancer is glioblastoma.

19. The method of claim 17, wherein the neurocancer is a diffuse endophytic pontine glioma (DIPG).

20. The method of any one of claims 17-19, wherein Akt is activated in the subject after administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

21. A method for increasing muscle strength in a subject identified or diagnosed with myasthenia gravis, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

22. A method for preventing or reversing myasthenia gravis in a subject, the method comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

23. The method of any one of claims 2-5 and 21-22, wherein after administering a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to the subject, magnetic resonance imaging (MRI) is used to assess the increase in muscle mass measured in the subject.

24. The method of any one of claims 2-5 and 21-23, comprising measuring the increase in grip strength using a force gauge.

25. The method of any one of claims 2-5 and 21-23, comprising measuring an increase in grip strength of at least 10% using a dynamometer.

26. A method for reducing or preventing cellular senescence in a subject, comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

27. The method of claim 26, wherein the method includes preventing cellular senescence of the object.

28. A method for increasing the motor capacity of a subject, comprising administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

29. The method of claim 28, wherein increasing the subject's motor capacity comprises increasing the number of voluntary muscle contractions of the subject prior to administering a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to the subject.

30. The method of any one of claims 28-29, wherein increasing the subject's motor capacity comprises increasing the duration of the subject's voluntary muscle contractions prior to administering therapeutically effective amounts of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to the subject.

31. The method of any one of claims 28-30, comprising determining an increase in walking distance during a 10-meter walking test after administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

32. The method of any one of claims 38-31, comprising determining a reduction in the time required for a subject to complete a 100-meter walk test after administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

33. The method of any one of claims 1-32, wherein, after administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof, an increase in AMPK activation, inhibition of mTORC1, inhibition of S6 kinase or any combination thereof is determined in the subject.

34. The method of any one of claims 1-33, wherein metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof are administered orally.

35. The method of any one of claims 1-34, wherein the metformin or a pharmaceutically acceptable salt thereof is administered daily.

36. The method of claim 35, wherein the dose of the metformin-based free base, metformin or a pharmaceutically acceptable salt thereof, is from about 500 mg to about 3000 mg.

37. The method of any one of claims 35-36, wherein the dose of the metformin-based free base, metformin or a pharmaceutically acceptable salt thereof, is from about 500 mg to about 1750 mg.

38. The method of any one of claims 1-37, wherein the rapamycin or a pharmaceutically acceptable salt thereof is administered daily.

39. The method of claim 38, wherein the dose of rapamycin based on the free base of rapamycin, or a pharmaceutically acceptable salt thereof, is from about 0.1 mg to about 2 mg.

40. The method according to any one of claims 38-39, wherein the dose of rapamycin based on the free base of rapamycin, or a pharmaceutically acceptable salt thereof, is from about 0.5 mg to about 1 mg.

41. The method according to any one of claims 38-40, wherein the dose of rapamycin based on the free base of rapamycin, or a pharmaceutically acceptable salt thereof, is about 0.7 mg.

42. The method of any one of claims 1-41, wherein metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof are administered simultaneously.

43. The method of claim 42, wherein metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof are administered simultaneously as a fixed dosage form.

44. The method of any one of claims 1-37, wherein the rapamycin or a pharmaceutically acceptable salt thereof is administered once weekly.

45. The method of claim 44, wherein the dose of rapamycin based on the free base of rapamycin, or a pharmaceutically acceptable salt thereof, is from about 1 mg to about 10 mg.

46. ​​The method according to any one of claims 44-45, wherein the dose of rapamycin based on the free base of rapamycin, or a pharmaceutically acceptable salt thereof, is from about 2 mg to about 10 mg.

47. The method according to any one of claims 44-46, wherein the dose of rapamycin based on the free base of rapamycin, or a pharmaceutically acceptable salt thereof, is about 5 mg.

48. The method of any one of claims 1-47, wherein the subject was not given cyclosporine, tacrolimus, and mycophenolate mofetil prior to administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof within one month of administration.

49. The method of any one of claims 1-48, wherein the subject has not been identified or diagnosed with a kidney-related disease.

50. The method of any one of claims 1-49, wherein the subject has not been identified or diagnosed with a liver-related disease.

51. The method of any one of claims 1-50, wherein the subject has not been identified or diagnosed with a heart-related disease.

52. The method of any one of claims 1-51, wherein the subject is not identified or diagnosed with diabetes.

53. The method of any one of claims 1-52, wherein the subject has not been identified or diagnosed as having abnormal endocrine function.

54. The method of any one of claims 1-53, wherein the subject has not been given a therapeutic agent that modulates the insulin transduction pathway prior to administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof within one year thereafter.

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