A combination of metformin and rapamycin for treating neurological, muscular, and proliferative disorders.

Abstract

This disclosure relates to a method for treating neurological, myocardial, and proliferative disorders using a combination of metformin and rapamycin. TIFF2026514863000009.tif86166

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 460,487, filed on April 19, 2023, which is hereby incorporated by reference in its entirety.

[0002] Technical Field The present disclosure relates to methods of treating disorders, such as neurological disorders, muscular disorders, and proliferative disorders, using combinations of metformin and rapamycin.

Background Art

[0003] Background The pathophysiology underlying some neurological, muscular, and proliferative disorders (such as myotonic dystrophy type 1 (DM1), Duchenne muscular dystrophy (DMD), and spinocerebellar ataxia type 3 (SCA - 3)) is associated with impaired activation of adenosine monophosphate - activated protein kinase (AMPK), dysfunction of the mammalian target of rapamycin complex 1 (mTORC1), and / or disruption of autophagic flux. There are no known treatments for neurological, muscular, and proliferative disorders such as DM1, DMD, and SCA - 3, and there are few existing treatment methods effective in treating these disorders that have a desirable profile for adverse effects. Moreover, many of such treatment methods only alleviate symptoms and improve the quality of life of patients. Treatment methods for DM1 include, for example, mexiletine, β - blockers, and angiotensin - converting enzyme (ACE) inhibitors. Treatment methods for DMD include, for example, prednisone and deflazacort. New treatment methods for neuromuscular diseases, including gene therapy, small - molecule therapy, and RNA - targeted therapy, are still in the experimental stage and require further testing before becoming available for clinical use.

[0004] Therefore, there is a need for a method to treat and / or prevent neurological, myocardial, and proliferative disorders associated with dysactivation of activated adenosine monophosphate-activated protein kinase (AMPK), dysfunction of the mammalian target of rapamycin complex 1 (mTORC1), and / or disruption of the autophagy flux, which not merely manages or improves the symptoms of such disorders, but also has fewer serious side effects and is conveniently administered in a single dosage form. [Overview of the Initiative]

[0005] overview Methods for treating or preventing neurological, myocardial, or proliferative disorders in subjects requiring such treatment are described herein, comprising the step of administering a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to the subject, wherein the neurological, myocardial, or proliferative disorder is selected from the group including 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 endogenous pontine glioma (DIPG).

[0006] In some embodiments, neurological, myocardial, or proliferative disorders constitute myotonic dystrophy type 1 (DM1). In some embodiments, a reduction in reactive oxygen species (ROS) is measured in subjects after a step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0007] In some embodiments, a neurological, muscular, or proliferative disorder constitutes Duchenne muscular dystrophy (DMD).

[0008] In some aspects, neurological, muscular, or proliferative disorders constitute spinocerebellar ataxia type 3 (SCA-3).

[0009] In some embodiments, a neurological, muscular, or proliferative disorder constitutes Alzheimer's disease.

[0010] In some embodiments, a neurological, myocardial, or proliferative disorder constitutes Parkinson's disease.

[0011] In some embodiments, vascular dementia involves neurological, myocardial, or proliferative disorders.

[0012] In some embodiments, neurological, myocardial, or proliferative disorders constitute Lewy body dementia (DLB).

[0013] In some embodiments, Huntington's disease (HD) is characterized by neurological, muscular, or proliferative disorders.

[0014] In some embodiments, a neurological, muscular, or proliferative disorder is amyotrophic lateral sclerosis (ALS).

[0015] In some embodiments, neurological, myocardial, or proliferative disorders constitute Lafora disease.

[0016] In some embodiments, the method includes a step of preventing injury.

[0017] In some embodiments, nerve damage, muscle damage, or proliferative disorder is glioblastoma.

[0018] In some embodiments, the neurological, myocardial, or proliferative disorder is diffuse endogenous pontine glioma (DIPG). In some embodiments, Akt is activated in the subject after a step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0019] Some embodiments provide a method for treating or preventing a neuronal cancer in a subject requiring such treatment, comprising the step of administering a therapeutically effective dose to the subject of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof, wherein the cancer is glioblastoma or diffuse endogenous pontine glioma (DIPG).

[0020] In some aspects, nerve cancer is glioblastoma.

[0021] In some embodiments, the cancer of the nerve is diffuse endogenous pontine glioma (DIPG). In some embodiments, Akt is activated in the subject after a step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0022] Some embodiments provide a method for increasing muscle strength in a subject who has been confirmed or diagnosed with muscle weakness, comprising the step of administering a therapeutically effective dose to the subject of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0023] Several embodiments provide a method for preventing or reversing muscle weakness in a subject in need, comprising the step of administering a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to the subject. In some embodiments, after the step of administering a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to the subject, an increase in muscle mass is measured in the subject using magnetic resonance imaging (MRI) evaluation.

[0024] In some embodiments, the method includes a step of preventing cellular senescence in a subject.

[0025] Some embodiments provide a method of increasing motility 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.

[0026] In some embodiments, the method comprises increasing the number of voluntary muscle contractions per minute in the subject as compared to before the therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof is administered to the subject.

[0027] In some embodiments, the method comprises increasing the duration of voluntary muscle contractions in the subject as compared to before the therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof is administered to the subject.

[0028] In some embodiments, the method comprises determining an increase in the distance walked by the subject during a 10-meter walk test after the step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0029] In some embodiments, the method comprises determining a decrease in the time taken for the subject to complete a 100-meter walk test after the step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0030] In some embodiments, an increase in AMPK activity, inhibition of mTORC1, inhibition of S6 kinase, or any combination thereof is determined in the subject after the step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0031] 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.

[0032] 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.

[0033] In some embodiments, rapamycin or a pharmaceutically acceptable salt thereof is administered daily.

[0034] 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.

[0035] 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.

[0036] In some embodiments, rapamycin or a pharmaceutically acceptable salt thereof is administered weekly.

[0037] In some embodiments, the dose of rapamycin or a 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 a 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 a pharmaceutically acceptable salt thereof is about 5 mg based on the free base of rapamycin.

[0038] In some embodiments, cyclosporine, tacrolimus, and mycophenolate mofetil were not administered to the subjects within one month prior to administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the subjects have not been confirmed or diagnosed with a kidney-related disease. In some embodiments, the subjects have not been confirmed or diagnosed with a liver-related disease. In some embodiments, the subjects have not been confirmed or diagnosed with a heart-related disease. In some embodiments, the subjects have not been confirmed or diagnosed with diabetes mellitus. In some embodiments, the subjects have not been confirmed or diagnosed with abnormal endocrine function. In some embodiments, the subjects have not been administered any insulin signaling pathway-modulating therapeutic agents within one year prior to administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0039] Details of one or more aspects of the present invention are described in the following description. Other features and advantages of the present invention will be apparent from this description and the drawings, as well as from the claims. [Brief explanation of the drawing]

[0040] [Figure 1] This figure shows the design and timeline of a study on the effects of rapamycin, metformin, and combinations thereof on the lifespan and healthy lifespan of C. elegans. [Figure 2]These are a series of survival curves for N2 wild-type worms treated with rapamycin, metformin, combination A, combination B, combination C, and combination D. [Figure 3] These are a series of survival curves up to day 17.5 (corresponding to approximately 50% survival in the negative control DMSO 1%) for N2 wild-type worms treated with rapamycin, metformin, combination A, combination B, combination C, and combination D. [Figure 4] These are a series of survival curves up to day 20 (corresponding to approximately 25% survival in the negative control DMSO 1%) for N2 wild-type worms treated with rapamycin, metformin, combination A, combination B, combination C, and combination D. [Figure 5] Figures 5A–5D are bar graphs showing the time (hours) from L4 larval injection to the laying of the first (Figure 5A) and last (Figure 5B) eggs, as well as the oviposition period (Figure 5C) and the average number of eggs laid per worm during the oviposition period (Figure 5D), observed in N2 wild-type worms treated with rapamycin, metformin, combination A, combination B, combination C, and combination D. [Figure 6] This is a bar graph showing the growth (area under the curve) of N2 wild-type worms treated with rapamycin, metformin, combination A, combination B, combination C, and combination D. [Figure 7A] Figures 7A–7D are plots of worm motility, showing N2 wild-type worms treated with rapamycin, metformin, combination A, combination B, combination C, and combination D (Figure 7A: head amplitude; Figure 7B: mid-section amplitude; Figure 7C: tail amplitude; Figure 7D: flexion frequency; Figure 7E: velocity). [Figure 7B] Refer to the explanation in Figure 7A. [Figure 7C] Refer to the explanation in Figure 7A. [Figure 7D] Refer to the explanation in Figure 7A. [Figure 7E] Refer to the explanation in Figure 7A. [Figure 8A]Figures 8A–8C are radar charts showing the worm motility (head amplitude, midsection amplitude, tail amplitude, velocity, and flexion frequency) at different stages (Stage 1: D0–D5; Stage 2: D6–D10; Stage 3: D11–D15; Stage 4: D16–D20) of N2 wild-type worms treated with rapamycin and metformin (Figure 8A), combination A and combination B (Figure 8B), and combination C and combination D (Figure 8C). [Figure 8B] See the explanation in Figure 8A. [Figure 8C] See the explanation in Figure 8A. [Modes for carrying out the invention]

[0041] Detailed explanation This specification describes (i) methods for treating and / or preventing neurological, myocardial, or proliferative disorders, or neurological, myocardial, or proliferative disorders, or neurological, myocardial, or proliferative conditions, and (ii) methods for reducing and / or preventing age-related processes (e.g., cellular senescence), the methods comprising the step of administering metformin or a pharmaceutically acceptable salt thereof in combination with rapamycin or a pharmaceutically acceptable salt thereof. The AMP-activated protein kinase (AMPK) pathway and the mammalian target of rapamycin (mTOR) pathway are two signaling pathways that play a role in cellular metabolism, growth, and proliferation. AMPK activation and mTOR inhibition are promising targets for the treatment of neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease, because they can (1) protect neurons from damage and improve cognitive function; (2) enhance muscle protein synthesis, making them promising treatments for muscle disorders, such as muscular dystrophy and myotonic dystrophy; and (3) suppress tumor growth and induce tumor cell death, making them promising strategies for cancer therapy. Surprisingly, the inventors have found that the aforementioned disorders can be treated synergistically with metformin (which, for example, activates AMPK) and rapamycin (which, for example, inhibits mTORC1), thus reducing the required dose and consequently reducing the adverse effects resulting from targeting undesirable metabolic pathways. Further benefits include, for example, reduced insulin and insulin growth factor 1 (IGF-1) concentrations, induction of energy stress, restoration of cerebral blood flow (CBF) and cerebral vascular density, reduced cerebral amyloid angiopathy and cerebral microhemorrhages, reduced inflammatory cytokines (e.g., IL-6), and reduced pro-inflammatory type 1 helper T cells (Th1) and type 17 helper T cells (Th17). In addition, the safety and efficacy profiles of metformin and rapamycin have been extensively studied.While we do not wish to be bound by theory, these two agents are thought to have complementary profiles in terms of side effects. For example, metformin may reduce the risk of hyperglycemia and hyperlipidemia caused by rapamycin, while rapamycin may reduce the gastrointestinal side effects of metformin. In addition, the combination of metformin and rapamycin may have synergistic effects on health and lifespan.

[0042] definition As used herein, the terms “about” and “approximately” are interchangeable and, when used to modify a number, encompass a range of uncertainty of that number, from 0% to 10%.

[0043] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context explicitly indicates otherwise.

[0044] As used herein, the terms “to treat” or “treatment” refer to therapeutic or palliative measures. Beneficial or desirable clinical outcomes include, but are not limited to, the relief of all or some symptoms associated with a disease or disorder or condition; a reduction in the severity of the disease; stabilization of the disease state (i.e., no worsening of the disease); delay or slowing of disease progression; improvement or reduction of the disease state (e.g., symptoms of one or more diseases); and remission (whether partial or complete). “Treatment” may also mean extending survival compared to the survival expected without treatment.

[0045] When used herein, the term "prevent" means preventing the occurrence, recurrence, or spread of all or part of the diseases or conditions described herein, or all or part of their symptoms. The term "preventive measures" means actions taken to prevent the occurrence, recurrence, or spread of all or part of the diseases or conditions described herein.

[0046] As used herein, the terms “subject,” “individual,” and “patient” are interchangeable and refer to any animal, which includes mammals such as mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses, primates, and humans. In some embodiments, the subject is a human. In some embodiments, the subject is experiencing and / or exhibiting symptoms of at least one disease or disorder to be treated and / or prevented.

[0047] As used herein, the term “adverse effect” refers to an undesirable effect resulting from an alteration of the normal physiological function of the subject.

[0048] When used herein, the term “pharmaceutically acceptable composition” is intended to encompass products comprising an active ingredient and an inert component constituting a support, as well as any products directly or indirectly resulting from any combination, complex formation, or aggregation of any two or more active ingredients, or from the dissociation of one or more active ingredients, or from other types of reactions or interactions of one or more active ingredients. Thus, the pharmaceutically acceptable compositions of the Disclosure include any compositions prepared by mixing the compounds of the Disclosure or pharmaceutically acceptable salts thereof with a pharmaceutically acceptable support.

[0049] The terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” refer to a carrier or adjuvant that can be administered to a patient together with the compounds of this disclosure or pharmaceutically acceptable salts thereof, and which does not impair their pharmacological activity and is nontoxic when administered in a dose sufficient to deliver a therapeutic amount of the compounds. In some embodiments, each component is “pharmaceutically acceptable” in the sense that it is compatible with other components of the pharmaceutical formulation and is suitable for use in contact with human and animal tissues or organs with a reasonable benefit / risk ratio without excessive toxicity, irritation, allergic reactions, immunogenicity, or other problems or complications. For example, see: "Remington: The Science and Practice of Pharmacy", 21st ed.; Lippincott Williams & Wilkins: Philadelphia, PA, 2005; "Handbook of Pharmaceutical Excipients", 6th ed.; Rowe et al., Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; "Handbook of Pharmaceutical Additives", 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007; "Pharmaceutical Preformulation and Formulation", 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009.

[0050] As used herein, the term “fixed dosage form” refers to the simultaneous administration of two or more therapeutic agents using a single dosage form (e.g., 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 as a fixed dosage form, both drugs are administered together as a single dosage form (e.g., an oral dosage form such as a pill, tablet, or capsule) containing both metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the fixed dosage form may include metformin, rapamycin, and one or more additional therapeutic agents.

[0051] The term “abnormally” means, for example, when used in terms such as “abnormally high,” “abnormally elevated,” or “abnormally low,” a deviation from the range of a reference parameter found in a healthy subject that can be recognized by a healthcare professional (e.g., a physician, nurse practitioner, clinical laboratory technician, nurse practitioner, physiotherapist, or physician assistant) and that can be considered to indicate or foreshadow a dysfunction or pathological condition. Furthermore, “abnormal” may, in some embodiments, refer to a physiological response that persists beyond the point from which a normal person recovers; or a physiological response that is excessive in degree and / or duration compared to what occurs in a normal, healthy subject.

[0052] As used herein, “autophagy” refers to a naturally conserved degrading mechanism in cells that breaks down, removes, and recycles unwanted or dysfunctional components within the cell. In some embodiments, promoting autophagy in a subject involves increasing the frequency of occurrence of the number of cells and / or cellular components undergoing autophagy and / or the autophagy process in the subject.

[0053] When used herein, the term “therapeutic effective dose” refers to the amount of one or more active chemical entities or pharmacokinetics (e.g., metformin and rapamycin) administered that elicits a biological or therapeutic response in the desired tissue, system, animal, individual, or human. In some embodiments, such response includes reduction and / or mitigation of signs, symptoms, or causes of a disease, or any other desirable change in a biological system. The appropriate “therapeutic effective dose” in any individual case is determined using any appropriate technique, such as a dose-escalation study.

[0054] The term "pharmaceutically acceptable" means a compound, material, composition, and / or dosage form that, within reasonable medical judgment, is suitable for use in contact with human and animal tissues in a reasonable benefit-to-risk ratio without excessive toxicity, irritation, allergic reactions, or other problems or complications. The term "pharmaceutically acceptable salt" means a salt formed by adding 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, etc. The term “pharmaceutically acceptable salt” may also refer to a pharmaceutically acceptable addition salt prepared by reacting a compound having an acidic group with a base to form a salt, such as: ammonium salts; alkali metal salts such as sodium or potassium salts; alkaline earth metal salts such as calcium or magnesium salts; organic bases such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine salts; and amino acids such as arginine, lysine, etc., or by other previously established methods. A pharmaceutically acceptable salt is not specifically limited as long as it is available for medicinal use. Examples of salts formed by the compounds described herein with bases include: salts with inorganic bases such as sodium, potassium, magnesium, calcium, and aluminum; salts with organic bases such as methylamine, ethylamine, and ethanolamine; salts with basic amino acids such as lysine and ornithine; and ammonium salts.The salt may be an acid addition salt, which is specifically exemplified by acid addition salts with the following: mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as 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 acidic amino acids such as aspartic acid and glutamic acid.

[0055] For the purposes of clarification, unless otherwise specified herein, if a variable in a subject (e.g., a condition, characteristic, state, parameter, score, evaluation, test, or statistic) increases, decreases, or improves, such increase, decrease, or improvement shall be measured, evaluated, or obtained, for example, in comparison to the same variable measured, evaluated, or obtained prior to the commencement of treatment (e.g., prior to the administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof), unless otherwise specified herein. The variable may be a single measurement, evaluation, or score; an average of multiple measurements, evaluations, or scores; or a daily average of multiple measurements, evaluations, or scores. Unless otherwise specified, measurements, evaluations, or scores are typically performed within one 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 administering metformin and / or rapamycin. For example, a decrease in the frequency of symptom occurrence in a subject may occur, for instance, when the number or average number of symptomatic episodes perceived by the subject during a certain period after administration of metformin and / or rapamycin is less than the number or average number of symptomatic episodes perceived by the subject during the same length of period before administration of metformin and / or rapamycin.

[0056] In this disclosure, methods are provided for treating or preventing neurological, myocardial, or proliferative disorders, or neurological, myocardial, or proliferative disorders, or neurological, myocardial, or proliferative conditions, in subjects where such treatment is needed. In some embodiments, the method includes a step of administering a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to a subject. In some embodiments, the neurological, myocardial, or proliferative disorders are selected from the group including 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 endogenous pontine glioma (DIPG).

[0057] In some embodiments, the method includes a step of determining whether the impairment is related to impaired activation of activated adenosine monophosphate-activated protein kinase (AMPK). In some embodiments, the method includes a step of determining whether the impairment is related to dysfunction of the mammalian target (mTORC1) of rapamycin complex 1. In some embodiments, the method includes a step of determining whether the impairment is related to disruption of the autophagy flux. In some embodiments, if it is determined that the impairment is related to impaired activation of activated adenosine monophosphate-activated protein kinase (AMPK), hyperactivation of the mammalian target (mTORC1) of rapamycin complex 1, and / or disruption of the autophagy flux, the method includes a step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to the target.

[0058] Some embodiments provide methods for treating or preventing neurological, myocardial, or proliferative disorders, or neurological, myocardial, or proliferative disorders, or neurological, myocardial, or proliferative conditions, in subjects requiring such treatment, the methods comprising the step of administering to a subject a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof, wherein the neurological, myocardial, or proliferative disorders are selected from the group including 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 endogenous pontine glioma (DIPG).

[0059] Several embodiments provide a method for treating neurological, myocardial, or proliferative disorders in subjects requiring such treatment, the method comprising the step of administering a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to a subject, wherein the neurological, myocardial, or proliferative disorder is selected from the group including 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 endogenous pontine glioma (DIPG).

[0060] Several embodiments provide a method for preventing neurological, myocardial, or proliferative disorders in subjects requiring such treatment, the method comprising the step of administering a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to a subject, wherein the neurological, myocardial, or proliferative disorder is selected from the group including 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 endogenous pontine glioma (DIPG).

[0061] Some embodiments provide methods for treating or preventing neurological, myocardial, or proliferative disorders in subjects requiring such treatment, the methods comprising: (a) determining whether the disorder is related to impaired activation of activated adenosine monophosphate-activated protein kinase (AMPK), dysfunction of the mammalian target of rapamycin complex 1 (mTORC1), and / or disruption of the autophagy flux; and (b) The step includes administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to the target when it is determined that the disorder is related to impaired activation of activated adenosine monophosphate-activated protein kinase (AMPK), hyperactivation of the mammalian target of rapamycin complex 1 (mTORC1), and / or disruption of the autophagy flux; wherein the neurological, myocardial, or proliferative disorder is selected from the group including 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 endogenous pontine glioma (DIPG).

[0062] In some embodiments, dysfunction of the mammalian target of rapamycin complex 1 (mTORC1) includes hyperactivation of the mammalian target of rapamycin complex 1 (mTORC1).

[0063] In some embodiments, subjects have neurological, myocardial, or proliferative disorders selected from the group including 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 endogenous pontine glioma (DIPG).

[0064] Some embodiments provide methods for treating or preventing neurological, myocardial, or proliferative disorders in subjects associated with impaired activation of activated adenosine monophosphate-activated protein kinase (AMPK), dysfunction of the mammalian target of rapamycin complex 1 (mTORC1), and / or disruption of autophagy flux, the methods being: A step to determine whether neurological, myocardial, or proliferative disorders are associated with impaired activation of activated adenosine monophosphate-activated protein kinase (AMPK), dysfunction of the mammalian target of rapamycin complex 1 (mTORC1), and / or disruption of the autophagy flux; and The step involves administering therapeutically effective doses of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to subjects determined to have neurological, myocardial, or proliferative disorders associated with impaired activation of activated adenosine monophosphate-activated protein kinase (AMPK), dysfunction of the mammalian target of rapamycin complex 1 (mTORC1), and / or disruption of autophagy flux. This includes, where neurological, myocardial, and proliferative disorders are selected from the group including 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 endogenous pontine glioma (DIPG).

[0065] Some embodiments provide methods for treating or preventing neurological, myocardial, or proliferative disorders in subjects associated with dysactivation of activated adenosine monophosphate-activated protein kinase (AMPK), dysfunction of the mammalian target of rapamycin complex 1 (mTORC1), and / or disruption of the autophagy flux, wherein the method has neurological or neuromuscular disorders associated with dysactivation of activated adenosine monophosphate-activated protein kinase (AMPK), dysfunction of the mammalian target of rapamycin complex 1 (mTORC1), and / or disruption of the autophagy flux. The step of administering a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to subjects who have been confirmed or diagnosed with the following conditions: where the neurological, myocardial, or proliferative disorder is selected from the group including 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 endogenous pontine glioma (DIPG).

[0066] Some embodiments provide a method for selecting a treatment or preventive measure suitable for a subject having a neurological disorder, muscle disorder, or proliferative disorder, the method is A step of determining whether the impairment in the subject is related to impaired activation of activated adenosine monophosphate-activated protein kinase (AMPK), dysfunction of the mammalian target of rapamycin complex 1 (mTORC1), and / or disruption of the autophagy flux, resulting in neurological, myocardial, or proliferative impairment; and a step of selecting treatment or prophylactic measures appropriate for the subject, including the administration of therapeutically effective doses of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. This includes, where neurological, myocardial, or proliferative disorders are selected from the group including 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 endogenous pontine glioma (DIPG).

[0067] Some embodiments provide a method for selecting a subject suitable for treatment or prophylaxis involving the administration of therapeutically effective doses of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof, the method A step to identify subjects having neurological, myocardial, or proliferative disorders associated with dysactivation of activated adenosine monophosphate-activated protein kinase (AMPK), dysfunction of the mammalian target of rapamycin complex 1 (mTORC1), and / or disruption of the autophagy flux; and The step of selecting subjects suitable for treatment or prophylaxis involving the administration of therapeutically effective doses of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. This includes, where neurological, myocardial, or proliferative disorders are selected from the group including 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 endogenous pontine glioma (DIPG).

[0068] Several embodiments provide a method for selecting a treatment or preventive measure suitable for a subject, the method being suitable for subjects confirmed or diagnosed with neurological, myocardial, or proliferative disorders associated with impaired activation of activated adenosine monophosphate-activated protein kinase (AMPK), dysfunction of the mammalian target of rapamycin complex 1 (mTORC1), and / or disruption of autophagy flux, and the method being suitable for subjects with metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. The procedure includes selecting a treatment or preventive measure comprising administering a therapeutically effective amount of the salt thereof, wherein the neurological, myocardial, or proliferative disorder is selected from the group including 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 endogenous pontine glioma (DIPG).

[0069] Some embodiments provide a method for selecting subjects having neurological, myocardial, or proliferative disorders that are suitable for treatment comprising the administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof, the method A step to determine whether the impairment in the subject is a neurological, myocardial, or proliferative disorder associated with AMPK deficiency and / or mTORC1 hyperactivation; and The step of selecting subjects who have been determined to have neurological, myocardial, or proliferative disorders associated with impaired activation of activated adenosine monophosphate-activated protein kinase (AMPK), dysfunction of the mammalian target of rapamycin complex 1 (mTORC1), and / or disruption of autophagy flux, and who are suitable for treatment involving the administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. This includes, where neurological, myocardial, or proliferative disorders are selected from the group including 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 endogenous pontine glioma (DIPG).

[0070] In some embodiments, the method includes a step of selecting a treatment appropriate for the subject. In some embodiments, the method includes a step of selecting a preventive measure appropriate for the subject.

[0071] In some embodiments, neurological, myocardial, or proliferative disorders are selected from a group that includes myotonic dystrophy type 1 (DM1), Duchenne muscular dystrophy (DMD), and spinocerebellar ataxia type 3 (SCA-3).

[0072] In some embodiments, neurological, myocardial, or proliferative disorders are selected from the group including Alzheimer's disease, Parkinson's disease, vascular dementia, Lewy body dementia (DLB), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and Lafora disease.

[0073] In some embodiments, neurological, myocardial, or proliferative disorders are selected from a group that includes glioblastoma and diffuse endogenous pontine glioma (DIPG).

[0074] In some embodiments, the method includes a step of addressing an impairment. In some embodiments, the method includes a step of preventing an impairment.

[0075] In some embodiments, the method includes a step of treating one or more symptoms. In some embodiments, the method includes a step of preventing one or more symptoms.

[0076] In some embodiments, neurological, muscular, or proliferative disorders constitute myotonic dystrophy type 1 (DM1). In some embodiments, treatment or prevention of myotonic dystrophy type 1 (DM1) includes treatment or prevention of one or more symptoms of myotonic dystrophy type 1 (DM1). In some embodiments, one or more symptoms are selected from the group including muscle weakness, muscle atrophy, myotonia, myalgia, ptosis, low blood oxygen saturation, intellectual disability, behavioral disturbances, fatigability, cataracts, retinal damage, shortness of breath, dyspnea, diabetes (e.g., type 1 or type 2 diabetes), sleep apnea, pneumonia, low testosterone, pilomatoma, hypogammaglobulinemia, erectile dysfunction, testicular dysfunction, gonadal atrophy, arrhythmia, cardiomyopathy, dysphagia, abdominal pain, irritable bowel syndrome (IBS), constipation, and diarrhea.

[0077] In some aspects, the symptom is muscle weakness.

[0078] In some embodiments, the symptom is muscle atrophy.

[0079] In some aspects, the symptom is muscle rigidity.

[0080] In some aspects, the symptom is muscle pain.

[0081] In some aspects, the symptom is ptosis (drooping eyelid).

[0082] In some aspects, the symptom is low blood oxygen saturation. In some aspects, low blood oxygen saturation is a blood oxygen saturation of less than 95% (for example, less than 94%, less than 93%, less than 92%, less than 91%, less than 90%, less than 89%, less than 88%, less than 87%, less than 86%, or less than 85%).

[0083] In some embodiments, the symptoms are intellectual disability. In some embodiments, the intellectual disability includes abnormally short attention duration. In some embodiments, the intellectual disability includes visuospatial cognitive impairment.

[0084] In some embodiments, the symptoms are behavioral disorders. In some embodiments, the behavioral disorders are autism spectrum disorder.

[0085] In some embodiments, the symptoms include easy fatigability. In some embodiments, easy fatigability includes excessive daytime sleepiness.

[0086] In some aspects, the symptom is cataracts.

[0087] In some aspects, the symptom is retinal damage.

[0088] In some aspects, the symptom is shortness of breath.

[0089] In some aspects, the symptom is shortness of breath.

[0090] In some aspects, the symptom is diabetes (for example, type 1 diabetes or type 2 diabetes).

[0091] In some forms, the symptom is sleep apnea.

[0092] In some aspects, the symptoms are those of pneumonia.

[0093] In some aspects, the symptom is low testosterone. In some aspects, low testosterone is defined as total testosterone, expressed as ng / dL, being below the 50th percentile (for example, 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) among men of the same age or age group.

[0094] In some forms, the symptom is a piloma.

[0095] In some aspects, the symptom is hypogammaglobulinemia.

[0096] In some aspects, the symptom is erectile dysfunction.

[0097] In some aspects, the symptom is testicular dysfunction.

[0098] In some embodiments, the symptom is gonadal atrophy.

[0099] In some aspects, the symptom is arrhythmia.

[0100] In some manifestations, the symptom is cardiomyopathy.

[0101] In some aspects, the symptom is difficulty swallowing.

[0102] In some aspects, the symptom is abdominal pain.

[0103] In some aspects, the symptoms are those of irritable bowel syndrome (IBS).

[0104] In some aspects, the symptom is constipation.

[0105] In some cases, the symptom is diarrhea.

[0106] In some embodiments, a reduction in reactive oxygen species (ROS) is measured in subjects after a step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0107] In some embodiments, the reduction in reactive oxygen species (ROS) is measured in the tissue sample of the subject. In some embodiments, the reduction in reactive oxygen species (ROS) is measured in the blood of the subject.

[0108] Various methods for measuring reactive oxygen species in a target are known in the art, and these methods include, but are not limited to, electron spin resonance (ESR) and spectrophotometric methods.

[0109] 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 spectrophotometrics.

[0110] In some embodiments, a neurological, muscular, or proliferative disorder constitutes Duchenne muscular dystrophy (DMD).

[0111] In some embodiments, treatment or prevention of Duchenne muscular dystrophy (DMD) includes treatment or prevention of one or more symptoms of Duchenne muscular dystrophy (DMD). In some embodiments, one or more symptoms are selected from a group including muscle weakness, muscle atrophy, difficulty walking, reduced ability to perform daily tasks, falls, learning disability, gastrocnemius hypertrophy, easy fatigability, reduced motor function, shortness of breath, and lumbar lordosis.

[0112] In some aspects, the symptom is muscle weakness.

[0113] In some embodiments, the symptom is muscle atrophy.

[0114] In some aspects, the symptom is difficulty walking. In some aspects, difficulty walking includes ataxia, spatial disorientation, balance impairment, or dizziness.

[0115] In some aspects, the symptom is a decline in the ability to perform daily tasks.

[0116] In some embodiments, the symptom is falls. In this context, falls are defined as falls of an abnormal frequency that do not occur in otherwise healthy individuals (for example, falls determined by healthcare professionals, e.g., physicians, nurse practitioners, nurses, or medical technicians).

[0117] In some embodiments, the symptoms are a learning disability. In some embodiments, the learning disability is a reading disability. In some embodiments, the learning disability is a dyscalculia. In some embodiments, the learning disability is a writing disability.

[0118] In some embodiments, the symptom is gastrocnemius muscle hypertrophy.

[0119] In some embodiments, the symptoms include easy fatigability. In some embodiments, easy fatigability includes excessive daytime sleepiness.

[0120] In some aspects, the symptom is reduced motor function.

[0121] In some aspects, the symptom is shortness of breath.

[0122] In some manifestations, the symptom is lumbar lordosis.

[0123] In some aspects, neurological, muscular, or proliferative disorders constitute spinocerebellar ataxia type 3 (SCA-3).

[0124] In some embodiments, treatment or prevention of spinocerebellar ataxia type 3 (SCA-3) includes treatment or prevention of one or more symptoms of spinocerebellar ataxia type 3 (SCA-3). In some embodiments, one or more symptoms are selected from a group including involuntary eye movements, impaired hand-eye coordination, impaired balance and coordination, slurred speech, learning impairment, memory impairment, and ataxic gait.

[0125] In some aspects, the symptom is involuntary eye movements.

[0126] In some aspects, the symptom is a decrease in hand-eye coordination.

[0127] In some aspects, the symptoms are a decline in balance and coordination.

[0128] In some forms, the symptom is slurred speech.

[0129] In some aspects, the symptoms are those of a learning disability.

[0130] In some aspects, the symptom is memory impairment.

[0131] In some aspects, the symptom is ataxic gait.

[0132] In some embodiments, a neurological, muscular, or proliferative disorder constitutes Alzheimer's disease.

[0133] In some embodiments, treatment or prevention of Alzheimer's disease includes treatment or prevention of one or more symptoms of Alzheimer's disease. In some embodiments, one or more symptoms are selected from a group including memory loss, lack of flexibility and hesitation to new challenges, confusion, disorientation, obsessive-compulsive behavior, repetitive behavior, impulsive behavior, delusions, speech disorders, aphasia, sleep disorders, frequent and abnormal mood swings, depression, anxiety, frustration, difficulty performing spatial tasks, difficulty judging distance, agnosia, difficulty changing position or moving without assistance, weight loss, weight gain, and loss of speech ability.

[0134] In some embodiments, the symptom is memory loss. In some embodiments, the symptom is short-term memory loss. In some embodiments, the symptom is long-term memory loss. In some embodiments, memory loss includes misplacing objects, forgetting places and names of objects, repeating things regularly, and asking the same questions repeatedly.

[0135] In some aspects, the symptoms are a lack of flexibility and hesitation when it comes to trying new things.

[0136] In some aspects, the symptom is confusion.

[0137] In some aspects, the symptom is disorientation.

[0138] In some aspects, the symptoms are compulsive behaviors.

[0139] In some aspects, the symptoms are repetitive behaviors.

[0140] In some aspects, the symptoms are impulsive behaviors.

[0141] In some forms, the symptoms are delusions.

[0142] In some aspects, the symptom is a speech disorder.

[0143] In some aspects, the symptom is aphasia.

[0144] In some forms, the symptom is sleep disturbance.

[0145] In some aspects, the symptoms are frequent and abnormal mood swings.

[0146] In some forms, the symptoms are depressive.

[0147] In some ways, the symptoms are anxiety-inducing.

[0148] In some aspects, the symptom is frustration.

[0149] In some embodiments, the symptom is difficulty performing spatial tasks.

[0150] In some aspects, the symptom is difficulty in judging distance.

[0151] In some aspects, the symptom is agnosia.

[0152] In some embodiments, the symptom is difficulty in changing position or moving. In some embodiments, the symptom is present without assistance.

[0153] In some forms, the symptom is weight loss.

[0154] In some aspects, the symptom is weight gain.

[0155] In some forms, the symptom is a loss of speech ability.

[0156] In some embodiments, the method includes a step of determining that a subject has an abnormal amount of extracellular amyloid plaques and / or tau protein in the brain before administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the method includes a step of determining that a subject has an abnormal amount of extracellular amyloid plaques in the brain before administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the method includes a step of determining that a subject has an abnormal amount of tau protein in the brain before administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the method includes a step of determining that the extracellular amyloid plaques and / or tau protein in the brain are reduced after the step of administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0157] In some embodiments, the method includes a step of determining that a subject has an abnormal amount of intracellular neurofibrillary tangles in the brain before administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the method includes a step of determining that the intracellular neurofibrillary tangles in the brain are reduced after the step of administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0158] In some embodiments, determining the amount of extracellular amyloid plaques, tau protein, and / or intracellular neurofibrillary tangles in the brain involves performing imaging techniques on the brain of interest. In some embodiments, the imaging technique is magnetic resonance imaging (MRI). In some embodiments, the imaging technique is positron emission tomography (PET).

[0159] In some embodiments, a neurological, myocardial, or proliferative disorder constitutes Parkinson's disease.

[0160] In some embodiments, treatment or prevention of Parkinson's disease includes treatment or prevention of one or more symptoms of Parkinson's disease. In some embodiments, one or more symptoms are selected from a group including tremor, arm stiffness, leg stiffness, trunk stiffness, bradykinesia, impaired balance and coordination, and dysarthria.

[0161] In some aspects, the symptom is a tremor.

[0162] In some forms, the symptom is stiffness in the arm.

[0163] In some forms, the symptom is stiffness in the legs.

[0164] In some forms, the symptom is stiffness of the trunk.

[0165] In some manifestations, the symptom is bradykinesia (slowness of movement).

[0166] In some aspects, the symptoms are a decline in balance and coordination.

[0167] In some aspects, the symptom is difficulty speaking.

[0168] In some embodiments, neurological, myocardial, or proliferative disorders constitute vascular dementia. In some embodiments, treatment or prevention of vascular dementia includes treatment or prevention of one or more symptoms of vascular dementia. In some embodiments, one or more symptoms are selected from a group including loss of short-term memory, getting lost in familiar environments, impaired concentration and planning, difficulty managing money, difficulty following instructions, urinary incontinence, fecal incontinence, delusions, and hallucinations.

[0169] In some aspects, the symptom is a loss of short-term memory.

[0170] In some aspects, the symptom is getting lost in a familiar environment.

[0171] In some aspects, the symptoms include a decline in concentration and planning ability.

[0172] In some cases, the symptom is difficulty managing finances.

[0173] In some aspects, the symptom is difficulty following instructions.

[0174] In some aspects, the symptom is urinary incontinence.

[0175] In some aspects, the symptom is fecal incontinence.

[0176] In some forms, the symptoms are delusions.

[0177] In some aspects, the symptoms are hallucinations.

[0178] In some embodiments, neurological, myocardial, or proliferative disorders constitute Lewy body dementia (DLB).

[0179] In some embodiments, neurological, myocardial, or proliferative disorders constitute Lewy body dementia (DLB). In some embodiments, treatment or prevention of Lewy body dementia (DLB) includes treatment or prevention of one or more symptoms of Lewy body dementia (DLB). In some embodiments, one or more symptoms are selected from a group including hallucinations, muscle rigidity, bradykinesia, shuffling gait or leg tremors, tremors, loss of balance, abnormally high or low blood pressure, sweating, digestion, confusion, difficulty concentrating, memory loss, and dyssomnia.

[0180] In some forms, the symptoms are visual hallucinations.

[0181] In some embodiments, the symptom is muscle rigidity.

[0182] In some forms, the symptom is bradykinesia (i.e., slowness of movement).

[0183] In some aspects, the symptoms include shuffling gait or leg tremors.

[0184] In some aspects, the symptom is a tremor.

[0185] In some aspects, the symptom is an inability to maintain balance.

[0186] In some aspects, the symptoms are abnormally high or abnormally low blood pressure.

[0187] In some forms, the symptom is sweating.

[0188] In some aspects, the symptom is digestion.

[0189] In some aspects, the symptom is confusion.

[0190] In some aspects, the symptom is difficulty concentrating.

[0191] In some aspects, the symptom is memory loss.

[0192] In some cases, the symptom is difficulty sleeping.

[0193] In some embodiments, neurological, myocardial, or proliferative disorders constitute Huntington's disease (HD). In some embodiments, treatment or prevention of Huntington's disease (HD) includes treatment or prevention of one or more symptoms of Huntington's disease (HD). In some embodiments, one or more symptoms are selected from the group including chorea, tremor, rigidity, brady or abnormal eye movements, gait disturbance, postural disturbance, balance disturbance, dysphagia, dysarthria, impaired organization, repetitive thoughts, repetitive behaviors, repetitive actions, lack of impulse control, lack of social or behavioral awareness, inability to organize thoughts, learning difficulties, difficulty choosing words to speak, dysgraphia, depression, mania, irritability, withdrawal, insomnia, fatigability, suicidal ideation or suicidal thoughts, and apathy.

[0194] In some forms, the symptom is chorea.

[0195] In some aspects, the symptom is a tremor.

[0196] In some embodiments, the symptom is muscle rigidity.

[0197] In some embodiments, the symptom is slow or abnormal eye movements.

[0198] In some aspects, the symptom is gait disturbance.

[0199] In some aspects, the symptom is postural dysfunction.

[0200] In some aspects, the symptom is a balance disorder.

[0201] In some aspects, the symptom is difficulty swallowing.

[0202] In some aspects, the symptom is difficulty speaking.

[0203] In some aspects, the symptom is impaired physiological function.

[0204] In some forms, the symptom is repetitive thinking.

[0205] In some aspects, the symptoms are repetitive behaviors.

[0206] In some aspects, the symptom is repetitive behavior.

[0207] In some aspects, the symptom is a lack of impulse control.

[0208] In some aspects, the symptom is a lack of social or behavioral awareness.

[0209] In some aspects, the symptom is an inability to organize one's thoughts.

[0210] In some aspects, the symptoms make learning difficult.

[0211] In some aspects, the symptom is difficulty in choosing the right words to speak.

[0212] In some aspects, the symptom is dysgraphia.

[0213] In some forms, the symptoms are depressive.

[0214] In some aspects, the symptoms are manic.

[0215] In some manifestations, the symptoms include irritability.

[0216] In some forms, the symptom is social withdrawal.

[0217] In some forms, the symptom is insomnia.

[0218] In some aspects, the symptoms include easy fatigue.

[0219] In some forms, the symptom is suicidal ideation or suicidal thoughts.

[0220] In some aspects, the symptom is apathy.

[0221] In some embodiments, a neurological, muscular, or proliferative disorder constitutes amyotrophic lateral sclerosis (ALS). In some embodiments, treatment or prevention of amyotrophic lateral sclerosis (ALS) includes treatment or prevention of one or more symptoms of amyotrophic lateral sclerosis (ALS). In some embodiments, one or more symptoms are selected from a group including muscle weakness, difficulty walking, difficulty performing activities of daily living, clumsiness, slurred speech, dysphagia, muscle spasms, monoconvulsions, postural abnormalities, and falls.

[0222] In some aspects, the symptom is muscle weakness.

[0223] In some aspects, the symptoms include difficulty walking.

[0224] In some cases, the symptoms involve difficulty performing daily living activities.

[0225] In some forms, the symptom is awkwardness.

[0226] In some forms, the symptom is slurred speech.

[0227] In some aspects, the symptom is difficulty swallowing.

[0228] In some forms, the symptom is muscle spasms.

[0229] In some aspects, the symptom is a single contraction.

[0230] In some aspects, the symptom is postural abnormalities.

[0231] In some aspects, the symptom is falling.

[0232] In some embodiments, a neurological, myocardial, or proliferative disorder constitutes Lafora disease. In some embodiments, treatment or prevention of Lafora disease includes treatment or prevention of one or more symptoms of Lafora disease. In some embodiments, one or more symptoms are selected from the group including seizures, myoclonus, dementia, headaches, and hallucinations (e.g., visual hallucinations).

[0233] In some aspects, the symptom is a seizure.

[0234] In some forms, the symptom is myoclonus.

[0235] In some aspects, the symptoms are those of dementia.

[0236] In some aspects, the symptom is a headache.

[0237] In some forms, the symptoms are hallucinations (e.g., visual hallucinations).

[0238] In some embodiments, a neurological, myocardial, or proliferative disorder is glioblastoma. In some embodiments, treatment or prophylaxis of glioblastoma includes treatment or prophylaxis of one or more symptoms of glioblastoma. In some embodiments, one or more symptoms are selected from the group including headache, nausea, vomiting, blurred vision, diplopia, and seizures.

[0239] In some aspects, the symptom is a headache.

[0240] In some cases, the symptom is nausea.

[0241] In some aspects, the symptom is vomiting.

[0242] In some forms, the symptom is blurred vision.

[0243] In some manifestations, the symptom is double vision.

[0244] In some aspects, the symptom is a seizure.

[0245] In some embodiments, a neuropathy, myopathy, or proliferative disorder constitutes diffuse endogenous pontine glioma (DIPG). In some embodiments, treatment or prophylaxis of diffuse endogenous pontine glioma (DIPG) includes treatment or prophylaxis of one or more symptoms of diffuse endogenous pontine glioma (DIPG). In some embodiments, one or more symptoms are selected from the group including blurred vision, diplopia, dysphagia, and headache.

[0246] In some forms, the symptom is blurred vision.

[0247] In some manifestations, the symptom is double vision.

[0248] In some aspects, the symptom is abnormal eye movements.

[0249] In some aspects, the symptom is a headache.

[0250] Some embodiments provide a method for increasing muscle strength in subjects who have been confirmed or diagnosed with muscle weakness, the method comprising the step of administering a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to the subject.

[0251] Some embodiments provide a method for preventing or reversing muscle weakness in subjects in need, the method comprising administering a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to the subject.

[0252] Some embodiments provide methods for preventing, delaying, or reversing sarcopenia in subjects requiring such treatment, the methods comprising the step of administering a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to a subject.

[0253] This specification discloses a method for treating or preventing myotonia in subjects requiring it, the method comprising administering a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to the subject.

[0254] In some embodiments, the method includes a step of determining improvement on the stiffness visual analogue scale (VAS). In some embodiments, the method includes a step of determining improvement of at least one point (e.g., one, two, three, or four points) on the stiffness visual analogue scale (VAS). Further information regarding the stiffness VAS can be found below, which is incorporated herein by reference in its entirety: Hammaren, Elisabet & Kjellby-Wendt, Gunilla & Lindberg, Christopher, (2005), "Quantification of mobility impairment and self-assessment of stiffness in patients with myotonia congenita by the physiotherapist", Neuromuscular disorders.

[0255] In some embodiments, the method includes a step of determining a decrease in electrical activity in the muscle of interest. In some embodiments, the method includes a step of determining a decrease in electrical activity in the muscle of interest (e.g., a decrease of at least 1%, at least 2%, at least 5%, at least 7%, at least 10%, at least 15%, at least 20%, at least 30%, or at least 40%) using electromyography (EMG).

[0256] This specification discloses a method for preventing muscle cell necrosis in a subject, the method comprising administering a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to the subject.

[0257] This specification discloses a method for improving balance and / or coordination in a subject, the method comprising administering therapeutically effective amounts of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to the subject.

[0258] In some embodiments, the method includes a step of determining improvement in a 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 includes a step of determining improvement of at least 1 point (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 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, the determination of improvement in the short physical performance battery (SPPB) includes determining improvement in a three-stage balance test. In some embodiments, the determination of improvement in the short physical performance battery (SPPB) includes determining improvement in a walking speed test. In some embodiments, the determination of improvement in the short physical performance battery (SPPB) includes determining improvement in a chair stand test. Further information on SPPB can be found, for example, in the following, which is incorporated herein by reference in its entirety: 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.

[0259] In some embodiments, the method includes a step of determining an increase in grip strength, as measured by a dynamometer, after a step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. Further information can be found, for example, in the following, which is incorporated herein by reference in its entirety: Journal of Bodywork and Movement Therapies, 2020, 24(1), 235-243.

[0260] In some embodiments, the method includes a step of determining at least 5% of the grip strength measured by a dynamometer (e.g., an increase of 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%, or at least 95%) after a step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0261] Several embodiments provide a method for increasing motility in a subject, the method comprising administering a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to the subject. In some embodiments, the increase in motility in a subject comprises increasing the number of voluntary muscle contractions per unit 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 compared to before the administration of a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to the subject. In some embodiments, the increase in motility in a subject comprises increasing the number of voluntary muscle contractions per minute in the subject. In some embodiments, increased mobility in a subject includes increasing the duration of voluntary muscle contractions in the subject compared to before administration of a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, increased mobility in a subject includes reducing the time required for the subject to perform tasks or activities requiring exercise and / or muscle contraction compared to before administration of a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the method includes a step of determining improvements on the DM1-Active scale for activity and social participation after the step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. Further information on the DM1-Active scale for activity and social participation can be found, for example, below, which is incorporated herein by reference in its entirety: PLoS One 2015, 10: e0139944.

[0262] In some embodiments, the method includes a step of determining improvement in the InQoL questionnaire for health-related quality of life, following a step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the determination of improvement in the InQoL questionnaire for health-related quality of life includes a determination of improvement in the fatigue subdomain. In some embodiments, the determination of improvement in the InQoL questionnaire for health-related quality of life includes a determination of improvement in the activity subdomain. In some embodiments, the determination of improvement in the InQoL questionnaire for health-related quality of life includes a determination of improvement in the autonomy subdomain. Further information regarding the InQoL questionnaire for health-related quality of life can be found, for example, below, which is incorporated herein by reference in its entirety: Taylor VR., "Measuring healthy days; population assessment of health-related quality of life", Atlanta: US Centers for Disease Control and Prevention; 2000.

[0263] In some embodiments, the method includes, after the step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof, a step of determining improvement in at least one of the clinical parameters selected from the group consisting of an index of myotonia, parameters of muscle function and strength, parameters of quality of life, parameters of gait, and total mechanical work of gait. Further information regarding the index of myotonia, parameters of muscle function and strength, parameters of quality of life, parameters of gait, and total mechanical work of gait can be found, for example, below, which is incorporated herein by reference in whole: Brain 2018: 141; 2855-2865.

[0264] In some embodiments, the method includes a step of determining an increase in the distance walked by the subject during a 6-minute walk test (6MWT), following a step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the method includes a step of 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 by the subject during a 6-minute walk test (6MWT), following a step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. Further information on the 6-minute walk test (6MWT) can be found, for example, in the following, which is incorporated herein by reference in its entirety: Endocrinology 2005; 146: 1328-1337.

[0265] In some embodiments, the method includes a step of determining a reduction in the time it takes a subject to complete a 10-meter walk test, following a step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the method includes a step of 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 it takes a subject to complete a 10-meter walk test, following a step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. Further information regarding the 10-meter walk test can be found, for example, in the following, which is incorporated herein by reference in its entirety: Journal of Neurologic Physical Therapy, 2018; 42(2):174-220. In some embodiments, the method includes a step of determining improvement in at least one of the following: an index of myotonicity, muscle function and strength, quality of life, and total mechanical work, after a step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the method includes a step of 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%) of the time it took the subject to complete the 10-meter walk test. Further information regarding the 10-meter walk test can be found, for example, in the following, which is incorporated herein by reference in its entirety: Journal of Neurologic Physical Therapy, 2018; 42(2):174-220.

[0266] In some embodiments, the method includes a step of determining a reduction in the time it takes a subject to complete a 100-meter walk test, following a step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the method includes a step of 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 it takes a subject to complete a 100-meter walk test, following a step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. Further information regarding the 100-meter walk test can be found, for example, in the following, which is incorporated herein by reference in its entirety: 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.

[0267] In some embodiments, the method includes a step of determining an increase in muscle mass in a subject using magnetic resonance imaging (MRI) after a step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the method includes a step of determining an increase of at least 1% in muscle mass in a subject using magnetic resonance imaging (MRI) after a step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. Further information can be found, for example, in the following, each of which is incorporated herein by reference in its entirety: Eur. J. Neurol. 2022 Mar;29(3):843-854, or Radiography, 2015, 21(1), e35-e39.

[0268] In some embodiments, the method includes a step of determining improvement on the Muscular Impairment Rating Scale (MIRS) after administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, improvement on the MIRS includes an improvement of at least 1 point (e.g., 1, 2, 3, or 4 points) on the scale. For further information on the MIRS, see, for example, the following, which is incorporated herein by reference in its entirety: Neurology, 2001 Feb 13;56(3):336-40.

[0269] In some embodiments, the method includes a step of determining a decrease in time measured when the subject performs a Timed Up and Go (TUG) test, following a step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, the method includes a step of determining a decrease of at least 2% (e.g., at least 3% decrease, at least 4% decrease, at least 5% decrease, at least 7% decrease, at least 9% decrease, at least 11% decrease, at least 13% decrease, at least 15% decrease, at least 20% decrease, at least 25% decrease, at least 30% decrease, at least 35% decrease, at least 40% decrease, at least 50% decrease, at least 60% decrease, or at least 70% decrease) in time measured when the subject performs a Timed Up and Go (TUG) test. Further information regarding the Timed Up and Go (TUG) test can be found, for example, in the following, which is incorporated herein by reference in its entirety: Osteosarcopenia, 2022, pages 181–204.Other tests that may be used to assess the mobility of an individual include, but are not limited to, the following: the Pick-up Weight Test (see, for example, the entire text of which is incorporated herein by reference: Reuben DB, Siu AL., "An objective measure of physical function of elderly outpatients. The Physical Performance Test", J Am Geriatr Soc, 1990, vol. 38 (pg. 1105-12)), the Half Turn Test (see, for example, the entire text of which is incorporated herein by reference: 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)), and the Alternate Step Test (see, for example, the entire text of which is incorporated herein by reference: Anne Tiedemann, Hiroyuki Shimada, (Catherine Sherrington, Susan Murray, Stephen Lord, Age and Ageing, Volume 37, Issue 4, July 2008, Pages 430–435), and the stair climbing test (see, for example, the following, which is incorporated herein by reference in its entirety: Anne Tiedemann, Hiroyuki Shimada, Catherine Sherrington, Susan Murray, Stephen Lord, Age and Ageing, Volume 37, Issue 4, July 2008, Pages 430–435).

[0270] Some embodiments provide methods for reducing or preventing genomic instability, telomere shortening, epigenetic changes (e.g., DNA methylation, histone modification, or regulation of gene activity by RNA and / or proteins), dysregulation or deregulation of proteostasis, deregulation of nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and / or changes in intercellular communication in subjects requiring such reduction, the methods comprising the step of administering to a subject a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0271] Some embodiments provide methods for preventing genomic instability, telomere shortening, epigenetic changes (e.g., DNA methylation, histone modification, or regulation of gene activity by RNA and / or proteins), dysregulation or deregulation of proteostasis, deregulation of nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and / or changes in intercellular communication in subjects requiring such prevention, the methods comprising administering to a subject a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0272] In some embodiments, the method includes a step of reducing or preventing cellular senescence. In some embodiments, the method includes a step of preventing cellular senescence.

[0273] In some embodiments, the method includes a step of reducing or preventing genomic instability.

[0274] In some embodiments, the method includes a step of reducing or preventing telomere shortening.

[0275] In some embodiments, the method includes a step of reducing or preventing epigenetic changes (e.g., DNA methylation, histone modification, or regulation of gene activity by RNA and / or proteins).

[0276] In some embodiments, the method includes steps to reduce or prevent proteostasis dysregulation or derepression.

[0277] In some embodiments, the method includes steps to reduce or prevent derepression of nutrient sensing.

[0278] In some embodiments, the method includes steps to reduce or prevent mitochondrial dysfunction.

[0279] In some embodiments, the method includes steps to reduce or prevent stem cell exhaustion.

[0280] In some embodiments, the method includes steps to reduce or prevent changes in cell - cell communication.

[0281] Some embodiments provide a method for promoting autophagy in a subject, the method including administering to the subject a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0282] Some embodiments provide a method for darkening the hair color of a subject, the method including 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 hair color of the subject before administration of the therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof is white or gray.

[0283] In some embodiments, the darkening includes a change in hair color to brown. In some embodiments, the darkening includes a change in hair color to black.

[0284] Several embodiments provide methods for improving memory in subjects requiring it, the methods comprising administering therapeutically effective doses of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to the subject. In some embodiments, the improvement in memory in the subject is determined by a digit span test, a phonetic ordering test, the California Language Learning Test, the Ray Auditory Language Learning Test, or the Wechsler Memory Scale. Further information on the digit span test, the phonetic ordering test, the California Language Learning Test, the Ray Auditory Language Learning Test, and the Wechsler Memory Scale can be found, for example, in: Am Fam Physician. 2019, 99(2), 101-108.

[0285] In some embodiments, the method includes a step of administering a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to a subject, followed by a step of measuring improvement in a span test in the subject.

[0286] In some embodiments, the method includes a step of administering a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to a subject, followed by a step of measuring improvement in a speech-to-speech alignment test in the subject.

[0287] In some embodiments, the method includes a step of administering a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to a subject, followed by a step of measuring improvement in the subject on the California Language Learning Test.

[0288] In some embodiments, the method includes a step of administering a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to a subject, followed by a step of measuring improvement in the subject on the Ray Auditory Language Learning Test.

[0289] In some embodiments, the method includes a step of administering a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to a subject, followed by a step of measuring improvement in the subject on the Wechsler Memory Scale.

[0290] Some embodiments provide a method for preventing memory loss in subjects in need, the method comprising administering a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to the subject.

[0291] Some embodiments provide a method for treating or preventing neuronal cancer in subjects in need thereof, the method comprising the step of administering a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to a subject, wherein the cancer is glioblastoma or diffuse endogenous pontine glioma (DIPG).

[0292] In some aspects, the cancer of the nerve is glioblastoma. In some aspects, the cancer of the nerve is diffuse endogenous pontine glioma (DIPG).

[0293] Some embodiments provide a method for inhibiting cell proliferation, the method comprising the step of administering a therapeutically effective amount of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to the target. In some embodiments, the cells are mammalian cells. In some embodiments, the mammalian cells are brain cells or spinal cord cells. In some embodiments, the brain cells or spinal cord cells are glial cells, astrocytes, oligodendrocyte progenitor cells, neurons, or neural stem cells. In some embodiments, the inhibition is carried out in vivo. In some embodiments, the inhibition is carried out in vitro.

[0294] Some embodiments provide a method for treating a cancer of the nerve and / or inhibiting metastases associated with the cancer of the nerve, the method comprising the step of administering a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to the target, wherein the cancer is glioblastoma or diffuse endogenous pontine glioma (DIPG).

[0295] Some embodiments provide a method for providing supportive care to a cancer patient, the method comprising the step of administering a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to the target, wherein the cancer is glioblastoma or diffuse endogenous pontine glioma (DIPG).

[0296] Some embodiments provide methods for counteracting or preventing acquired resistance to anticancer drugs in subjects having a neurosurgical cancer, the method comprising the step of administering a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to the subject, wherein the cancer is glioblastoma or diffuse endogenous pontine glioma (DIPG).

[0297] Some embodiments provide a method for treating subjects having a neurosurgical cancer that is likely to develop resistance to anticancer drugs, the method comprising the step of administering a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to the subject, wherein the cancer is glioblastoma or diffuse endogenous pontine glioma (DIPG).

[0298] In some aspects, subjects have been identified or diagnosed with a neurological cancer selected from a group including glioblastoma and diffuse endogenous pontine glioma (DIPG).

[0299] In the field of clinical oncology, it is common practice to treat individual patients with cancer by using a combination of various forms of treatment. In clinical oncology, other elements of such a combination of treatment or therapy, added to metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof, may include, for example, surgery, radiotherapy, and chemotherapeutic agents such as other kinase inhibitors, signaling inhibitors, and monoclonal antibodies. For example, surgery may 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 may also be useful as an adjuvant for cancer treatment, that is, the combination may be used in combination with one or more additional therapies or therapeutic agents, such as chemotherapeutic agents acting by the same or different mechanisms of action.

[0300] In some embodiments of any method 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 including: 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, clopharabine, cytarabine, floxuridine, flu) darabine, 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, liposomal doxorubicin, epirubicin, idarubicin, barurubicin). Some therapeutic agents useful for treating glioblastoma include, but are not limited to, cyclophosphamide, danisza (naxitamab), dinutuximab, doxorubicin, naxitamab, unituxin (dinutuximab), vincristine sulfate, busulfan, and melphalan. Some useful therapies for treating diffuse endogenous pontine glioma (DIPG) include, but are not limited to, fractionated radiotherapy 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, at least one additional therapeutic agent is selected from the group including: 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, clopharabine, cytarabine, phloxuridine, 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), as well as antitumor antibiotics (e.g., daunorubicin, doxorubicin, liposomal doxorubicin, epirubicin, idarubicin, barurubicin), cyclophosphamide, danisza (naxitamab), dinutuximab, doxorubicin, naxitamab, unituxin (dinutuximab), vincristine sulfate, busulfan, melphalan, anti-GD2 CAR T cell agents, and anti-EGFR agents (e.g., nimotuzumab, gefitinib, and erlotinib).

[0301] Accordingly, methods for treating nerve cancers are also provided herein, comprising the step of administering to a patient in need a pharmaceutically acceptable combination for treating cancer, 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, the pharmaceutically acceptable combination for simultaneous, separate, or sequential use to treat the 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 amounts of the additional therapeutic agent, together are effective to treat the cancer; wherein the cancer is glioblastoma or diffuse endogenous pontine glioma (DIPG)).

[0302] Some embodiments provide a method for inhibiting the metastasis of nerve cancer, preventing the metastasis, assisting in the prevention of the metastasis, or reducing the symptoms of the metastasis in a patient who requires it, 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). The term "metastasis" is a term known in the art and this means the formation of additional tumors (eg solid tumors) at sites distant from the primary tumor in a subject or patient, where the additional tumors contain cancer cells that are the same or similar to the primary tumor.

[0303] In some embodiments, after the step of administering 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. In some embodiments, after the step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof, an increase in AMPK activation, inhibition of mTORC1, and inhibition of S6 kinase are determined in the subject.

[0304] In some embodiments, after the step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof, p70 S6 kinase is inhibited or inactivated in the subject.

[0305] In some embodiments, after the step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof, Akt is activated in the subject.

[0306] In some embodiments, the metformin or a pharmaceutically acceptable salt thereof is metformin hydrochloride.

[0307] In some embodiments, rapamycin or a pharmaceutically acceptable salt thereof is rapamycin.

[0308] In some embodiments, cyclosporine, tacrolimus, and mycophenolate mofetil were not administered to the subjects within one month prior to the administration of metformin or a pharmaceutically acceptable salt thereof, and rapamycin or a pharmaceutically acceptable salt thereof.

[0309] In some aspects, the subjects have not been identified or diagnosed with any kidney-related disease.

[0310] In some aspects, the subjects have not been identified or diagnosed with any liver-related disease.

[0311] In some aspects, the subjects have not been identified or diagnosed with any heart-related disease.

[0312] In some aspects, the subjects have not been confirmed or diagnosed with diabetes.

[0313] In some aspects, the subjects have not been identified or diagnosed with abnormal endocrine function.

[0314] In some embodiments, subjects have not received any insulin signaling pathway-modulating therapeutic agents within one year prior to administration of metformin or a pharmaceutically acceptable salt thereof, or rapamycin or a pharmaceutically acceptable salt thereof.

[0315] The dosage varies depending on the compound formulation, route of administration, the disorder being treated or prevented, the desired therapeutic outcome, etc., and is generally determined experimentally. Variations are inevitable depending on the target, host, and route of administration. Generally, the amount of the active compound in a unit dose of the preparation may be changed or adjusted to suit the specific application. For convenience, the total daily dose may be divided and administered in small amounts throughout the day.

[0316] In some embodiments, metformin or a pharmaceutically acceptable salt thereof is administered monthly, every three weeks, every two weeks, every ten days, every nine days, every eight days, every seven days, every six days, every five days, every four days, every three days, every two days, daily (i.e., every day), three times a week, twice 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., an "initial loading dose") is administered to the subject as the first dose of treatment. In some embodiments, the initial dose of metformin is higher than any subsequent doses administered to the subject.

[0317] In some aspects, each dose of metformin or a pharmaceutically acceptable salt thereof is approximately 100 mg to approximately 5000 mg based on the free base of metformin (for example, approximately 100 mg to approximately 500 mg, approximately 100 mg to approximately 800 mg, approximately 100 mg to approximately 1000 mg, approximately 500 mg to approximately 1000 mg, approximately 1000 mg to approximately 1500 mg, approximately 1500 mg to approximately 2250 mg, approximately 1500 mg to approximately 2000 mg, approximately 2000 mg to approximately 2500 mg, approximately 2250 mg to approximately 3000 mg, approximately 2500 mg to approximately 3000 mg, approximately 3000 mg to approximately 3500 mg, approximately 3500 mg to approximately 4000 mg, approximately 4000 mg to approximately 5000 mg, approximately 500 mg to approximately 3000 mg, approximately 500 to approximately 1750 mg The dosages are approximately mg, 500 mg to 1125 mg, 1125 mg to 2250 mg, 1500 mg to 3000 mg, 500 mg, 650 mg, 750 mg, 850 mg, 1000 mg, 1750 mg, 2000 mg, or 3000 mg. In some embodiments, the dose of metformin or a pharmaceutically acceptable salt thereof is approximately 500 to 3000 mg based on the free base of metformin. In some embodiments, the dose of metformin or a pharmaceutically acceptable salt thereof is approximately 1500 to 3000 mg based on the free base of metformin. In some embodiments, the dose of metformin or a pharmaceutically acceptable salt thereof is approximately 500 to 1750 mg based on the free base of metformin.

[0318] In some embodiments, the method comprises a step for treating or preventing myotonic dystrophy type 1 (DM1), wherein metformin or a pharmaceutically acceptable salt thereof is administered in a daily dose of approximately 1500 mg to approximately 3000 mg based on the free base of metformin.

[0319] In some embodiments, the method comprises a step of treating or prophylactically treating Duchenne muscular dystrophy, wherein metformin or a pharmaceutically acceptable salt thereof is administered in a daily dose of approximately 1500 mg to approximately 3000 mg based on the free base of metformin.

[0320] In some embodiments, the method comprises a step for treating or preventing spinocerebellar ataxia type 3 (SCA-3), wherein metformin or a pharmaceutically acceptable salt thereof is administered in a daily dose of approximately 1500 mg to approximately 3000 mg based on the free base of metformin.

[0321] In some embodiments, the method comprises a step for treating or preventing Alzheimer's disease, and metformin or a pharmaceutically acceptable salt thereof is administered in a daily dose of approximately 500 mg to approximately 1750 mg based on the free base of metformin.

[0322] In some embodiments, the method includes a step that promotes autophagy, and metformin or a pharmaceutically acceptable salt thereof is administered in a daily dose of approximately 500 mg to approximately 1750 mg based on the free base of metformin.

[0323] In some embodiments, the method comprises steps to reduce or prevent genomic instability, telomere shortening, epigenetic changes (e.g., DNA methylation, histone modification, or regulation of gene activity by RNA and / or proteins), dysregulation or deregulation of proteostasis, deregulation of nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and / or changes in intercellular communication, wherein metformin or a pharmaceutically acceptable salt thereof is administered in a daily dose of approximately 500 mg to approximately 1750 mg based on the free base of metformin.

[0324] In some embodiments, rapamycin or a pharmaceutically acceptable salt thereof is administered monthly, every three weeks, every two weeks, every ten days, every nine days, every eight days, every seven days, every six days, every five days, every four days, every three days, every two days, daily (i.e., every day), three times a week, twice a week, twice a day, or three times a day. In some embodiments, rapamycin or a pharmaceutically acceptable salt thereof is administered daily. In some embodiments, rapamycin or a pharmaceutically acceptable salt thereof is administered weekly. In some embodiments, rapamycin or a pharmaceutically acceptable salt thereof is administered daily, as well as weekly in a different dose. In some embodiments, an initial dose of rapamycin (i.e., an "initial loading dose") is administered to the subject as the first dose of treatment. In some embodiments, the initial dose of rapamycin is higher than any subsequent doses administered to the subject.In some aspects, the dose of rapamycin or a pharmaceutically acceptable salt thereof is approximately 0.1 mg to approximately 25 mg based on the free base of rapamycin (for example, approximately 0.1 mg to approximately 20 mg, approximately 0.1 mg to approximately 18 mg, approximately 0.1 mg to approximately 15 mg, approximately 0.1 mg to approximately 13 mg, approximately 0.1 mg to approximately 10 mg, approximately 0.1 mg to approximately 7 mg, approximately 0.1 mg to approximately 5 mg, approximately 0.1 mg to approximately 3 mg, approximately 0.1 mg to approximately 2 mg, approximately 0.1 mg to approximately 1 mg, approximately 0.5 mg to approximately 20 mg, approximately 0.5 mg to approximately 18 mg, approximately 0.5 mg to approximately 15 mg, approximately 0.5 mg to approximately 13 mg, approximately 0.5 mg to approximately 10 mg, approximately 0.5 mg to approximately 7 mg, approximately 0.5 mg to approximately 5 mg, approximately 0.5 mg to approximately 3 mg, approximately 0.5 mg to approximately 2 mg, approximately 0.5 mg ~ 1 mg, 1 mg ~ 20 mg, 1 mg ~ 18 mg, 1 mg ~ 15 mg, 1 mg ~ 13 mg, 1 mg ~ 10 mg, 1 mg ~ 7 mg, 1 mg ~ 5 mg, 1 mg ~ 3 mg, 1 mg ~ 2 mg, 2 mg ~ 12 mg, 4 mg ~ 10 mg, 4 mg ~ 8 mg, 10 mg ~ 30 mg, approximately 13 mg to approximately 17 mg, approximately 2 mg to approximately 4 mg, approximately 2 mg to approximately 10 mg, approximately 1 mg to approximately 3 mg, approximately 3 mg to approximately 7 mg, approximately 4 mg to approximately 6 mg, approximately 15 mg to approximately 25 mg, approximately 18 mg to approximately 22 mg, approximately 10 mg to approximately 14 mg, approximately 0.5 mg, approximately 1 mg, approximately 2 mg, approximately 2.5 mg, approximately 3 mg, approximately 4 mg, approximately 5 mg, approximately 6 mg, about 7 mg, about 8 mg, about 9 The doses are approximately 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, or 15 mg. In some embodiments, the dose of rapamycin or a pharmaceutically acceptable salt thereof is approximately 0.5 mg to 1 mg based on the free base of rapamycin. In some embodiments, if the subject is 12 years of age or younger, the dose is approximately 50% of the dose administered to subjects older than 12 years of age. In some embodiments, the dose of rapamycin or a pharmaceutically acceptable salt thereof is approximately 0.7 mg based on the free base of rapamycin.In some embodiments, the dose of rapamycin or a 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 a 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 a pharmaceutically acceptable salt thereof is about 5 mg based on the free base of rapamycin.

[0325] In some embodiments, rapamycin or a pharmaceutically acceptable salt thereof is administered as a daily dose of about 0.1 mg to about 2 mg based on the free base of rapamycin; and as a further weekly dose of about 1 mg to about 10 mg based on the free base of rapamycin.

[0326] In some embodiments, rapamycin or a pharmaceutically acceptable salt thereof is administered as a daily dose of about 0.5 mg to about 1 mg based on the free base of rapamycin; and as a further weekly dose of about 2 mg to about 10 mg based on the free base of rapamycin.

[0327] In some embodiments, rapamycin or a pharmaceutically acceptable salt thereof is administered as a daily dose of about 0.7 mg based on the free base of rapamycin; and as a further weekly dose of about 5 mg based on the free base of rapamycin.

[0328] In some embodiments, the method comprises a step for treating or prophylactically treating myotonic dystrophy type 1 (DM1), wherein rapamycin or a pharmaceutically acceptable salt thereof is administered as a daily dose of about 0.5 mg to about 1 mg based on the free base of rapamycin; and as a further weekly dose of about 2 mg to about 10 mg based on the free base of rapamycin.

[0329] In some embodiments, the method comprises a step of treating or preventing myotonic dystrophy type 1 (DM1), wherein rapamycin or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 0.5 mg to about 1 mg based on the free base of rapamycin.

[0330] In some embodiments, the method comprises a step of treating or prophylactically treating myotonic dystrophy type 1 (DM1), wherein rapamycin or a pharmaceutically acceptable salt thereof is administered as a weekly dose of approximately 2 mg to approximately 10 mg based on the free base of rapamycin.

[0331] In some embodiments, the method comprises a step of treating or prophylactically treating Duchenne muscular dystrophy, wherein rapamycin or a pharmaceutically acceptable salt thereof is administered as a daily dose of about 0.5 mg to about 1 mg based on the free base of rapamycin; and as a further weekly dose of about 2 mg to about 10 mg based on the free base of rapamycin.

[0332] In some embodiments, the method comprises a step of treating or prophylactically treating Duchenne muscular dystrophy, wherein rapamycin or a pharmaceutically acceptable salt thereof is administered as a daily dose of about 0.5 mg to about 1 mg based on the free base of rapamycin.

[0333] In some embodiments, the method comprises a step of treating or prophylactically treating Duchenne muscular dystrophy, wherein rapamycin or a pharmaceutically acceptable salt thereof is administered as a weekly dose of about 2 mg to about 10 mg based on the free base of rapamycin.

[0334] In some embodiments, the method comprises a step for treating or preventing spinocerebellar ataxia type 3 (SCA-3), wherein rapamycin or a pharmaceutically acceptable salt thereof is administered as a daily dose of about 0.5 mg to about 1 mg based on the free base of rapamycin; and as a further weekly dose of about 2 mg to about 10 mg based on the free base of rapamycin.

[0335] In some embodiments, the method comprises a step of treating or prophylactically treating spinocerebellar ataxia type 3 (SCA-3), wherein rapamycin or a pharmaceutically acceptable salt thereof is administered in a daily dose of approximately 0.5 mg to approximately 1 mg based on the free base of rapamycin.

[0336] In some embodiments, the method includes a step of treating or preventing spinocerebellar ataxia type 3 (SCA-3), wherein rapamycin or a pharmaceutically acceptable salt thereof is administered as a weekly dose of approximately 2 mg to approximately 10 mg based on the free base of rapamycin.

[0337] In some embodiments, the method comprises a step for treating or preventing Alzheimer's disease, wherein rapamycin or a pharmaceutically acceptable salt thereof is administered as a daily dose of about 0.5 mg to about 1 mg based on the free base of rapamycin; and as a further weekly dose of about 1 mg to about 10 mg based on the free base of rapamycin.

[0338] In some embodiments, the method comprises a step for treating or preventing Alzheimer's disease, and rapamycin or a pharmaceutically acceptable salt thereof is administered as a daily dose of about 0.5 mg to about 1 mg based on the free base of rapamycin.

[0339] In some embodiments, the method comprises a step for treating or preventing Alzheimer's disease, and rapamycin or a pharmaceutically acceptable salt thereof is administered as a weekly dose of about 1 mg to about 10 mg based on the free base of rapamycin.

[0340] In some embodiments, the method comprises a step that promotes autophagy, and rapamycin or a pharmaceutically acceptable salt thereof is administered as a daily dose of about 0.5 mg to about 1 mg based on the free base of rapamycin; and as a further weekly dose of about 1 mg to about 10 mg based on the free base of rapamycin.

[0341] In some embodiments, the method includes a step that promotes autophagy, and rapamycin or a pharmaceutically acceptable salt thereof is administered as a daily dose of about 0.5 mg to about 1 mg based on the free base of rapamycin.

[0342] In some embodiments, the method includes a step that promotes autophagy, and rapamycin or a pharmaceutically acceptable salt thereof is administered as a weekly dose of about 1 mg to about 10 mg based on the free base of rapamycin.

[0343] In some embodiments, the method includes steps to reduce or prevent genomic instability, telomere shortening, epigenetic changes (e.g., DNA methylation, histone modification, or regulation of gene activity by RNA and / or proteins), dysregulation or deregulation of proteostasis, deregulation of nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and / or changes in intercellular communication, wherein rapamycin or a pharmaceutically acceptable salt thereof is administered as a daily dose of approximately 0.5 mg to approximately 1 mg based on the free base of rapamycin; and as a further weekly dose of approximately 1 mg to approximately 10 mg based on the free base of rapamycin.

[0344] In some embodiments, the method includes steps to reduce or prevent genomic instability, telomere shortening, epigenetic changes (e.g., DNA methylation, histone modification, or regulation of gene activity by RNA and / or proteins), dysregulation or deregulation of proteostasis, deregulation of nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and / or changes in intercellular communication, wherein rapamycin or a pharmaceutically acceptable salt thereof is administered as a daily dose of approximately 0.5 mg to approximately 1 mg based on the free base of rapamycin.

[0345] In some embodiments, the method includes steps to reduce or prevent genomic instability, telomere shortening, epigenetic changes (e.g., DNA methylation, histone modification, or regulation of gene activity by RNA and / or proteins), dysregulation or deregulation of proteostasis, deregulation of nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and / or changes in intercellular communication, wherein rapamycin or a pharmaceutically acceptable salt thereof is administered as a weekly dose of approximately 1 mg to approximately 10 mg based on the free base of rapamycin.

[0346] In some embodiments, metformin or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 1500 mg to about 3000 mg; and rapamycin or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 0.5 mg to about 1 mg based on the free base of rapamycin.

[0347] In some embodiments, metformin or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 1500 mg to about 3000 mg; and rapamycin or a pharmaceutically acceptable salt thereof is administered in a weekly dose of about 2 mg to about 10 mg based on the free base of rapamycin.

[0348] In some embodiments, metformin or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 1500 mg to about 3000 mg; and rapamycin or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 0.5 mg to about 1 mg based on the free base of rapamycin; and in a further weekly dose of about 2 mg to about 10 mg based on the free base of rapamycin.

[0349] In some embodiments, metformin or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 1500 mg to about 3000 mg; and rapamycin or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 0.5 mg to about 1 mg based on the free base of rapamycin; and in a further weekly dose of about 2 mg to about 10 mg based on the free base of rapamycin; where 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 (for example, as a fixed dosage form).

[0350] 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 is administered in a daily dose of about 0.5 mg to about 1 mg based on the free base of rapamycin; and in a further weekly dose of about 1 mg to about 10 mg based on the free base of rapamycin.

[0351] 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 is administered in a daily dose of about 0.5 mg to about 1 mg based on the free base of rapamycin; and in a further weekly dose of about 1 mg to about 10 mg based on the free base of rapamycin; where 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 (for example, as a fixed dosage form).

[0352] In some embodiments, when metformin or a pharmaceutically acceptable salt thereof is administered on the same day as rapamycin or a pharmaceutically acceptable salt thereof, both are administered simultaneously (for example, as a fixed dosage form (for example, as 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 daily, both 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 as a fixed dosage form together with the daily dose of metformin or a pharmaceutically acceptable salt thereof, where the daily dose of metformin or a pharmaceutically acceptable salt thereof is administered on the same day as the weekly dose of rapamycin or a pharmaceutically acceptable salt thereof.

[0353] In some embodiments, the method comprises a step of treating or prophylactically treating myotonic dystrophy type 1 (DM1), wherein metformin or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 1500 mg to about 3000 mg based on the free base of metformin; and rapamycin or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 0.5 mg to about 1 mg based on the free base of rapamycin (for example, as a fixed dosage form with metformin or a pharmaceutically acceptable salt thereof); and as a further weekly dose of about 2 mg to about 10 mg based on the free base of rapamycin.

[0354] In some embodiments, the method comprises a step of treating or prophylactically treating myotonic dystrophy type 1 (DM1), wherein metformin or a pharmaceutically acceptable salt thereof is administered at a daily dose of approximately 1500 mg to approximately 3000 mg based on the free base of metformin; and rapamycin or a pharmaceutically acceptable salt thereof is administered at a daily dose of approximately 0.5 mg to approximately 1 mg based on the free base of rapamycin (for example, as a fixed dosage form with metformin or a pharmaceutically acceptable salt thereof).

[0355] In some embodiments, the method comprises a step of treating or prophylactically treating myotonic dystrophy type 1 (DM1), wherein metformin or a pharmaceutically acceptable salt thereof is administered at a daily dose of about 1500 mg to about 3000 mg based on the free base of metformin; and rapamycin or a pharmaceutically acceptable salt thereof is administered at a weekly dose of about 2 mg to about 10 mg based on the free base of rapamycin (for example, as a fixed dosage form with metformin or a pharmaceutically acceptable salt thereof).

[0356] In some embodiments, the method comprises a step of treating or prophylactically treating Duchenne muscular dystrophy, wherein metformin or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 1500 mg to about 3000 mg based on the free base of metformin; and rapamycin or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 0.5 mg to about 1 mg based on the free base of rapamycin (for example, as a fixed dosage form with metformin or a pharmaceutically acceptable salt thereof); and as a further weekly dose of about 2 mg to about 10 mg based on the free base of rapamycin.

[0357] In some embodiments, the method comprises a step of treating or prophylactically treating Duchenne muscular dystrophy, wherein metformin or a pharmaceutically acceptable salt thereof is administered at a daily dose of about 1500 mg to about 3000 mg based on the free base of metformin; and rapamycin or a pharmaceutically acceptable salt thereof is administered at a daily dose of about 0.5 mg to about 1 mg based on the free base of rapamycin (for example, as a fixed dosage form with metformin or a pharmaceutically acceptable salt thereof).

[0358] In some embodiments, the method comprises a step of treating or prophylactically treating Duchenne muscular dystrophy, wherein metformin or a pharmaceutically acceptable salt thereof is administered at a daily dose of about 1500 mg to about 3000 mg based on the free base of metformin; and rapamycin or a pharmaceutically acceptable salt thereof is administered at a weekly dose of about 2 mg to about 10 mg based on the free base of rapamycin (for example, as a fixed dosage form with metformin or a pharmaceutically acceptable salt thereof).

[0359] In some embodiments, the method comprises a step of treating or prophylactically treating spinocerebellar ataxia type 3 (SCA-3), wherein metformin or a pharmaceutically acceptable salt thereof is administered in a daily dose of approximately 1500 mg to approximately 3000 mg based on the free base of metformin; and rapamycin or a pharmaceutically acceptable salt thereof is administered in a daily dose of approximately 0.5 mg to approximately 1 mg based on the free base of rapamycin (for example, as a fixed dosage form with metformin or a pharmaceutically acceptable salt thereof); and as a further weekly dose of approximately 2 mg to approximately 10 mg based on the free base of rapamycin.

[0360] In some embodiments, the method comprises a step of treating or prophylactically treating spinocerebellar ataxia type 3 (SCA-3), wherein metformin or a pharmaceutically acceptable salt thereof is administered at a daily dose of approximately 1500 mg to approximately 3000 mg based on the free base of metformin; and rapamycin or a pharmaceutically acceptable salt thereof is administered at a daily dose of approximately 0.5 mg to approximately 1 mg based on the free base of rapamycin (for example, as a fixed dosage form with metformin or a pharmaceutically acceptable salt thereof).

[0361] In some embodiments, the method comprises a step of treating or prophylactically treating spinocerebellar ataxia type 3 (SCA-3), wherein metformin or a pharmaceutically acceptable salt thereof is administered at a daily dose of approximately 1500 mg to approximately 3000 mg based on the free base of metformin; and rapamycin or a pharmaceutically acceptable salt thereof is administered at a weekly dose of approximately 2 mg to approximately 10 mg based on the free base of rapamycin (for example, as a fixed dosage form with metformin or a pharmaceutically acceptable salt thereof).

[0362] In some embodiments, the method comprises a step for treating or preventing Alzheimer's disease, wherein metformin or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 500 mg to about 1750 mg based on the free base of metformin; and rapamycin or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 0.5 mg to about 1 mg based on the free base of rapamycin (for example, as a fixed dosage form with metformin or a pharmaceutically acceptable salt thereof); and as a further weekly dose of about 1 mg to about 10 mg based on the free base of rapamycin.

[0363] In some embodiments, the method comprises a step of treating or preventing Alzheimer's disease, wherein metformin or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 500 mg to about 1750 mg based on the free base of metformin; and rapamycin or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 0.5 mg to about 1 mg based on the free base of rapamycin (for example, as a fixed dosage form with metformin or a pharmaceutically acceptable salt thereof).

[0364] In some embodiments, the method comprises a step for treating or preventing Alzheimer's disease, wherein metformin or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 500 mg to about 1750 mg based on the free base of metformin; and rapamycin or a pharmaceutically acceptable salt thereof is administered in a weekly dose of about 1 mg to about 10 mg based on the free base of rapamycin (for example, as a fixed dosage form with metformin or a pharmaceutically acceptable salt thereof).

[0365] In some embodiments, the method comprises a step to promote autophagy, where metformin or a pharmaceutically acceptable salt thereof is administered in a daily dose of approximately 500 mg to approximately 1750 mg based on the free base of metformin; and rapamycin or a pharmaceutically acceptable salt thereof is administered in a daily dose of approximately 0.5 mg to approximately 1 mg based on the free base of rapamycin (for example, as a fixed dosage form with metformin or a pharmaceutically acceptable salt thereof); and as a further weekly dose of approximately 1 mg to approximately 10 mg based on the free base of rapamycin.

[0366] In some embodiments, the method includes a step to promote autophagy, in which metformin or a pharmaceutically acceptable salt thereof is administered at a daily dose of about 500 mg to about 1750 mg based on the free base of metformin; and rapamycin or a pharmaceutically acceptable salt thereof is administered at a daily dose of about 0.5 mg to about 1 mg based on the free base of rapamycin (for example, as a fixed dosage form with metformin or a pharmaceutically acceptable salt thereof).

[0367] In some embodiments, the method includes a step to promote autophagy, in which metformin or a pharmaceutically acceptable salt thereof is administered at a daily dose of approximately 500 mg to approximately 1750 mg based on the free base of metformin; and rapamycin or a pharmaceutically acceptable salt thereof is administered at a weekly dose of approximately 1 mg to approximately 10 mg based on the free base of rapamycin (for example, as a fixed dosage form with metformin or a pharmaceutically acceptable salt thereof).

[0368] In some embodiments, the method comprises steps of reducing or preventing genomic instability, telomere shortening, epigenetic changes (e.g., DNA methylation, histone modification, or regulation of gene activity by RNA and / or proteins), dysregulation or deregulation of proteostasis, deregulation of nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and / or alterations in intercellular communication, wherein metformin or a pharmaceutically acceptable salt thereof is administered in a daily dose of approximately 500 mg to approximately 1750 mg based on the free base of metformin; and rapamycin or a pharmaceutically acceptable salt thereof is administered in a daily dose of approximately 0.5 mg to approximately 1 mg based on the free base of rapamycin (e.g., as a fixed dosage form with metformin or a pharmaceutically acceptable salt thereof); and in a further weekly dose of approximately 1 mg to approximately 10 mg based on the free base of rapamycin.

[0369] In some embodiments, the method includes steps to reduce or prevent genomic instability, telomere shortening, epigenetic changes (e.g., DNA methylation, histone modification, or regulation of gene activity by RNA and / or proteins), dysregulation or deregulation of proteostasis, deregulation of nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and / or changes in intercellular communication, wherein metformin or a pharmaceutically acceptable salt thereof is administered at a daily dose of approximately 500 mg to approximately 1750 mg based on the free base of metformin; and rapamycin or a pharmaceutically acceptable salt thereof is administered at a daily dose of approximately 0.5 mg to approximately 1 mg based on the free base of rapamycin (e.g., as a fixed dosage form with metformin or a pharmaceutically acceptable salt thereof).

[0370] In some embodiments, the method includes steps to reduce or prevent genomic instability, telomere shortening, epigenetic changes (e.g., DNA methylation, histone modification, or regulation of gene activity by RNA and / or proteins), dysregulation or deregulation of proteostasis, deregulation of nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and / or changes in intercellular communication, wherein metformin or a pharmaceutically acceptable salt thereof is administered at a daily dose of approximately 500 mg to approximately 1750 mg based on the free base of metformin; and rapamycin or a pharmaceutically acceptable salt thereof is administered at a weekly dose of approximately 1 mg to approximately 10 mg based on the free base of rapamycin (e.g., as a fixed dosage form with metformin or a pharmaceutically acceptable salt thereof).

[0371] 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.

[0372] In some embodiments, the duration of administration 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 longer. In some embodiments, the duration of administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof is approximately 1 day to approximately 1 month. In some embodiments, the duration of administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof is approximately 1 day to approximately 2 weeks. In some embodiments, the duration of administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof is approximately 2 weeks. In some embodiments, the duration of administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof is approximately 12 days. In some embodiments, the duration of administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof is approximately 1 week. In some embodiments, the duration of administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof is at least approximately 1 month. In some embodiments, the duration of administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof is at least about one year.In some embodiments, the period during which administration is discontinued may be 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 longer than 12 months. In some embodiments, the period during which administration is discontinued 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, metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof are administered to an individual for a period of time, followed by another period. In another embodiment, metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof are administered for a first period, followed by a second period during which administration is suspended, and then in a third period, administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof is initiated, and thereafter, in a fourth period following the third period, administration is suspended. In one aspect of this embodiment, periods of administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof, followed by periods of suspension of administration, are repeated over a specified period or an unspecified period.In one further embodiment, the duration 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 longer. In one further embodiment, the period during which administration is discontinued may be 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 longer.

[0373] In some embodiments, after administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof (e.g., after oral administration), subjects experience improvement of gastrointestinal symptoms by consuming food before administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. In some embodiments, subjects consume food up to approximately 6 hours before administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. For example, subjects consume food up to approximately 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, 1 minute, 30 seconds, or 5 seconds before administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof. For example, the subject ingests food at the same time as being administered metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

[0374] In some embodiments, the target age is under 90 years, which is, for example, under 80, under 70, under 60, under 50, under 40, under 30, under 15, under 10, under 5, approximately 1 week to approximately 5 years, approximately 5 to approximately 12 years, approximately 12 to approximately 21 years, approximately 21 to approximately 34 years, approximately 34 to approximately 45 years, approximately 45 to approximately 55 years, approximately 55 to approximately 65 years, approximately 65 to approximately 75 years, or approximately 75 to approximately 90 years. In some embodiments, the target age is under 80 years. In some embodiments, the target age is under 70 years. In some embodiments, the target age is under 60 years.

[0375] The solid dosage forms of this pharmaceutical 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 inert and pharmaceutically acceptable excipient or carrier, for example, sodium citrate or dicalcium phosphate, and / or with the following: a) fillers or bulking agents such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; c) humectants such as glycerol; d) disintegrants such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) dissolution retarders such as paraffin; f) absorption enhancers such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay; and i) Lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also include a buffer.

[0376] Similar types of solid pharmaceutical compositions can also be used as fillers in soft or hard filled gelatin capsules, using excipients such as lactose or milk sugar and high molecular weight polyethylene glycol.

[0377] Solid dosage forms of this pharmaceutical composition, such as tablets, sugar-coated tablets, capsules, pills, and granules, may be prepared to have a coating or shell, such as enteric coatings and other pharmaceutical coatings. These may optionally contain opacifying agents and may also be formulations that release the active ingredient only in a specific part of the intestinal tract, or preferentially in such a specific part, and this may optionally be in the form of delayed release. Examples of materials that may be used to embed the pharmaceutical composition include polymeric substances and waxes.

[0378] The active compound may also be in a microencapsulated form, which may be microencapsulated together with one or more of the excipients described above, if appropriate.

[0379] Liquid dosage forms of this pharmaceutical composition for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art, such as: water or other solvents, solubilizers, and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, as well as mixtures thereof.

[0380] The suspension of this compound may contain, in addition to the active compound, a suspending agent, such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum methhydroxyl, bentonite, agar, tragacanth, and mixtures thereof.

[0381] The pharmaceutically acceptable compositions of this disclosure for injection include pharmaceutically acceptable, sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution immediately before use as sterile injectable solutions or dispersions. Examples of suitable carriers, diluents, solvents, or vehicles, both aqueous and non-aqueous, include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters, such as ethyl oleate. Adequate fluidity may be maintained, for example, by using a coating substance such as lecithin, by maintaining the required particle size in the case of dispersions, and by using a surfactant.

[0382] In addition to inert diluents, these pharmaceutical compositions may also contain adjuvants, such as preservatives, humectants, emulsifiers, dispersants, sweeteners, flavorings, and fragrances. Prevention of microbial action can be ensured by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. The inclusion of isotonic agents may also be desirable, such as sugars and sodium chloride. Extending the absorption of injectable dosage forms can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin. Compounds may be incorporated into slow-release or targeted systems, such as polymer matrices, liposomes, and microspheres. Such formulations may result in more effective distribution of the compound.

[0383] Pharmaceutical compositions that are injectable formulations can be sterilized, for example, by filtration through a filter that retains bacteria, or by introducing a sterilizing agent into a pharmaceutical composition in the form of a sterile solid that is soluble or dispersible in sterile water or other sterile injectable medium before use.

[0384] Topical dosage forms of the compounds or pharmaceutical compositions of this disclosure include powders, patches, sprays, ointments, and inhalants. The active compounds are mixed under sterile conditions with a pharmaceutically acceptable carrier, as well as any preservatives, buffers, or propellants as may be required.

[0385] The compounds and compositions described herein may be administered, for example, orally, parenterally (e.g., subcutaneously, intradermally, intravenously, or intramuscularly), topically, rectally, nasally, sublingually, or buccally, in which case the dosage may range from about 0.01 milligrams / kilogram (mg / kg) to about 1000 mg / kg (e.g., about 0.01 to about 100 mg / kg, about 0.1 to about 100 mg / kg) every 4 to 120 hours, or the dosage may be in accordance with the requirements of a particular drug, a particular dosage form, and / or a particular route of administration. Other routes of administration include enteral, intra-arterial, intraperitoneal, and intrathecal administration. The correlation of dosage between humans and animals (based on milligrams per square meter of body surface area) is described below: Freireich et al., Cancer Chemother. Rep. 50, 219-244 (1966). Body surface area can be approximately determined from the patient's height and weight. See, for example, Scientific Tables, Geigy Pharmaceuticals, Ardsley, NY, 537 (1970). In some embodiments, the composition is administered orally or by injection. The methods described herein are intended to administer a therapeutically effective amount of the compound or compound composition to achieve a desired or specified effect. Typically, the pharmaceutical compositions of this disclosure are administered about 1 to 6 times per day, or as continuous infusions. Such administrations may be used as treatments for chronic or acute phases.

[0386] Lower or higher doses than those mentioned above may be required. Specific dosages and treatment regimens for any particular patient will vary depending on a variety of factors, including the activity of the specific compound used, age, weight, overall health, sex, dietary habits, time of treatment, excretion rate, drug combinations, severity and course of the disease or condition or symptoms, the patient's outcome to the disease, and the judgment of the physician administering the treatment.

[0387] In this disclosure, dosage forms are provided, including: approximately 100 mg to approximately 5000 mg based on the free base of metformin (for example, approximately 100 mg to approximately 500 mg, approximately 100 mg to approximately 800 mg, approximately 100 mg to approximately 1000 mg, approximately 500 mg to approximately 1000 mg, approximately 1000 mg to approximately 1500 mg, approximately 1500 mg to approximately 2250 mg, approximately 1500 mg to approximately 2000 mg, approximately 2000 mg to approximately 2500 mg, approximately 2250 mg to approximately 3000 mg, approximately 2500 mg to approximately 3000 mg, approximately 3000 mg to approximately 3500 mg, approximately 3500 mg to approximately 4000 mg, approximately 4000 mg to approximately 5000 mg, approximately 500 mg to approximately 3000 mg, approximately 500 to approximately 1750 mg, approximately 500 mg Metformin or a pharmaceutically acceptable salt thereof in amounts of approximately 1125 mg, 2250 mg, 3000 mg, 500 mg, 650 mg, 750 mg, 850 mg, 1000 mg, 1750 mg, 2000 mg, or 3000 mg, and approximately 0.1 mg to 25 mg of free rapamycin (for example, approximately 0.1 mg to 20 mg, 18 mg to 15 mg, 13 mg to 10 mg, 7 mg to 0.1 mg, 5 mg to 0.1 mg, 3 mg to 0.1 mg, 2 mg to 0.1 mg, 1 mg to 0.5 mg) based on the free base of rapamycin. 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~30 mg, 13 mg~17 mg, 2 mg~4 mg, 2 mg~10 mg, 1 mg~3 mg, 3 mg~7 mg, 4 mg~6 mg, 15 mg~25 mg, 18 mg~22 mg, 10 mg~14 mg, 0.5 mg, 1 mg, 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, about 6 mg, about 7 mg, about 8 Rapamycin or a pharmaceutically acceptable salt thereof in amounts of (approximately 9 mg, approximately 10 mg, approximately 11 mg, approximately 12 mg, approximately 13 mg, approximately 14 mg, or approximately 15 mg). The dosage form may further contain a pharmaceutically acceptable carrier and / or additional therapeutic agent.

[0388] In some embodiments, the dosage form comprises metformin or a pharmaceutically acceptable salt thereof in an amount of about 1500 mg to about 3000 mg based on the free base 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 of rapamycin.

[0389] In some embodiments, the dosage form comprises metformin or a pharmaceutically acceptable salt thereof in an amount of about 1500 mg to about 2250 mg based on the free base 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 of rapamycin.

[0390] In some embodiments, the dosage form comprises metformin or a pharmaceutically acceptable salt thereof in an amount of about 2250 mg to about 3000 mg based on the free base 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 of rapamycin.

[0391] In some embodiments, the dosage form comprises about 1500 mg of metformin or a pharmaceutically acceptable salt thereof, based on the free base of metformin, and about 0.5 mg of rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin.

[0392] In some embodiments, the dosage form comprises about 3000 mg of metformin or a pharmaceutically acceptable salt thereof, based on the free base of metformin, and about 0.5 mg of rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin.

[0393] In some embodiments, the dosage form comprises about 1500 mg of metformin or a pharmaceutically acceptable salt thereof, based on the free base of metformin, and about 1 mg of rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin.

[0394] In some embodiments, the dosage form comprises approximately 3,000 mg of metformin or a pharmaceutically acceptable salt thereof, based on the free base of metformin, and approximately 1 mg of rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin.

[0395] 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 based on the free base 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 of rapamycin.

[0396] 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 based on the free base 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 of rapamycin.

[0397] 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 based on the free base 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 of rapamycin.

[0398] In some embodiments, the dosage form comprises about 500 mg of metformin or a pharmaceutically acceptable salt thereof, based on the free base of metformin, and about 0.5 mg of rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin.

[0399] In some embodiments, the dosage form comprises approximately 1750 mg of metformin or a pharmaceutically acceptable salt thereof, based on the free base of metformin, and approximately 0.5 mg of rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin.

[0400] In some embodiments, the dosage form comprises about 500 mg of metformin or a pharmaceutically acceptable salt thereof, based on the free base of metformin, and about 1 mg of rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin.

[0401] In some embodiments, the dosage form comprises approximately 1750 mg of metformin or a pharmaceutically acceptable salt thereof, based on the free base of metformin, and approximately 1 mg of rapamycin or a pharmaceutically acceptable salt thereof, based on the free base of rapamycin.

[0402] In some aspects, the weight ratio of metformin to rapamycin is approximately 10,000:1 to approximately 100:1 (for example, approximately 10,000:1 to approximately 300:1, approximately 5,000:1 to approximately 400:1, approximately 4,000:1 to approximately 500:1, approximately 3,500:1 to approximately 750:1, approximately 3,500:1 to approximately 900:1, approximately 3,500:1 to approximately 1,000:1, approximately 2,500:1 to approximately 1,000:1, approximately 2,500:1 to approximately 1,700:1, approximately 3,000:1 to approximately 2,000:1, approximately 2,000:1 to approximately 1,500:1, approximately 1,300:1 to approximately 700:1, approximately 3,500:1, approximately 2,500:1, approximately 1,750:1, or approximately 1,000:1). In some embodiments, the weight ratio of metformin to rapamycin is approximately 3500:1. In some embodiments, the weight ratio of metformin to rapamycin is approximately 2500:1. In some embodiments, the weight ratio of metformin to rapamycin is approximately 1000:1. In some embodiments, the weight ratio of metformin to rapamycin is approximately 1750:1.

[0403] The appropriate dosage level can be determined by any suitable method. Preferably, the active substance is administered topically at a frequency of 1 to 4 times per day, or less frequently if a drug delivery system is used. However, the actual dosage level and duration of administration of the active ingredient in the pharmaceutical compositions of this disclosure may be varied to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, a particular composition, and a particular mode of administration without causing unacceptable toxicity to the patient. In some cases, particularly depending on the patient's age, sex, weight, dietary habits, and overall health, the route of administration, the individual response to the active ingredient, the nature of the preparation, and the duration or interval over which administration takes place, the dosage may deviate from the specified amount. Thus, in some cases, a smaller amount than the minimum amount described above may be sufficient, while in other cases, it may exceed the specified upper limit. If a larger dose is to be administered, it may be recommended to divide the dose into several individual doses spread out over the days of administration.

[0404] In some embodiments of the methods disclosed herein, metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof are administered orally, parenterally, transdermally, intranasally, sublingually, spinally, or orally. In some embodiments, metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof are administered orally.

[0405] All disclosures of publications cited herein are explicitly incorporated herein by reference to the same extent as each publication is incorporated by reference individually. [Examples]

[0406] Example 1: Evaluation of a mouse model of combination therapy with metformin and rapamycin for neurological, muscular, and proliferative disorders such as myotonic dystrophy type 1 (DM1). material and method: The DM1 mouse model is available as described in Brockhoff et al., J Clin Invest, 2017, 127(2):549-563, which is incorporated in its entirety by reference. The available DM1 mouse model is a transgenic mouse model that overexpresses a mutant DMPK gene containing CTG repeat elongation, characteristic of DM1. These mice exhibit skeletal muscle weakness, as well as histological hallmarks of DM1, such as muscle fiber atrophy, nuclear centralization, and increased fibrosis. The mice can be bred to obtain DM1 mice that are either wild-type AMPKα2 (AMPKα2WT) or have a skeletal muscle-specific AMPKα2 deletion (AMPKα2MKO). The genotype of the mice can be determined by PCR using tail DNA. Insulin resistance, hyperglycemia, glucose uptake, and HbAIc can be measured.

[0407] In vitro studies can be performed using myoblasts derived from the quadriceps muscles of control and DM1 mice. Myoblasts can be cultured in growth medium, differentiated in differentiation medium, and treated with AICAR or rapamycin. Western blotting can be performed to evaluate the levels of protein expression and phosphorylation of various markers in the AMPK and mTORC1 pathways.

[0408] Combination therapy may involve treating DM1 mice with a combination therapy of metformin and rapamycin to target the deregulated AMPK / mTORC1 pathway in muscle cells. Metformin may be used to activate AMPK, while rapamycin may be used to inhibit mTORC1. The effects of this combination therapy can be evaluated by measuring muscle function and gene expression in the treated mice. Dosage 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 a non-limiting example, metformin may be administered, for example, in drinking water at a dose of 300 mg / kg / day for two weeks, and rapamycin may be administered, for example, at 3 mg / kg / day by intraperitoneal injection for two weeks.

[0409] Data analysis: In the DM1 model, the effectiveness of the metformin and rapamycin combination drug on relevant disease parameters, such as blood glucose levels, body weight, insulin sensitivity, or other relevant biomarkers, can be measured by comparing the effects of the combination drug with the effects of each drug alone.

[0410] Blood glucose levels: Blood glucose levels can be analyzed by taking blood samples from mice at regular intervals and measuring the glucose concentration using a glucose meter, or by performing an enzyme-linked immunosorbent assay (ELISA) on the collected serum samples.

[0411] Body weight: Body weight can be measured at regular intervals using a digital scale during the trial period. The data can then be analyzed using statistical methods such as analysis of variance or linear regression to determine any significant differences between treatment groups.

[0412] Insulin sensitivity: Insulin sensitivity can be assessed using an insulin tolerance test (ITT) or a glucose tolerance test (GTT). These tests involve administering insulin or glucose to mice and measuring the resulting change in blood glucose levels. The data can then be analyzed using statistical methods such as analysis of variance or linear regression to determine any significant differences between treatment groups.

[0413] Other relevant biomarkers: Other relevant biomarkers can be analyzed by taking blood or tissue samples from mice at the end of the study and performing various assays, such as ELISA, Western blotting, or PCR. The data can then be analyzed using statistical methods such as analysis of variance or linear regression to determine any significant differences between treatment groups.

[0414] The degree of drug synergy can be evaluated using quantitative methods, such as the combination index (CI) or the Bliss independence model. These models allow for precise determination of the degree of synergistic effect of metformin and rapamycin in the treatment of myotonic dystrophy type I. Further experiments, such as dose-response curves, time-series studies, or genetic engineering, can be conducted to validate the results of in vivo experiments.

[0415] Example 2: A microfluidic-based assay for automated in vivo evaluation of rapamycin, metformin, and combinations thereof in relation to lifespan and healthy lifespan using the nematode Caenorhabditis elegans. Abstract the purpose The objective of this study was to investigate the roles of rapamycin (referred to herein as "Compound 1" or "Cpd 1"), metformin (referred to herein as "Compound 2" or "Cpd 2"), and combinations thereof, on the lifespan and healthy lifespan of C. elegans. Microfluidic-based techniques were used to achieve this objective.

[0416] Outline of the method To achieve the objectives of this study, the two compounds described above, along with four combinations thereof, were tested on a microfluidic platform. Worms were injected into a dedicated microfluidic chamber at the L4 larval stage, sealed within the chamber, and continuously exposed to the presence of the two compounds, either alone or in combination, from the L4 larval stage until the end of the experiment (corresponding to the day the worms died). A series of short videos of each microfluidic chamber containing worms were automatically acquired every six hours throughout the duration of the assay. Motility measurements, as well as worm size measurements to address the toxicity of the compounds (1), and reproductive capacity measurements to assess longevity (2), were performed using algorithms in the acquired series of videos (3,4). Worm viability was automatically determined based on motility parameters. Each condition was tested on four technical replicates on separate microfluidic cartridges. The experiment was performed once.

[0417] Test design and method The proposed experiment was carried out according to the design and timeline shown in Figure 1. N2 wild-type C. elegans were grown to the adult stage on solid Nematode Growth Medium (NGM) agar plates and collected in complete S medium. Eggs derived from the adult population were incubated overnight in S medium, and the resulting L1 offspring were collected using a dedicated filter. L1 larvae were then collected by filtration in S medium, seeded on NGM agar plates, and grown at 20°C for 48 hours (h). After 48 hours, the worm population reached the L4 larval stage. L4 larvae were then collected by filtration in 5 mL of S medium. 20–30 L4 larvae were injected into microfluidic chips for each condition. The worms were then continuously fed on the chips via bacterial culture medium throughout the entire duration of the experiment. Videos of each microchamber were taken every 6 hours after feeding throughout the entire duration of the experiment.

[0418] Preparation of the test substance Compounds 1 and 2 were suspended in DMSO at stock concentrations of 100 mM and 2.5 M, respectively (Table 1). The compounds were then mixed into bacterial culture media, either alone or in combination, to reach target concentrations (Table 2). For all tested conditions, the final concentration of the vehicle (DMSO) was 1%. For the negative control condition, nematodes were fed bacterial culture media containing only the vehicle solution (1% DMSO). All stock solutions were stored at -20°C, and dilutions were freshly prepared on the first day of the on-tip experiment and subsequently replaced with compound / bacterial culture media solutions every 72 hours.

[0419] (Table 1) List of chemical substances used in this test TIFF2026514863000002.tif21149

[0420] (Table 2) Conditions tested in this examination TIFF2026514863000003.tif56149

[0421] Preparation of bacteria The worms were continuously fed on tips with bacterial medium (lyophilized Escherichia coli OP50 in complete S medium) at a concentration of 8.05E+9 cells per milliliter. Briefly, 500 mg of lyophilized OP50 was dissolved in 5 mL of complete S medium and carefully homogenized using a vortex mixer. The bacterial concentration was monitored using a spectrophotometer and adjusted to 8.05E+9 as needed (5).

[0422] Worm preparation Eggs of C. elegans (N2 wild type, provided by Caenorhabditis Genetics Center) were incubated on solid Nematode Growth Medium (NGM) agar plates seeded with E. coli OP50 and grown under these conditions at 20°C for 4 days. After 4 days, N2 larvae of various stages appeared on the NGM agar plates, mainly consisting of adults and eggs. The worm populations were collected and filtered in complete S medium to isolate adults from populations of various other stages. Isolated adults were incubated overnight at room temperature. L1 larvae hatched from eggs laid by adults, and these were subsequently collected by filtering in S medium, seeded on NGM agar plates, and grown at 20°C for 48 hours. After 48 hours, the worm populations reached the L4 larval stage. L4 larvae were then collected by filtering in 5 mL h, and the initial solution of bacteria and compounds was replaced with a fresh solution containing bacteria. 4 μL of fresh feed / compound solution was injected every 60 minutes into microfluidic chips corresponding to each test condition. Videos of each microchamber were taken after feeding.

[0423] Phenotype analysis Five time-resolved phenotypic readouts were obtained during the assay, and these included: - Worm size (area): This parameter was measured to evaluate the potential effect of the test compound on delayed growth or resting larval phenotype (7). - Sexual maturity: This parameter was measured to assess the potential effect of the test compound on the timing required for the worms to reach sexual maturity and was determined by observing the first embryos (i.e., eggs) produced in each microfluidic chamber. This phenotype also indicated a general infertility phenotype (i.e., failure to lay eggs) (7). - Worm reproductive capacity: This parameter was measured to assess the potential effect of the test compound on the average number of eggs laid by each worm during the breeding period and was calculated using images acquired during the assay (7). - Reproductive period: This parameter was measured to evaluate the potential effect of the test compound on the period during which the worms are capable of reproduction, and was determined by observing the last embryo born in each microfluidic chamber. - Worm longevity: This parameter was measured to evaluate the potential effect of the test compound on the survival time of worms.

[0424] Worm motility is a further phenotypic leadout, in which five motility parameters were evaluated every six hours from the acquired video (each video being 2 seconds long, 10 frames per second). Values ​​recorded for dead worms were excluded at each time point. The five motility parameters monitored were as follows (3): ○ Amplitude of head movement ○ Amplitude of movement of the middle part of the worm's body ○ Amplitude of tail movement 〇 Worm bending frequency 〇 Worm speed

[0425] These kinetic parameters were analyzed period by period as defined below (calculation of area under the curve followed by a two-way ANOVA): 〇 1st period: 1st to 5th day 〇 2nd period: 6th to 10th day 〇 3rd period: 11th to 15th day 〇 4th period: 16th to 20th day

[0426] statistical analysis Raw values ​​were normalized and compared (where shown) to their respective negative controls. Survival analysis was performed using the Kaplan-Meier method, and the significance of differences between survival curves was calculated using the log-rank test. Two-way ANOVA was performed to compare interactions between groups. For comparisons of more than two groups, ANOVA evaluated by Bonferroni's multiple comparison test was used. GraphPad Prism 5 (GraphPad Software) was used for all other statistical analyses, including the calculation of standard deviation (sd), standard error (sem), and area under the curve (AUC). All p-values ​​< 0.05 were considered significant. The radar charts shown in Figures 8A-8C are: 2 The statistical values ​​were compared to a negative control (1% DMSO).

[0427] Summary and interpretation of lifespan monitoring results: For example, combination D showed a +14% improvement compared to the negative control. All compounds, whether used alone or in combination, had a survival-extending effect; however, none of the tested conditions reached statistical significance for the overall survival of N2 worms under current conditions (Figure 2 and Table 3). Treatment with combination D showed a significant effect (+56.0%; p<0.05) on worm 50% survival compared to the negative control (Figure 3 and Table 4). Treatment with compound 1, combination A, and combination B showed some improvement in worm 50% survival, but it was not statistically significant (0.05). <p<0.1) Treatment with combination A showed a significant effect (+77.8%; p<0.05) on worm survival rate compared to the negative control (Figure 4 and Table 5).

[0428] Monitoring of reproduction and growth observation: Figures 5A–5D are bar graphs showing the elapsed time (hours) from L4 larval injection to the first oviposition (Figure 5A) and the last oviposition (Figure 5B), as well as the oviposition period (Figure 5C) and the average number of eggs laid per worm during the oviposition period (Figure 5D). These were observed for N2 wild-type N2 worms treated under the conditions summarized in Table 2. The bar graphs represent the mean + / - sem. The p-value was obtained by comparing the treatment with the negative control (blue bars) using a one-way ANOVA followed by a Bonferroni multiple comparison test (p<0.05). * indicates a statistical value compared to the negative control (1% DMSO). Tables 6–9 summarize the data from Figures 5A–5D.

[0429] (Table 6) Summary of results in Figure 5A TIFF2026514863000004.tif64149n C This corresponds to the number of chambers in which the start of spawning was detected. The start of spawning within a chamber was determined at the collective level, not at the individual worm level.

[0430] (Table 7) Summary of results in Figure 5B TIFF2026514863000005.tif61149n C This corresponds to the number of chambers in which the start of spawning was detected. The start of spawning within a chamber was determined at the collective level, not at the individual worm level.

[0431] (Table 8) Summary of results in Figure 5C TIFF2026514863000006.tif67149n C This corresponds to the number of chambers in which the start of spawning was detected. The start of spawning within a chamber was determined at the collective level, not at the individual worm level.

[0432] (Table 9) Summary of results in Figure 5D TIFF2026514863000007.tif61149n CThis corresponds to the number of chambers in which the start of spawning was detected. The start of spawning within a chamber was determined at the collective level, not at the individual worm level.

[0433] Figure 6 is a bar graph showing worm growth (area under the curve) for N2 wild-type worms treated under the conditions summarized in Table 2. The p-values ​​were obtained by comparing the treatment with the negative control (blue line) using a two-way ANOVA to assess the overall difference in the curves, followed by a Bonferroni multiple comparison test. ** p<0.01; *** p<0.001; **** p<0.0001. Table 10 summarizes the data from Figure 6.

[0434] (Table 10) Summary of results regarding worm area shown in Figure 6 TIFF2026514863000008.tif61149

[0435] Summary and interpretation of the results of monitoring reproduction and growth: All tested conditions (test compound alone or in combination) significantly reduced the number of eggs laid per worm compared to the negative control (Figure 5A and Table 9). Treatment in combination A had a slight effect on the time of last spawning compared to the negative control (+9.9%, p<0.05), and consequently extended the overall length of the spawning period by 12.8% (p<0.05) (Figures 5B-5C and Table 8). All tested conditions (test compounds alone or in combination) had a significant effect on worm size compared to the negative control (Figure 6 and Table 10): Compared to the negative control, treatment with compound 1, combination A, combination C, and combination D showed a significant improvement in worm size, while compound 2 and combination B showed a significant decrease in worm size (Figure 6 and Table 10).

[0436] Monitoring of motor skills observation: Figures 7A–7D are plots of worm motility (Figure 7A: head amplitude; Figure 7B: mid-section amplitude; Figure 7C: tail amplitude; Figure 7D: flexion frequency; Figure 7E: velocity), for N2 wild-type worms treated under the conditions summarized in Table 2. The p-values ​​were obtained by comparing the treatment with the negative control (blue line) using a two-way ANOVA to assess the difference across the curves, followed by Bonferroni multiple comparisons (p<0.05). Error bars indicate SEM. Up: Significant increase in the analyzed phenotype compared to the negative control (blue curve) (comparison of curves using two-way ANOVA (p<0.05)), Down: Significant decrease in the analyzed phenotype compared to the negative control (blue curve) (comparison of curves using two-way ANOVA (p<0.05)).

[0437] Figures 8A-8C are a series of radar charts showing the worm motility (head amplitude, midsection amplitude, tail amplitude, velocity, and flexion frequency), which are N2 wild-type worms treated with compound 1 (final concentration 100 μM) and compound 2 (final concentration 25 mM) (Figure 8A); worms treated with combination A (100 μM compound 1 + 25 mM compound 2) and combination B (33 μM compound 1 + 25 mM compound 2) (Figure 8B); worms treated with combination C (100 μM compound 1 + 8.5 mM compound 2) and combination D (50 μM compound 1 + 12.5 mM compound 2) These are the results for worms treated with mM compound 2) at different time points (Figure 8C) (as shown clockwise in each radar chart, the period represented by the number "1" corresponds to days 0-5; the period represented by the number "2" corresponds to days 6-10; the period represented by the number "3" corresponds to days 11-15; and the period represented by the number "4" corresponds to days 16-20). The values ​​shown in the radar charts correspond to the ratio to the AUC of the negative control (1% DMSO), calculated from the curve shown in Figure 7. ↓: Significant decrease in the analyzed phenotype compared to the negative control (1% DMSO, blue line) (comparison of curves by two-way ANOVA (p<0.05)); ↑: Significant increase in the analyzed phenotype compared to the negative control (1% DMSO, blue line) (comparison of curves by two-way ANOVA (p<0.05)).

[0438] Summary and interpretation of the results of monitoring mobility and behavior during basic conditions: The overall motility of worms treated with compound 1 (all motility parameters improved by an average of 14%), compound 2 (all motility parameters improved by an average of 22%), combination A (all motility parameters improved by an average of 19%), combination B (one of five motility parameters improved by 14%), and combination D (four of five motility parameters improved by an average of 16%) was significantly increased compared to the negative control (Figures 7A-7E). No effect on motility was observed when worms were treated with combination C (Figures 7A-7E). The overall improvement in worm motility in the groups treated with compound 1, compound 2, and combination A (Figures 7A-7E) is explained by the significant increase in almost all motility parameters only during the first phase (days 0-5) and second phase (days 6-10) (Figures 8A-8C). No effect on motility was observed during the third phase (days 11-15) and fourth phase (days 16-20). The overall improvement of 16% in worm motility (average) in the population treated with combination D (Figures 7A-7E) is explained by significant increases in most motility parameters during Phase 1 (Days 0-5), Phase 2 (Days 6-10), and Phase 3 (Days 11-15) (Figures 8A-8C). No effect on motility was observed during Phase 4 (Days 16-20). Treatment in combination B showed significant improvement in all motor parameters during the first phase (days 0-5) and the second phase (days 6-10) (Figures 8A-8C). However, motor function was significantly impaired in the later stages (phases 3 and 4), which explains the weak overall effect of this particular condition on motor function (Figures 7A-7E). Treatment in combination C showed significant improvement in all motor parameters only during the first phase (days 0-5) (Figures 8A-8C). Motor function was significantly impaired during the third phase (days 11-15), which explains the absence of an overall effect on motor function from this particular condition (Figures 7A-7E).

[0439] conclusion Exam conclusions The biological effects of compound 1 and compound 2, as well as combinations thereof, on aging and associated aging-related phenotypes were analyzed and profiled. Overall, this study suggests that high-dose combinations of compound 1 and compound 2 (100 μM and 25 mM, respectively, corresponding to combination A) and combinations at half the maximum dose (50 μM and 12.5 mM, respectively, corresponding to combination D) have the ability to produce significant synergistic effects on the lifespan and motility of the nematode C. elegans. Further conclusions are summarized below: Treatment with combination D (50 μM compound 1 + 12.5 mM compound 2) showed a significant effect (+56.0%; p<0.05) on 50% worm survival compared to the negative control, but no effect was observed in the later stages of life (25% worm survival). Furthermore, treatment with combination A (100 μM compound 1 + 25 mM compound 2) showed a significant effect (+77.8%; p<0.05) on 25% worm survival compared to the negative control, while some improvement was observed for 50% worm survival (p<0.1). In conclusion, synergistic effects on survival were observed for combinations A and D, but no significant effect was detected when the two test compounds were used individually. These results indicate that the combination of rapamycin and metformin synergistically extends the lifespan of C. elegans. In general, combination A tended to improve the overall lifespan of the worms (+14.4%; p=0.2), but none of the conditions tested had a significant effect on the overall survival of the worms compared to the negative control. • All tested conditions significantly reduced the number of eggs laid per worm compared to the negative control. No synergistic effect was observed when combinations of the two compounds were tested (i.e., the combination did not result in a reduction in the number of eggs laid per worm). While we do not wish to be bound by theory, it is thought that some interventions, particularly calorie restriction, reduce reproductive capacity while promoting beneficial effects on longevity across species (2). Several compounds known to mimic the effects of calorie restriction also produce similar effects on reproductive capacity. However, treatment in combination A had a slight effect on the time of last spawning (+9.9%, p<0.05) and extended the overall length of the spawning period by 12.8% (p<0.05) compared to the negative control, which can be interpreted as having a beneficial effect on the reproductive system of C. elegans. Regarding worm size, treatment with compound 1, combination A, combination C, and combination D showed significant improvement in worm size compared to the negative control, while compound 2 and combination B showed a significant decrease in worm size. Based on these observations, it became clear that high doses of compound 2 (alone or in combination as observed in combination B) negatively affect worm size; high doses (100 μM) of compound 1 have the ability to reverse this harmful effect on size, as observed under the conditions of combination A (a +4.9% increase compared to compound 2 alone at 25 mM). The overall motility of worms treated with compound 1, compound 2, combination A, combination B, and combination D was significantly increased compared to the negative control. Interestingly, the overall improvement in worm motility in the groups treated with compound 1, compound 2, and combination A is explained by the significant increase in almost all motility parameters only during periods 1 (days 0-5) and 2 (days 6-10). No effect on motility was observed in periods 3 (days 11-15) and 4 (days 16-20) for these specific conditions. Furthermore, the overall improvement in worm motility in the population treated with combination D is explained by the significant increases in most motility parameters during Phase 1 (Days 1-5), Phase 2 (Days 6-10), and Phase 3 (Days 11-15). This observation indicates that treatment with combination D has the ability to maintain and improve the health and motility of worms in the later stages of their life. Furthermore, during the later stages of the worm's life cycle (stages 3 and 4), several detrimental effects on the worm's motility were observed in combination B. However, these negative effects were reversed in combinations A and D, which were either due to a decrease in the concentration of compound 2 (combination D) or an increase in the concentration of compound 1 (combination A).

[0440] Overall, this study suggests that high-dose combinations of compound 1 and compound 2 (100 μM and 25 mM, respectively), as well as combinations at half the maximum dose (50 μM and 12.5 mM, respectively), have the ability to produce significant synergistic effects on both life extension and improved motility in the nematode C. elegans.

[0441] References 1) Hunt PR., "The C. elegans model in toxicity testing", J Appl Toxicol. 2017 Jan;37(1). 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. 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. 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. 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. 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. 7) Mouchiroud L et al., "Worm-on-Chip technology as a new alternative for early toxicity studies", Toxicology Letters. 2021 350, S118-S119.

[0442] Other embodiments Although the present invention has been described in detail, it should be understood that the above description is intended to illustrate the scope of the invention, not to limit it, and that the scope of the invention is determined by the appended claims. Other aspects, advantages, and modifications are also included in the appended claims.

Claims

1. A method for treating or preventing a neurological, myocardial, or proliferative disorder in a subject requiring such treatment, comprising the step of administering a therapeutically effective dose to the subject of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof, wherein the neurological, myocardial, or proliferative disorder 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 endogenous pontine glioma (DIPG).

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

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

4. The method according to claim 1, wherein the neurological, muscular, or proliferative disorder is Duchenne muscular dystrophy (DMD).

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

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

7. The method according to claim 1, wherein the neurological, myocardial, or proliferative disorder is Parkinson's disease.

8. The method according to claim 1, wherein the neurological, myocardial, or proliferative disorder is vascular dementia.

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

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

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

12. The method according to claim 1, wherein the neurological, muscular, or proliferative disorder is Lafora disease.

13. The method according to any one of claims 6 to 12, further comprising the step of preventing the aforementioned ailment.

14. The method according to claim 1, wherein the neurological, myocardial, or proliferative disorder is glioblastoma.

15. The method according to claim 1, wherein the neurological, myocardial, or proliferative disorder is diffuse endogenous pontine glioma (DIPG).

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

17. A method for treating or preventing a neuronal cancer in a subject requiring such treatment, comprising the step of administering a therapeutically effective dose to the subject of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof, wherein the cancer is glioblastoma or diffuse endogenous pontine glioma (DIPG).

18. The method according to claim 17, wherein the nerve cancer is glioblastoma.

19. The method according to claim 17, wherein the nerve cancer is diffuse endogenous pontine glioma (DIPG).

20. The method according to any one of claims 17 to 19, wherein Akt is activated in the subject after the step of administering 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 who has been confirmed or diagnosed with muscle weakness, comprising the step of administering a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to the subject.

22. A method for preventing or reversing muscle weakness in a subject in need thereof, comprising the step of administering a therapeutically effective dose to the subject of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

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

24. The method according to any one of claims 2 to 5 and 21 to 23, comprising the step of determining an increase in grip strength measured by a grip strength meter.

25. The method according to any one of claims 2 to 5 and 21 to 23, comprising the step of determining an increase of at least 10% in grip strength as measured by a grip strength meter.

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

27. The method according to claim 26, further comprising the step of preventing cellular senescence in the subject.

28. A method for increasing motility in a subject, comprising the step of administering a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof to the subject.

29. The method according to claim 28, wherein the increase in motility in the subject is an increase in the number of voluntary muscle contractions per minute in the subject compared to before the subject was administered a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

30. The method according to any one of claims 28 to 29, wherein the increase in motility in the subject increases the duration of voluntary muscle contraction in the subject compared to before the subject was administered a therapeutically effective dose of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

31. The method according to any one of claims 28 to 30, comprising the step of determining the increase in the distance walked by a subject during a 10-meter walk test, after the step of administering metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

32. The method according to any one of claims 38 to 31, further comprising the step of determining the reduction in the time it takes for a 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.

33. The method according to any one of claims 1 to 32, wherein, after the step of administering 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 according to any one of claims 1 to 33, wherein metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof are administered orally.

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

36. The method according to claim 35, wherein 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.

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

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

39. The method according to claim 38, wherein 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.

40. The method according to any one of claims 38 to 39, wherein 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.

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

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

43. The method according to 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 according to any one of claims 1 to 37, wherein rapamycin or a pharmaceutically acceptable salt thereof is administered weekly.

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

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

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

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

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

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

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

52. The method according to any one of claims 1 to 51, wherein the subject has not been confirmed or diagnosed with diabetes.

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

54. The method according to any one of claims 1 to 53, wherein the subject has not received any insulin transport pathway modulating therapeutic agents within one year prior to administration of metformin or a pharmaceutically acceptable salt thereof and rapamycin or a pharmaceutically acceptable salt thereof.

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