Aminothiols for treating conditions characterized by protein misfolding or aggregation, mitochondrial dysfunction or chronic inflammation

Abstract

The present invention relates to aminothiols for use in the treatment of (1) diseases and conditions characterized by misfolding and / or aggregation of endogenous proteins, (2) diseases and conditions associated with mitochondrial dysfunction, and (3) diseases and conditions associated with chronic inflammation in a subject in need thereof.

Description

Technical Field

[0001] This article provides a technique in which PrC-210 aminothiol or its analogues have been shown to be very effective, when applied, in treating (i) diseases and conditions characterized by misfolding and / or aggregation of endogenous proteins, (2) diseases and conditions associated with mitochondrial dysfunction, and (3) diseases and conditions associated with chronic inflammation. Background Technology

[0002] Glutathione (GSH) is the most abundant non-protein thiol in human cells. Although GSH is synthesized solely from its constituent amino acids in the cytosol, it is distributed across various compartments, including mitochondria, where its concentration in the matrix is ​​equal to its concentration in the cytosol. It plays a crucial role in the detoxification of lipid hydroperoxides and electrophilic agents. Furthermore, given the central strategic role of mitochondria in the activation and patterning of cell death, mitochondrial GSH has been shown to decisively regulate sensitization levels to secondary damage that induces mitochondrial membrane permeability and releases proteins confined in the intermembrane space. These proteins, once in the cytosol, participate in the molecular mechanisms of cell death (see Vicent Ribas et al., Glutathione and mitochondria, Front. Pharmacol., July 1, 2014, Sec. Experimental Pharmacology and Drug Discovery, Vol. 5–2014). One function of GSH is to form transient disulfide bonds with cysteine ​​residues on many proteins, thereby inhibiting protein misfolding and aggregation. Cysteine ​​is primarily found in critical functional and structural regions of proteins, playing a central role in protein folding or, in excess, a central role in protein aggregation. GSH binding (“occupying”) of protein cysteine ​​residues can protect them by inhibiting the formation of unnatural disulfide bonds, thereby protecting the surrounding protein structure from damage and loss of function. Importantly, GSH-cysteine ​​binding is reversible (see, e.g., Koji Aoyama, Int. J. Mol. Sci. 2021, 22(9), 5010). Studies have shown that proteins form intramolecular unnatural disulfide bonds, leading to the formation of misfolded proteins (see, e.g., Borges et al., July 20, 2014; 21(3): 511–531). While GSH confers this protection on cysteine ​​residues, the GSH pool in each cell is finite, and GSH cannot cross the blood-brain barrier; therefore, once the GSH pool is depleted, it cannot be replaced when redox balance is disrupted in vivo. Because i) the GSH pool in brain cells and other body organs is finite, and ii) it is known to be depleted under oxidative stress, alternative interventions are needed to provide the necessary stability, biodistribution, and redox properties required to protect cellular structure and inhibit protein misfolding and aggregation.There is a need for therapeutic interventions superior to GSH in order to i) protect cellular structures (such as proteins, lipids, sugars, DNA and RNA) from damage by reactive molecules, ii) inhibit protein misfolding and aggregation, and iii) enter and reach all cells in the body. Furthermore, there is a need for new approaches to treat (i) diseases and conditions characterized by the misfolding and / or aggregation of endogenous proteins, (2) diseases and conditions associated with mitochondrial dysfunction, and (3) diseases and conditions associated with chronic inflammation. Summary of the Invention

[0003] The present invention is characterized by PrC-210 aminothiol or similar analogues thereof, which are used to treat (i) diseases and conditions characterized by misfolding and / or aggregation of endogenous proteins, (2) diseases and conditions associated with mitochondrial dysfunction, and (3) diseases and conditions associated with chronic inflammation.

[0004] In a first aspect, the present invention is characterized by a method for inhibiting the aggregation of proteins in a desired subject, the method comprising administering to the subject an effective amount of a compound of formula (I),

[0005]

[0006] Or a pharmaceutically acceptable acid addition salt thereof, in an amount sufficient to inhibit the aggregation of proteins or peptides, wherein (i) A is -CH2NHR' and B is -CH2NHR, or A = -NRR' and B = H; and (ii) each of R and R' is independently selected from H, C1-C6 alkyl and C1-C6 heteroalkyl.

[0007] The prerequisite is that if B = H, then R and R' cannot both be H. In a particular embodiment, the compound is...

[0008]

[0009] Or a pharmaceutically acceptable salt thereof. In some embodiments, the protein whose aggregation is inhibited is selected from TDP-43, α-synuclein, τ, amyloid-β, PolyQhtt, islet amyloid polypeptide, and prions, or their non-aggregated precursors. For example, the subject may suffer from a neurodegenerative disease characterized by misfolding and / or aggregation of endogenous proteins. Neurodegenerative diseases that can be treated using the methods of the present invention include, but are not limited to, Parkinson's disease, prions, Alzheimer's disease, multiple system atrophy, diffuse Lewy body disease, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia and other Poly-Q diseases, hereditary cerebral amyloid angiopathy, and any other neurodegenerative disease described herein. In some embodiments, the neurodegenerative disease is a prion disease selected from the following: Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob disease, hereditary human prion disease, bovine spongiform encephalopathy (BSE), and scrapie. In some embodiments, the neurodegenerative disease is a synucleinopathy (e.g., Parkinson's disease (PD), Lewy body dementia (DLB), and multiple system atrophy (MSA)). In certain embodiments, the subject has a disease or condition characterized by amyloidosis. Amyloidosis may be selected from primary systemic amyloidosis (AL amyloidosis), reactive systemic amyloidosis (AA amyloidosis), type II diabetes mellitus, injection-induced localized amyloidosis, β-2 microglobulin amyloidosis, hereditary non-neuropathic amyloidosis, and Finnish hereditary systemic amyloidosis. In other embodiments, the subject has a disease or condition characterized by ocular protein aggregation. For example, diseases or conditions characterized by the accumulation of proteins in the eye can be cataracts or presbyopia.

[0010] In another aspect, the invention is characterized by a method for treating a subject with a condition related to mitochondrial dysfunction, the method comprising administering to the subject an effective amount of a compound of formula (I),

[0011]

[0012] Or a pharmaceutically acceptable acid addition salt thereof, in an amount sufficient to inhibit the aggregation of proteins or peptides, wherein (i) A is -CH2NHR' and B is -CH2NHR, or A = -NRR' and B = H; and (ii) each of R and R' is independently selected from H, C1-C6 alkyl and C1-C6 heteroalkyl.

[0013] The prerequisite is that if B = H, then R and R' cannot both be H. In a particular embodiment, the compound is...

[0014]

[0015] Or a pharmaceutically acceptable salt thereof. In some embodiments, the condition associated with mitochondrial dysfunction is a neurodegenerative disorder, a neuropsychiatric disorder, diabetes, a metabolic disease, an eye disorder associated with mitochondrial dysfunction, an ischemia-related condition, aging, mitochondrial toxicity associated with the therapeutic agent, or a migraine. For example, the condition associated with mitochondrial dysfunction may be a neurodegenerative disorder selected from the following: Friedrich's ataxia; amyotrophic lateral sclerosis; mitochondrial myopathy, encephalopathy, lactic acidosis, stroke (MELAS); myoclonic epilepsy with broken red fibers (MERFF); epilepsy; Parkinson's disease; Alzheimer's disease and Huntington's disease. In some embodiments, the condition associated with mitochondrial dysfunction is a neuropsychiatric disorder selected from the following: bipolar disorder, schizophrenia, depression, addiction disorder, anxiety disorder, attention deficit disorder, personality disorder, autism (i.e., autism spectrum disorder), and Asperger's syndrome. In some embodiments, the method reduces the mitochondrial toxicity of the therapeutic agent administered to the subject. In some embodiments, the condition associated with mitochondrial dysfunction is selected from the following ocular disorders associated with mitochondrial dysfunction: glaucoma, diabetic retinopathy, and age-related macular degeneration. In specific embodiments, the condition associated with mitochondrial dysfunction is an ischemia-related condition caused by the following: vascular occlusion, arteriosclerosis, valvular heart disease, tachycardia, or hypotension or hypertension.

[0016] In another aspect, the invention is characterized by a method for treating chronic inflammatory conditions in a subject of need, the method comprising administering to the subject an effective amount of a compound of formula (I),

[0017]

[0018] Or a pharmaceutically acceptable acid addition salt thereof, in an amount sufficient to inhibit the aggregation of proteins or peptides, wherein (i) A is -CH2NHR' and B is -CH2NHR, or A = -NRR' and B = H; and (ii) each of R and R' is independently selected from H, C1-C6 alkyl and C1-C6 heteroalkyl.

[0019] The prerequisite is that if B = H, then R and R' cannot both be H. In a particular embodiment, the compound is...

[0020]

[0021] Or a pharmaceutically acceptable salt thereof. In some embodiments, the chronic inflammatory condition is selected from sinusitis, arthritis, dermatitis, vasculitis, acute malaria, sickle cell disease, gastrointestinal inflammatory conditions, or inflammatory lung conditions. In certain embodiments, the chronic inflammatory condition is selected from the following inflammatory lung conditions: asthma, chronic obstructive pulmonary disease (COPD), lung conditions caused by physical trauma, emphysema, bronchitis, sarcoidosis, histiocytosis, lymphangioleiomyomatosis, acute lung injury, chronic lung disease, bronchopulmonary dysplasia, pneumonia, airway deterioration, and acute respiratory distress syndrome (ARDS). In some embodiments, the chronic inflammatory disease is selected from the following gastrointestinal inflammatory conditions: inflammatory bowel disease (IBD), ulcerative colitis (UC), Crohn's disease (CD), and colitis. In some embodiments, the chronic inflammatory condition is an autoimmune disease. For example, the method of the present invention can be used to treat autoimmune diseases selected from the following: multiple sclerosis, type 1 diabetes, rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, pancreatitis, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, paraneoplastic autoimmune diseases, autoimmune hepatitis, bullous pemphigoid, myasthenia gravis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, celiac disease, thyroiditis, Sjögren's syndrome, Guillain-Barré syndrome, Raynaud's phenomenon, Addison's disease, primary biliary cirrhosis, primary sclerosing cholangitis, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis, and diabetes. In some embodiments, chronic inflammation is associated with lipid deposition, such as in the context of arteriosclerosis, valvular heart disease or arterial leakage or endothelial injury, vascular injury during dialysis, and metabolic syndrome. In certain embodiments, the chronic inflammatory condition is a neuroinflammatory disease. Neuroinflammatory diseases that can be treated using the methods of the present invention include multiple sclerosis, neuromyelitis optica, antimyelin oligodendrocyte glycoprotein antibody disorder, autoimmune encephalitis, transverse myelitis, optic neuritis, and neurosarcoidosis, and also include all neurodegenerative diseases, such as, but not limited to, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease.

[0022] definition

[0023] To facilitate understanding of this invention, several terms are defined below. The terms defined herein have meanings commonly understood by one of ordinary skill in the art related to this invention. Terms such as “an,” “a,” and “the” are not intended to refer to a single entity only, but rather to encompass general categories that can be illustrated using specific examples. The terms herein are used to describe specific embodiments of the invention, but their use does not limit the invention unless set forth in the claims.

[0024] As used herein, the term “about” refers to a value within 10% above or below the stated value.

[0025] As used in this article, any value provided in the range includes the upper and lower limits, as well as any value contained within the upper and lower limits.

[0026] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a described compound that, to the extent of reasonable medical judgment, is suitable for contact with tissues in humans and animals without excessive toxicity, irritation, allergic reactions, etc., and is proportionate to a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and Handbook of Pharmaceutical Salts: Properties, Selection, and Use, (edited by P.H. Stahl and C. G. Germuth), Wiley-VCH, 2008. These salts can be acid addition salts involving inorganic or organic acids. These salts can be prepared in situ during the final isolation and purification of the compounds described herein, or prepared separately by reacting a free base group with a suitable acid.

[0027] As used herein, the term "effective amount" refers to an amount sufficient to achieve a beneficial or desired outcome (such as clinical results), and therefore, a "therapeutic effective amount" depends on the context of its application. For example, in the context of administering a compound disclosed herein to inhibit protein misfolding, protein aggregation, or peptide aggregation, an effective amount of the compound is, for example, an amount sufficient to improve the symptoms or progression of a disease characterized by such misfolding or aggregation. In the context of treating a condition characterized by mitochondrial dysfunction, an effective amount of the compound is, for example, an amount sufficient to improve the symptoms or progression of a disease characterized by such mitochondrial dysfunction. In the context of treating a condition characterized by chronic inflammation, an effective amount of the compound is, for example, an amount sufficient to improve the symptoms or progression of a disease characterized by such chronic inflammation. Depending on the nature of the condition being treated, the methods of the present invention may include systemic administration (e.g., intravenous) or local administration (e.g., topical or local injection).

[0028] As used herein and as is well known in the art, “to treat” a condition or “treatment” of various diseases and disorders is a method of achieving a beneficial or desired outcome (such as clinical results). A beneficial or desired outcome may include, but is not limited to, relief of one or more symptoms or conditions; reduction of the severity of a disease, disorder, or condition; stabilization (i.e., non-worsening) of the state of a disease, disorder, or condition; delay or slowing the progression of a disease, disorder, or condition; improvement or relief of a disease, disorder, or condition; and relief (partial or complete), whether detectable or undetectable. “Relief” of a disease, disorder, or condition means a reduction and / or a slowing or prolonging of the severity and / or undesirable clinical manifestations of a disease, disorder, or condition compared to its severity or time course without treatment.

[0029] As used herein, the term "subject" can refer to a human, a non-human primate, or other mammal, such as, but not limited to, dogs, cats, horses, cows, pigs, goats, monkeys, rats, mice, and sheep. In a preferred embodiment, the subject is a human.

[0030] As used herein, the term "pharmaceutical composition" refers to an active compound formulated with one or more pharmaceutically acceptable excipients. In some embodiments, the compounds of the present invention are present in unit doses suitable for administration in treatment regimens that, when administered to relevant populations, show a statistically significant probability of achieving a predetermined therapeutic effect. In some embodiments, the pharmaceutical composition may be specifically formulated for administration in solid or liquid form, including forms suitable for: oral administration, e.g., infusions (aqueous or non-aqueous solutions or suspensions), tablets, or capsules; and parenteral administration, e.g., via subcutaneous, intramuscular, or intravenous injection.

[0031] As used herein, the term "pharmaceuticalally acceptable excipient" refers to any inactive ingredient (e.g., a medium capable of suspending or dissolving an active compound) that is biocompatible and suitable for administration to a subject. Typical excipients include, for example: anti-adhesion agents, antioxidants, binders, coatings, compression aids, disintegrants, dyes, softeners, emulsifiers, diluents, film-forming agents or coatings, flavorings, fragrances, flow aids, lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, or hydrated water. Excipients include, but are not limited to: butylated optional substituted hydroxytoluene (e.g., BHT), calcium carbonate, calcium hydrogen phosphate, calcium stearate, croscarmellose, croscarmellose, citric acid, croscarmellose, cysteine, ethyl cellulose, gelatin, optional substituted hydroxypropyl cellulose, optional substituted hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silica, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch, stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol. A variety of reagents and materials that can be used as excipients are familiar to those skilled in the art.

[0032] As used herein, the term "alkyl" refers to a branched or straight-chain monovalent saturated aliphatic group containing only C and H when unsubstituted. The monovalent nature of an alkyl group does not include optional substituents on the alkyl group. For example, if an alkyl group is attached to a compound, the monovalent nature of the alkyl group refers to its attachment to the compound and does not include any additional substituents that may be present on the alkyl group. In some embodiments, the alkyl group may contain, for example, 1 to 6, 1 to 4, or 1 to 2 carbon atoms (e.g., C1-C6, C1-C4, or C1-C2). Examples include, but are not limited to, methyl, ethyl, isobutyl, sec-butyl, tert-butyl, 2-methylpropyl, and 2,2-dimethylpropyl.

[0033] As used herein, the term “C1-C6 heteroalkyl” refers to a branched or straight-chain monovalent saturated aliphatic group containing C, H and one or three N atoms.

[0034] Other features and advantages of the present invention will become apparent from the following detailed description, drawings and claims. Attached Figure Description

[0035] Figure 1 (A) and Figure 1 (B) shows PrC-210 at pH 3.0 ( Figure 1 a) or pH 9.1 Figure 1 b) After being placed in deuterated water for 24 hours1 1H NMR spectra. These spectra show that PrC-210 does not change its charged structure and remains stable for at least 24 hours within this pH range.

[0036] Figure 2 The results show the effects of PrC-210 after 1 hour, 24 hours, and 72 hours in deuterated water at pH 9.0. 1 1H NMR spectra. These spectra show that PrC-210 exhibits some instability at high pH values ​​after 72 hours.

[0037] Figure 3 The graph shows that the pKa of the two amines of PrC-210 is much larger than (i.e., 3 pH units) the physiological pH (7.2).

[0038] Figure 4 The results show the effects of PrC-210 after 1 hour and 24 hours in deuterated water containing ammonium acetate at pH 7.0. 1 1H NMR spectrum. The figure shows that PrC-210 began to undergo significant structural changes within hours, which likely indicates that a disulfide bond was formed between two PrC-210 molecules in the presence of an acetate anion.

[0039] Figure 5 The results show the effects of PrC-210 on deuterated water containing ammonium acetate and the thiol blocker TCEP at pH 7.0 for 1 hour and 24 hours. 1 HNMR spectrum. The figure shows that PrC-210 remains stable for 24 hours in the presence of TCEP and acetate anion.

[0040] Figure 6 (A) is a graph depicting the half-life of PrC-210 in acetate buffer at pH 7.2 as 3.5 hours; previously, a half-life of PrC-210 in water was observed to be several days.

[0041] Figure 6 (B) shows a reaction scheme describing the reaction between the anionic ammonium acetate and PrC-210 thiol that induces the formation of a PrC-210-PrC-210 disulfide bond. In short, (1) the free electron pair of the negatively charged acetate attacks the proton of the thiol on PrC-210, (2) and then the deprotonated, activated thiol anion on the PrC-210 molecule can (3) react with the PrC-210 thiol molecule to form a PrC-210 dimer.

[0042] Figure 6 (C) describes the situation in the presence of proteins with one or more cysteine ​​residues (such as TDP-43). Figure 6(B) Diagram of the disulfide bond formation reaction.

[0043] Figure 7 The table depicts the percentage of

[14] C-labeled PrC-210 recovered after intravenous administration to male rats. Very low metabolism of PrC-210 was observed at 48 hours (1.07%).

[0044] Figure 8 The graph shows that adding ammonium acetate a few seconds before adding hydrogen peroxide can significantly increase (“accelerate”) the consumption of PrC-210 thiols in a dose-dependent manner by adding 0.5 mM hydrogen peroxide.

[0045] Figure 9 The levels of (A) 8-oxo-2'-deoxyguanosine (8-oxo-dG), (B) p53, (C) cytochrome C, (D) caspase 8, and (E) caspase 3 / 7 markers in the mouse brain after irradiation with 8.68 Gy are shown. (F) The levels of caspase 3 / 7 in mouse plasma after irradiation with 8.68 Gy are shown. Caspase levels were elevated 30 minutes before irradiation with 8.68 Gy in the absence of PrC-210 treatment, and were inhibited to background levels by PrC-210 upon administration of 0.5 MTDP.

[0046] Figure 10 Three SOD1s were displayed G93A Changes in mouse body weight over time. Weight gradually decreased with the onset of ALS motor symptoms and hind limb paralysis. Systemic administration of PrC-210 (0.1 MTD, IP) was associated with an immediate plateau in weight loss.

[0047] Figure 11 The levels of (A) cytochrome C and (B) caspase 3 / 7 markers in the heart of mice after irradiation with 8.68 Gy are shown. PrC-210 (0.3 MTD) was administered 24 hours after irradiation with 8.68 Gy.

[0048] Figure 12 The levels of (A) caspase 1, (B) complement 3, and (C) caspase 3 / 7 markers in the mouse brain after irradiation with 8.68 Gy are shown. PrC-210 (0.3 MTD) was administered 24 hours after irradiation with 8.68 Gy.

[0049] Figure 13 The paralysis scores in EAE mouse models are shown, where (A) different intraperitoneal PrC-210 doses were started twice a week 24 hours before immunization with MOG peptides, and (B) different intraperitoneal PrC-210 doses were started twice a week when paralysis symptoms were first observed.

[0050] Figure 14 The paw swelling fractions of DBA / 1J mice are shown, where i) intraperitoneal PrC-210 doses were started twice weekly at the first appearance of paw swelling symptoms, and ii) local application of 370 mM PrC-210 was started when the paw swelling fraction was higher than 10. Detailed Implementation

[0051] The present invention is characterized by the use of aminothiols to protect cellular structures from damage and to inhibit protein misfolding and aggregation in subjects in need. The applicant has discovered that the aminothiols of the present invention, in the presence of a negative charge, have the ability to form disulfide bonds with cysteine ​​residues of endogenous proteins, thereby inhibiting the formation of intraprotein Cys-Cys disulfide bonds and the associated acceleration of protein misfolding and aggregation in a variety of cellular proteins.

[0052] aminothiols

[0053] The aminothiol used in the methods of the present invention can be synthesized, for example, as described in U.S. Patent 7,314,959.

[0054] Dosage

[0055] The dosage of the disclosed compounds depends on a variety of factors, including the route of administration, the disease to be treated, and the subject's physical characteristics, such as age, weight, and overall health. Typically, the amount of the disclosed compound (e.g., PrC-210) contained in a single dose or in multiple doses over a longer period can be an amount that effectively treats the disease without inducing significant toxicity. Clinicians can adjust the dosage based on routine factors such as the severity of the disease and different parameters of the subject. Typically, the pharmaceutical compositions of this disclosure can be administered in amounts ranging from about 0.001 mg / kg / day to up to about 500 mg / kg / day of aminothiols (such as PrC-210).

[0056] Treatment

[0057] This invention relates to the systemic administration of PrC-210 aminothiol or its analogues to protect proteins, lipids, sugars, RNA, and DNA, as well as cysteine ​​residues within proteins, thereby preventing protein misfolding and aggregation, and by doing so, preventing or significantly inhibiting a variety of disease states. In this invention, we have demonstrated that both amines of aminothiol PrC-210 are positively charged over a very wide physiological pH range, which results in a strong attraction between them and the negatively charged surfaces of proteins, lipids, sugars, RNA, and DNA via electrostatic interactions. DNA, RNA, lipids, sugars, proteins, lipoproteins, and glycoproteins all possess a certain amount of negative charge on their surfaces. The two positive charges on PrC-210 will generate attraction through charge complementarity with these negatively charged surfaces on cellular biomolecules. Although the PrC-210 thiol group exhibits very low reactivity in water in the absence of a negative charge, and is highly stable as a monomeric aminothiol, in this invention we have demonstrated that the electrostatic interaction of PrC-210 with the negative charge of the closely adjacent aminothiol significantly alters the stability of the aminothiol and increases (“activates”) the PrC-210 thiol group. This “activated thiol” then reacts more readily with other thiols or reactive structures. Furthermore, the activated aminothiol of this invention subsequently forms disulfide bonds with cysteine ​​residues of endogenous proteins, suggesting that similar reactions of PrC-210 with other cellular proteins will inhibit the formation of intraprotein Cys-Cys disulfide bonds, as well as their associated protein misfolding and aggregation.

[0058] The present invention is characterized by the application of PrC-210 aminothiol or similar substances thereof to treat (i) diseases and conditions characterized by misfolding and / or aggregation of endogenous proteins, (2) diseases and conditions associated with mitochondrial dysfunction, and (3) diseases and conditions associated with chronic inflammation.

[0059] Neurodegenerative diseases

[0060] The method of the present invention can be used to treat neurodegenerative diseases characterized by misfolding and / or aggregation of endogenous proteins, such as Parkinson's disease, prions, Alzheimer's disease, multiple system atrophy, diffuse Lewy body disease, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia and other Poly-Q diseases and hereditary cerebral amyloid angiopathy.

[0061] In some implementations, the prion disease to be treated is selected from Crout-Jacques disease, variant Crout-Jacques disease, hereditary human prion disease, bovine spongiform encephalopathy (BSE), and scrapie.

[0062] In a particular implementation, the neurodegenerative disease to be treated is synucleinosis. Synucleinosis is characterized by the intracellular accumulation of protein aggregates, oligomers, profibrils, and fibrils, primarily containing α-synuclein. In the case of synucleinosis, the pathological effects on nerve cells are believed to result from the formation of oligomers of α-synuclein and the subsequent formation of membrane pores. Examples of synucleinosis include Parkinson's disease (PD), Lewy body dementia (DLB), and multiple system atrophy (MSA).

[0063] The method of the present invention can be used to treat conditions involving amyloidosis. Amyloidosis is a general term to describe a variety of diseases characterized by the presence of a pathological form of amyloid protein, which typically involves extracellular deposition of protein fibrils, forming numerous “amyloid deposits” or “amyloid plaques” that can occur locally or systemically.

[0064] In some implementations, the amyloidosis to be treated is selected from primary systemic amyloidosis (AL amyloidosis), reactive systemic amyloidosis (AA amyloidosis), type II diabetes, injection-induced localized amyloidosis, β-2 microglobulin amyloidosis, hereditary non-neuropathic amyloidosis, and Finnish hereditary systemic amyloidosis.

[0065] ocular protein aggregation disease

[0066] The method of the present invention can be used to treat eye diseases and conditions characterized by misfolding and / or aggregation of endogenous proteins, including cataracts and presbyopia.

[0067] Presbyopia is the loss of the eye's accommodative ability, leading to an inability to focus on near objects. Hardening of the lens and changes in the elasticity of the lens capsule are common causes of presbyopia. Therefore, agents that can prevent or reverse lens hardening would offer a promising avenue for novel non-invasive treatments for presbyopia. At the molecular level, proteins called lens proteins play a crucial role in the hardening of the eye's lens. Lens proteins include three isotypes: a, b, and g, accounting for 90% of the protein content of the eye's lens. α-lens (AC) is a non-ATP-dependent molecular chaperone and a member of the small heat shock protein (sHsp) family, accounting for 40% of the lens proteins. It exists as a heterooligomeric form of two subunits (aA-lens protein (AAC) and aB-lens protein (ABC)), and its expression is primarily confined to the eye's lens. It identifies exposed conformational features in partially unfolded lens proteins and isolates them from each other, thereby reducing the amount of easily aggregated material that would otherwise contribute to various age-related vision impairments. The conversion of soluble AC to HMW aggregates is accompanied by a significant increase in lens stiffness.

[0068] Mitochondrial dysfunction

[0069] The method of this invention can be used to treat diseases and conditions characterized by mitochondrial dysfunction. Conditions associated with mitochondrial dysfunction can result in a progressive mosaic appearance of cells in muscle with defective electron transport activity, cells with little to no cytochrome c oxidase (COX) activity randomly scattered among normally active cells, and a high incidence of COX-negative cells in biopsies (e.g., in elderly subjects). Therefore, during aging or in various conditions associated with mitochondrial dysfunction, organisms face a situation where irreplaceable postmitotic cells (e.g., neurons, skeletal muscle, and cardiac muscle) must be preserved and their function largely maintained, while facing the inevitable gradual decline in mitochondrial respiratory chain function. Neurons with mitochondrial dysfunction become increasingly sensitive to damage such as excitotoxic injury. Mitochondrial failure is a contributing factor to most age-related degenerative diseases, particularly neurodegeneration. Congenital mitochondrial diseases often involve early-onset neurodegeneration, with underlying mechanisms similar to those experienced during aging in individuals born with normal mitochondria. The methods and compositions of the present invention can improve dysfunctional mitochondria in these irreplaceable postmitotic cells, thereby rescuing or improving mitochondrial function in these cells.

[0070] Conditions associated with mitochondrial dysfunction include pathophysiological conditions in which defects in mitochondrial respiratory chain activity lead to such diseases or disorders in mammals. These defects include: (1) congenital genetic defects in the activity of one or more components of the mitochondrial respiratory chain or electron transport chain; and (2) acquired defects in the level or activity of one or more components of the mitochondrial respiratory chain, wherein such defects are caused by: (a) oxidative damage during aging; (b) elevated intracellular calcium; (c) exposure of affected cells to nitric oxide; (d) hypoxia or ischemia; (e) microtubule-related defects in mitochondrial axonal transmission; or (f) expression of mitochondrial uncoupling proteins.

[0071] Common symptoms of mitochondrial dysfunction include cardiomyopathy, muscle weakness and atrophy, developmental delay (involving motor, language, cognitive, or executive functions), ataxia, epilepsy, renal tubular acidosis, peripheral neuropathy, optic neuropathy, autonomic neuropathy, neurogenic bowel dysfunction, sensorineural hearing loss, neurogenic bladder dysfunction, dilated cardiomyopathy, migraine, liver failure, lactic acidosis, and diabetes.

[0072] Neuropsychiatric disorders

[0073] The brain requires an average of ten times more energy than other parts of the body. Specifically, many neuropsychiatric disorders can be associated with abnormal energy production or mitochondrial dysfunction. Neuropsychiatric disorders include, but are not limited to, bipolar disorder (BD), schizophrenia, depression, anxiety disorders, attention deficit disorder, addiction disorders, personality disorders, autism, and Asperger's syndrome. The methods and compositions of this invention can be used to treat neuropsychiatric disorders.

[0074] Neurodegenerative disorders

[0075] The methods and compositions of this invention can be used to treat neurodegenerative disorders. Many progressive neurological diseases are caused by neuronal apoptosis in mitochondria. Friedrich's ataxia is caused by a genetic defect in the frataxin gene, which is involved in mitochondrial iron transport (Babcock et al., Science 276:1709 (1997)); human deafness dystonia is caused by a small component defect in the mitochondrial protein import mechanism (Koehler et al., Proc. Natl. Acad. Sci. USA 96:2141 (1999)); a well-established cause of amyotrophic lateral sclerosis is a deficiency in Cu-Zn superoxide dismutase, which is located in the mitochondrial intermembrane space and cytoplasm (Deng et al., Science 261:1047 (1993)). Multiple environmental toxins have been found to contribute to Parkinson's disease by inhibiting respiratory complex I and promoting the production of reactive oxygen species, making this complex a focus of research in Parkinson's disease (Dawson et al., Science 302:819 (2003)). Recently, mitochondrial proteins encoded by PINK 1 have provided a direct link between mitochondria and Parkinson's disease (Valente et al., Science 304:1158 (2004)). Alzheimer's disease is also associated with mitochondrial toxicity because the mitochondrial protein ABAD is a target of amyloid protein (Lustbader et al., Science 304:448 (2004)). Huntington's disease is associated with a persistent energy metabolism defect affecting both the brain and peripheral tissues, and is caused by mitochondrial dysfunction (Leegwater-Kim et al., NeuroRx 1:128 (2004)). The pathogenesis of bipolar disorder (BD) is believed to involve fundamental abnormalities related to energy production, particularly mitochondrial activity. Evidence from multiple sources (including autopsy, genetics, brain imaging, and peripheral cell studies) supports that energy deficiency and mitochondrial dysfunction are important pathogenic factors in the development of BD (see Hough et al., Bipolar Disord. 2:145 (2000), Fattal et al., Psychosomatics 47:1 (2006), and Kato et al., Bipolar Disord. 2:180 (2000)).

[0076] Diabetes and metabolic diseases

[0077] The methods and compositions of this invention can be used to treat diabetes and metabolic diseases. The central role of mitochondria in carbohydrate and fatty acid metabolism makes this organelle crucial in diabetes (Maechler et al., Nature 414:807 (2001)). Knockout of abundant mitochondrial transcription factors in mice provides a model for 13-cell ablation in juvenile diabetes (Silva et al., Nat. Genet. 26:335 (2000)). Mutations in mtDNA and PPARγ (major regulators of mitochondrial biosynthesis) are associated with type 2 diabetes. Insulin release depends on mitochondrial function, which is influenced by the expression of the membrane transporter UCP2 (Petersen et al., Science 300:1140 (2003); Zhang et al., Cell 105:745 (2001)). The activity of thiazolidinediones as antidiabetic drugs appears to depend on their ability to control the expression of nuclear genes of mitochondrial gene products as ligands of PPARγ and its coactivator PGC-1 (Mootha et al., Nature Genet 34:267 (2003); Puigserver et al., Endocr. Rev. 24:78 (2003)).

[0078] migraine

[0079] The methods and compositions of this invention can be used to treat migraines. Metabolic studies in patients with recurrent migraines have shown that mitochondrial activity defects are often associated with this impairment, manifested as impaired oxidative phosphorylation and excessive lactate production. This defect is not necessarily due to a genetic defect in mitochondrial DNA. Migraine patients are highly sensitive to nitric oxide (an endogenous inhibitor of cytochrome c oxidase). Furthermore, patients with mitochondrial cell diseases (such as MELAS) often suffer from recurrent migraines.

[0080] Ocular disorders associated with mitochondrial dysfunction

[0081] The methods and compositions of this invention can be used to treat eye disorders such as glaucoma, diabetic retinopathy, and age-related macular degeneration (AMD). Retinal damage results from free radical-induced reactions in glaucoma, diabetic retinopathy, and AMD. The eye is part of the central nervous system and has limited regenerative capacity. The retina is composed of many nerve cells containing the highest concentrations of polyunsaturated fatty acids (PFAs) and is prone to oxidation. Ultraviolet light entering the eye and the mitochondria in rod and cone cells generates free radicals, producing the energy needed to convert light into visual impulses. Free radicals cause PFAs to undergo peroxidative translation via hydroxyl or superoxide radicals, subsequently generating additional free radicals. Free radicals can cause temporary or permanent damage to retinal tissue.

[0082] Glaucoma is generally considered a barrier that causes elevated intraocular pressure (IOP), leading to permanent damage to retinal nerve fibers. However, one in six glaucoma cases does not present with elevated IOP. Currently, this barrier is thought to be a result of reduced vascular perfusion and increased neurotoxicity. Recent studies have shown that elevated levels of glutamate, nitric oxide, and peroxynitrite in the eye are contributing factors to retinal ganglion cell death.

[0083] Diabetic retinopathy occurs when microvascular abnormalities (primarily microaneurysms and intraretinal hemorrhage) appear in the basal vessels. Oxidative metabolites are directly involved in the pathogenesis of diabetic retinopathy, and increased free radicals lead to the production of growth factors with enhanced proliferative activity. Nitric oxide produced by vascular endothelial cells can also cause relaxation of smooth muscle cells and vasodilation in vascular segments. Retinal ischemia and hypoxia occur after thickening of the arterial basement membrane, endothelial proliferation, and loss of pericytes. Insufficient oxygenation leads to capillary occlusion or no perfusion, arteriole-venule shunting, slow blood flow, and impaired oxygen release from erythrocytes. Lipid peroxidation in retinal tissue also results from free radical damage.

[0084] Ischemia-related diseases

[0085] The methods and compositions of this invention can be used to treat ischemia-related conditions. Oxygen deficiency inhibits mitochondrial respiratory chain activity both directly (by depriving cytochrome c of its terminal electron acceptor at complex IV for re-oxidation) and indirectly (particularly in the nervous system, through excitotoxicity and nitric oxide formation following secondary hypoxia). In conditions such as cerebral hypoxia and angina crisis, tissues are relatively hypoxic. In such cases, increased mitochondrial activity protects affected tissues from the harmful effects of hypoxia, reduces secondary delayed cell death, and accelerates recovery from hypoxic tissue stress and injury. The methods and compositions of this invention can be used to prevent delayed cell death following, for example, cerebral ischemia or hypoxic injury (apoptosis occurring in regions such as the hippocampus or cortex approximately 2 to 5 days after an ischemic attack).

[0086] Muscle function

[0087] The methods and compositions of the present invention can be used to enhance muscle performance. For example, the methods and compositions of the present invention can be used to improve physical endurance (e.g., the ability to perform physical tasks such as exercise, manual labor, sports activities, etc.), inhibit or delay physical fatigue, increase blood oxygen levels, enhance energy in healthy individuals, improve work capacity and endurance, reduce muscle fatigue, reduce stress, enhance cardiac and cardiovascular function, improve sexual function, increase muscle ATP levels and / or reduce lactic acid in the blood.

[0088] Enhanced athletic performance, strength, speed, and endurance are typically measured by increased muscle contraction strength, increased muscle contraction amplitude, shortened muscle response time between stimulus and contraction, ability to overcome muscle fatigue, and ability to sustain activity for longer periods. In addition to muscle performance during endurance training, free radical and oxidative stress parameters are also influenced by pathophysiological states. Extensive data suggest that oxidative stress is a contributing factor to wasting or atrophy in pathophysiological states (see Clarkson, Crit. Rev. Food Sci. Nutr. 35:31 (1995); and Powers et al., Proc. Nutr. Soc. 58:1025 (1999)). For example, in muscular dystrophy, defects in the dystrophin-glycoprotein complex (DGC) suggest that one mechanism of cellular damage is functional ischemia associated with altered cellular NOS and disruption of normal NO protective function. Rando (Microsc.Res.Tech.55:223(2001)) demonstrated that oxidative damage precedes pathological changes, and that defective muscle cells in DGCs are more sensitive to oxidative stress. Excessive lipid peroxidation caused by free radicals has also been shown to be a cause of myopathy (such as McCardell's disease) (see Russo et al., Med.Hypotheses.39:147(1992)). Furthermore, mitochondrial dysfunction is known to be associated with age-related muscle atrophy (sarcopenia), and although this is not well-studied, free radical damage is considered a contributing factor (see Navarro et al., Front.Biosci.6:D26(2001)). Other indications include acute sarcopenia, such as muscle atrophy and / or cachexia associated with burns, bed rest, limb immobilization, or major chest, abdominal, and / or orthopedic surgery. The method of this invention can effectively treat muscle-related pathological conditions.

[0089] senescence

[0090] The methods and compositions of this invention can be used to treat aging and related conditions. During normal aging, the function of the mitochondrial respiratory chain gradually declines. Starting around age 40, the accumulation of mitochondrial DNA defects in humans increases exponentially, and the nuclear regulatory factors of mitochondrial respiratory activity also decline synchronously. Many mitochondrial DNA defects have a selective advantage during mitochondrial turnover, especially in post-mitotic cells. The proposed mechanism is that mitochondria with defective respiratory chains produce less oxidative damage (mitochondrial respiration is a major source of free radicals in the body) than mitochondria with intact functional respiratory chains. Therefore, oxidative damage to membrane lipids accumulates in normally functioning mitochondria more quickly than in defective mitochondria, and is thus “tagged” for degradation by the autophagy and lysosomal systems. The inevitable decline in mitochondrial function with age is a contributing factor to age-related neurodegenerative diseases and type II diabetes. Just as oxidative stress is the root cause of certain specific diseases, it is also considered a contributing factor to generalized aging (Harman, Proc. Natl. Acad. Sci. USA 78:7124 (1981)). Studies have shown that mutations in *C. elegans* and *D. melanogaster* that reduce mitochondrial oxidative stress extend the lifespan of these organisms (Hekimi et al., Science 299:1351 (2003)). Furthermore, mammals maintained on calorie-restricted diets have lower metabolic rates, which is thought to contribute significantly to extended lifespan. Numerous studies have demonstrated that point mutations and deletions in mtDNA increase with age. In addition, Trifunovic et al. recently demonstrated that mice engineered to express error-prone mitochondrial DNA polymerase can serve as a good model of premature aging (Nature 429:417 (2004)). The method of this invention can be used to treat progeria, Werner syndrome, Wiedemann-Rautenstrauch syndrome, and symptoms of aging (e.g., wrinkles, amnesia, arthritis, age-related muscle atrophy).

[0091] In certain embodiments, the method of the present invention is used to treat conditions characterized by mitochondrial dysfunction selected from the following: neurodegenerative disorders (e.g., Friedrich's ataxia; amyotrophic lateral sclerosis; mitochondrial myopathy, encephalopathy, lactic acidosis, stroke (MELAS); myoclonic epilepsy with broken red fibers (MERFF); epilepsy; Parkinson's disease; Alzheimer's disease or Huntington's disease), neuropsychiatric disorders (e.g., bipolar disorder, schizophrenia, depression, addiction disorder, anxiety disorder, attention deficit disorder, personality disorder, autism or Asperger's syndrome), diabetes, metabolic diseases, eye disorders associated with mitochondrial dysfunction (e.g., glaucoma, diabetic retinopathy or age-related macular degeneration), ischemia-related conditions (e.g., conditions caused by vascular occlusion, arteriosclerosis, valvular heart disease, tachycardia or hypotension), aging, mitochondrial toxicity associated with therapeutic agents, or migraine.

[0092] Chronic inflammation

[0093] The methods and compositions of this invention can be used to treat chronic inflammation and related conditions. Exemplary inflammatory conditions that can be treated using the methods of this invention include asthma (e.g., aspirin-sensitive / exacerbated asthma, atopic asthma, severe asthma, mild asthma, moderate to severe asthma, asthma not treated with corticosteroids, chronic asthma, corticosteroid-resistant asthma, corticosteroid-refractory asthma, newly diagnosed and untreated asthma, smoking-induced asthma, asthma uncontrolled by corticosteroids, etc.), airway hyperresponsiveness, airway hypersensitivity, sinusitis, sinusitis with polyps, nasal polyposis, arthritis (e.g., osteoarthritis, rheumatoid arthritis, arthritic joint inflammation due to injury, etc.), and seronegative osteomyelitis. Spot disease and joint disease (SEA) syndrome, acute malaria, sickle cell disease, osteoporosis, eosinophilic esophagitis, dermatitis, atopic dermatitis, allergic rhinitis, bullous pemphigoid, chronic urticaria, chondritis, polymyalgia rheumatica, polyarteritis nodosa, Wegener's granulomatosis, Behcet's disease, myelitis, polymyositis, dermatomyolitis, dermatomyositis, vasculitis, arteritis, diabetic nephropathy, interstitial cystitis, gastrointestinal inflammatory diseases (e.g., inflammatory... Enteropathy (IBD), ulcerative colitis (UC), Crohn's disease (CD), colitis (e.g., colitis caused by environmental pollution (e.g., colitis caused by or related to treatment regimens such as chemotherapy, radiation therapy, etc.)), chronic hepatitis (e.g., viral hepatitis, alcohol-related hepatitis) and chronic hepatitis (e.g., viral hepatitis, alcohol-related hepatitis, non-alcoholic fatty liver disease, etc.), ischemic colitis, collagenous colitis or lymphocytic colitis, colitis in conditions such as chronic granulomatous disease or celiac disease, food allergies, and gastritis, and inflammatory lung diseases (e.g., chronic obstructive pulmonary disease). Lung diseases include chronic respiratory diseases such as COPD, allergen-induced lung conditions, pollutant-induced lung conditions (e.g., asbestosis, silicosis, or beryllium poisoning), aspiration-induced lung conditions, immune dysregulation, genetically predisposed inflammatory diseases (such as cystic fibrosis), traumatic lung conditions (e.g., ventilator injury), emphysema, bronchitis, sarcoidosis, histiocytosis, lymphangioleiomyomatosis, acute lung injury, chronic lung disease, bronchopulmonary dysplasia, pneumonia (e.g., community-acquired pneumonia, hospital-acquired pneumonia, ventilator-associated pneumonia, and severe pneumonia), airway deterioration, and acute respiratory distress syndrome (ARDS). Other examples of chronic inflammatory conditions include diseases with neuroinflammatory components, such as multiple sclerosis and neurodegenerative diseases.

[0094] Autoimmune diseases

[0095] The methods and compositions of the present invention can be used to treat autoimmune diseases, including multiple sclerosis, type 1 diabetes, rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, pancreatitis, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, paraneoplastic autoimmune diseases, autoimmune hepatitis, bullous pemphigoid, myasthenia gravis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, celiac disease, thyroiditis (e.g., Graves' disease), Sjögren's syndrome, Guillain-Barré syndrome, Raynaud's phenomenon, Addison's disease, liver diseases (e.g., primary biliary cirrhosis, primary sclerosing cholangitis, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis), and diabetes (e.g., type 1 diabetes).

[0096] Related symptoms

[0097] The methods and compositions of the present invention can be used to treat conditions exacerbated by one or more of protein aggregation, mitochondrial dysfunction, and / or chronic inflammation, including encephalitis, meningitis, nephritis, myocarditis, pneumonia, gangrene and non-healing wounds, chronic hepatitis, endotoxin-induced liver or organ damage, acne, inflammatory bowel disease, ulcerative colitis, Crohn's disease, psoriasis, vasculitis, glomerulonephritis, pancreatitis, tendinopathy, thyroiditis, myasthenia gravis, pernicious anemia, dermatomyositis, Sjögren's syndrome, aplastic anemia, celiac disease, eczema, arteriosclerosis, hypertension, myocardial ischemia, nerve ischemia, renal ischemia, chronic heart failure and cardiac hypertrophy, cardiomyopathy, chronic kidney disease, coronary heart disease, stroke, aortic disease, varicose veins, angina pectoris, organ failure (e.g., renal failure, liver failure, and / or lung failure), fatty streaks, atherosclerosis, atherosclerotic plaques, fetal growth restriction, and preeclampsia.

[0098] The following examples are intended to illustrate the present invention. They are not intended to limit the invention in any way.

[0099] Example

[0100] Example 1: Mode of Operation of the Invention

[0101] We established the chemical structure spectrum of PrC-210 using nuclear magnetic resonance (NMR) spectroscopy. We found that when titrated at pH values ​​between pH 3 and pH 9.1, PrC-210 does not change its charged structure and remains stable (see [link to NMR spectroscopy]). Figure 1 a and Figure 1 b). The structural stability of this PrC-210 monothiol form was maintained for at least 24 hours within this pH range. We did indeed observe a significant change after 72 hours at pH 9.0 ( Figure 2The addition of the thiol blocker TCEP to water did not affect the stability of PrC-210 at pH 9.0 for 72 hours (data not shown), meaning that the thiol groups are stable and non-reactive (or “activating”) under these conditions. In pH titrations between pH 9.0 and pH 13.0, we observed significant structural changes in PrC-210; from these asymptotic changes, we calculated that the pKa of both PrC-210 amines were significantly higher than that of physiological conditions (pH 7.2). Figure 3 ).

[0102] Interestingly, when ammonium acetate with an anionic charge was added at pH 7, PrC-210 began to undergo significant structural changes within hours. Figure 4 This indicates that a disulfide bond has formed between the two PrC-210 molecules. We verified this hypothesis by adding the thiol blocker TCEP to ammonium acetate incubation, and the single PrC-210 remained stable again for 24 hours. Figure 5 ).exist Figure 6 In A, when ammonium acetate was added to an aqueous solution of PrC-210 monothiol, we determined the half-life at pH 7.2 to be 3.1 hours. These observations indicate that the addition of anionic ammonium acetate molecules to an aqueous solution of PrC-210 monothiol significantly increases the reactivity of the thiol and forms a PrC-210 disulfide molecule; that is, 1) the free electron pair of the negatively charged acetate ion attacks the protons of the thiol on PrC-210, and 2) the deprotonated, activated thiol anion on the PrC-210 molecule subsequently exhibits affinity, 3) reacting with another PrC-210 thiol to form a PrC-210 disulfide dimer. Figure 6 B).

[0103] We anticipate that, in the presence of negatively charged ammonium acetate, thiol activation reflects a reaction that occurs when a single PrC-210 is in close proximity to any negatively charged molecule or site. The thiol is then activated, thereby reacting with any of a number of reactive sites, including electrophiles, other thiol residues on the PrC-210 molecule, or other thiols on Cys residues of cellular proteins (forming disulfides). Figure 6 C)), carbon structure (thioether), oxygen structure (forming sulfoxide (1xO), hyposulfonic acid ((1xO)H), sulfinic acid ((2xO)H) and sulfonic acid ((3xO)H)), nitrogen structure (forming S-nitrosothiol or non-free radical (such as hydrogen peroxide)).

[0104] We also wanted to observe whether the formation of PrC-210-cysteine ​​disulfide was temporary or permanent; the formation of permanent PrC-210 isodisulfide with cellular proteins was expected to affect protein function and could translate into undesirable toxic side effects, and should be considered as PrC-210 residues appearing as urinary or fecal metabolites. Multiple experimental results indicated no clear evidence of permanent formation of PrC-210-cysteine / protein disulfide bonds, including: i) the absence of toxic side effects of PrC-210 within its therapeutic window (e.g., see Soref et al., Int J Radiation Oncol Biol Phys, Vol. 82, No. 5, p. e701ee707, 2012), and ii) in pharmacokinetic studies using PrC-210 labeled with

[14] C, we found that PrC-210 was metabolized very little (1.07%) only after 48 hours. Figure 7 (Data not published). It has been well described that i) disulfide bond binding is reversible (see, e.g., Koji Aoyama, Int. J. Mol. Sci. 2021, 22(9), 5010); and ii) two efficient thiol reduction systems exist in the cell (see, e.g., Cumming et al., The Journal of Biological Chemistry, Vol. 279, No. 21, May 21, pp. 21749–21758, 2004), which reduce the disulfide bond between glutathione and proteins under non-oxidative stress conditions (see, e.g., Koji Aoyama, Int. J. Mol. Sci. 2021, 22(9), 5010). Based on our experimental results and published literature, it is logical that the binding of PrC-210 disulfide bonds to proteins is controlled by the same chemical and enzymatic processes.

[0105] We believe that the reactivity of PrC-210-thiols increases in the presence of negatively charged compounds (such as ammonium acetate) when they are in close proximity to other reactive structures (such as hydrogen peroxide). We investigated hydrogen peroxide because it is a non-radical oxygen compound and has a much longer half-life than superoxide. We added hydrogen peroxide to an aqueous solution of 5 mM PrC-210-thiols at pH 7. Hydrogen peroxide caused a dose-dependent consumption of free PrC-210-thiols. Figure 8 Adding 5 mM ammonium acetate to a 5 mM PrC-210 thiol aqueous solution at pH 7 a few seconds before adding hydrogen peroxide significantly accelerates the consumption of hydrogen peroxide by PrC-210 thiol (“accelerator effect”).

[0106] To investigate the protective effects conferred by PrC-210 in an animal model, we irradiated mice with 8.68 Gy and treated them with a 0.5 MTD dose of PrC-210 30 minutes prior to irradiation. Typically, lethal radiation of 8.68 Gy results in significant DNA damage and activation of intrinsic apoptosis pathways. When we examined the brains of the irradiated mice… Figure 9 A to Figure 9 C), the levels of 8-oxo-dG, cytochrome C, and p53 damage biomarkers were not significantly increased. This means that radiation damage does not trigger significant activation of the intrinsic apoptotic pathway, nor does it trigger mitotic arrest due to p53 activation. The most likely reason is that neurons are already in the most active phase of mitosis, thus DNA is highly protected from radiation damage. Interestingly, caspase 8 and caspase 3 / 7 were also activated in the absence of PrC-210, and when PrC-210 was administered systemically 24 hours before radiation damage, they were suppressed to background levels, meaning that in the presence of PrC-210, proteins are protected from damage. Figure 9 D、 Figure 9 E). This inhibition and protection against apoptosis persisted throughout the six-day observation period, indicating that PrC-210 remained active after six days. When we observed mouse plasma in the same experiment, we found that caspase 3 / 7 levels were also subject to the same inhibition of apoptosis as in the brain over six days. Figure 9 F).

[0107] In cells, the positively charged amine on PrC-210 typically i) causes the PrC-210 molecule to concentrate and “suspend” around negatively charged biomolecules, and ii) the negative charge of the biomolecules also increases the reactivity of the PrC-210 thiol, thereby promoting its reaction with reactive structures (such as hydroperoxides) and other thiol groups on cellular proteins. We conclude that for the treatment of disease: i) the amount of PrC-210 required at a given time depends on the activity of the disease; ii) the amount of PrC-210 required in a steady state may be relatively small, thus the PrC-210 available in the cell acts as a reservoir; and iii) once PrC-210 enters the cell, it remains there for a long time as a stable, low-reactivity molecule until activated through the described mechanisms. Therefore, PrC-210 dosing regimens need to take into account both acute and chronic nature of the disease and the severity of the injury.

[0108] Example 2: Using the present invention to reduce the progression of diseases involving protein aggregation, such as amyotrophic lateral sclerosis (ALS).

[0109] The experiment was designed to elucidate whether PrC-210 could halt the progression of protein aggregation-based diseases, such as amyotrophic lateral sclerosis (ALS). Figure 10 SOD1 was displayed G93A The changes in mouse body weight over time. Body weight gradually decreased, exhibiting an exponential decline with the onset of ALS motor symptoms. Administration of systemic PrC-210 (0.1 MTD, intraperitoneal) was associated with an immediate plateau in body weight loss, i.e., the cessation of weight loss in ALS mice. Since ALS pathology is based on protein aggregation, the results of this experiment support our inventive concept that PrC-210 can inhibit protein misfolding, thereby suppressing the pathology of these diseases. This invention is applicable to all neuronal, organ-specific, and systemic diseases caused by protein aggregation, such as neurodegenerative diseases, focal and systemic amyloidosis, type II diabetes, cataracts, etc.

[0110] Example 3: Using the present invention to protect the mitochondrial membrane

[0111] Figure 11 A shows a second cytochrome C peak 48 hours after mouse myocardial irradiation with 8.68 Gy. Unlike the first peak 12 hours later, this peak was not due to DNA damage or activation of intrinsic apoptosis pathways, as there was no increase in either p53 or 8-oxo-dG (data not shown) or activation of intrinsic apoptosis pathways at 48 hours post-irradiation. Figure 11 B). This peak indicates severe disruption of respiratory chain complex IV located in the inner mitochondrial membrane. PrC-210 administered 24 hours after the first cytochrome C release was able to penetrate mitochondria and inhibit the release of cytochrome C into the background. Figure 11 A). This discovery teaches us that PrC-210 enters the mitochondria and protects the proteins and lipids of the mitochondrial membrane from damage that leads to the release of cytochrome C. This invention is applicable to all diseases caused by mitochondrial damage, such as aging and all age-related diseases, neurodegenerative diseases, cardiovascular diseases (such as arteriosclerosis, damage to heart valves or major arteries), and diseases generally caused by damage to the mitochondrial membrane, mitochondrial DNA, and proteins.

[0112] Example 4: Using this invention to protect the brain from direct damage by neuroinflammation and to directly inhibit inflammation caused by chronic inflammation and autoimmune diseases.

[0113] To observe the effects of radiation on the immune response of the mouse brain, ICR mice were subjected to 8.68 Gy of whole-body radiation at time "0". Figure 10 0.3 MTD, intraperitoneal. PrC-210 is administered 24 hours after irradiation. Figure 12As shown in Figure A, the inflammasome marker caspase-1 began to be significantly inhibited eight days after irradiation. The same applies to the inflammasome marker complement 3 (C3). Figure 12 B). Treatment with PrC-210 significantly suppressed two inflammatory markers. Most importantly, this suppression was a direct inhibition of the immune response, rather than an indirect inhibition through protecting proteins and lipids from damage. Radiation-induced protein damage triggers apoptosis, such as… Figure 12 The second peak of caspase 3 / 7 in C is shown. Therefore, this experiment teaches that PrC-210 can enter the brain and directly inhibit post-radiation inflammation.

[0114] To demonstrate that PrC-210 can inhibit neuroinflammation induced by disease-induced autoimmunity, C57 mice were immunized with MOG peptides 24 hours prior to immunization. Figure 13 A) and PrC-210 was administered immediately after the first paralysis symptom appeared due to autoimmunity against myelin. PrC-210 significantly inhibited the development and severity of paralysis symptoms in both administration models.

[0115] To further demonstrate that this suppression of chronic inflammation generally applies to organs other than the brain, DBA / 1J mice immunized against collagen III were treated with systemic and local PrC-210. Twice-weekly intraperitoneal injections of 0.075 mTp PrC-210 and local application of 370 mMp PrC-210 significantly reduced the typical paw swelling induced in this mouse model.

[0116] This invention is applicable to the treatment and prevention of all diseases caused by neuroinflammation, such as i) neurodegenerative diseases and multiple sclerosis, and the use of Pr-210 for the treatment of neuroinflammation after brain injury caused by trauma, irradiation, neurotoxins, ischemia and ischemia-reperfusion injury and microbial diseases, and ii) all diseases caused by inflammation, such as autoimmune diseases, including but not limited to type I diabetes, rheumatoid arthritis, colitis, psoriasis, lupus erythematosus; pancreatitis, hepatitis, and inflammation caused by lipid deposition (such as arteriosclerosis, damage to heart valves or major arteries, vascular damage during dialysis and metabolic syndrome).

[0117] Other implementation plans

[0118] Various modifications and variations to the compositions, methods, and uses of the invention described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the method has been described in conjunction with specific embodiments, it should be understood that the claimed method should not be unduly limited to such specific embodiments. In fact, the various modifications to the manner of carrying out the invention that are obvious to those skilled in the art are intended to be included within the scope of the invention.

[0119] Other embodiments are within the scope of the claims.

Claims

1. A method for inhibiting the aggregation of proteins in a subject in need, the method comprising administering to the subject an effective amount of a compound of formula (I), Or a pharmaceutically acceptable acid addition salt thereof, in an amount sufficient to inhibit the aggregation of the protein or peptide, and wherein (i) A is -CH2NHR' and B is -CH2NHR, or A = -NRR' and B = H; and (ii) Each of R and R' is independently selected from H, C1-C6 alkyl, and C1-C6 heteroalkyl. The prerequisite is that if B = H, then R and R' cannot both be H.

2. The method of claim 1, wherein the compound is Or its pharmaceutically acceptable salt.

3. The method of claim 1 or claim 2, wherein the protein is selected from TDP-43, α-synuclein, τ, amyloid β, PolyQhtt, pancreatic amyloid polypeptide, and prions.

4. The method of claim 1 or claim 2, wherein the subject suffers from a neurodegenerative disease characterized by misfolding and / or aggregation of endogenous proteins.

5. The method of claim 4, wherein the neurodegenerative disease is Parkinson's disease, prions, Alzheimer's disease, multiple system atrophy, diffuse Lewy body disease, frontotemporal dementia, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia and other Poly-Q diseases or hereditary cerebral amyloid angiopathy.

6. The method of claim 5, wherein the neurodegenerative disease is selected from the following prion diseases: Kreutzfeldt-Jacob disease, variant Kreutzfeldt-Jacob disease, hereditary human prion disease, bovine spongiform encephalopathy (BSE), and scrapie.

7. The method of claim 4, wherein the neurodegenerative disease is synucleinosis.

8. The method of claim 7, wherein the synucleinosis is selected from Parkinson's disease (PD), Lewy body dementia (DLB), and multiple system atrophy (MSA).

9. The method of claim 1 or claim 2, wherein the subject suffers from a disease or condition characterized by amyloidosis.

10. The method of claim 9, wherein the amyloidosis is selected from primary systemic amyloidosis (AL amyloidosis), reactive systemic amyloidosis (AA amyloidosis), type II diabetes, injection-induced localized amyloidosis, β-2 microglobulin amyloidosis, hereditary non-neuropathic amyloidosis, and Finnish hereditary systemic amyloidosis.

11. The method of claim 1 or claim 2, wherein the subject suffers from a disease or condition characterized by protein aggregation in the eye.

12. The method of claim 11, wherein the disease or condition is selected from cataracts and presbyopia.

13. A method for treating a subject in need of a condition related to mitochondrial dysfunction, the method comprising administering to the subject an effective amount of a compound of formula (I), Or a pharmaceutically acceptable acid addition salt thereof, in an amount sufficient to inhibit the aggregation of the protein or peptide, and wherein (i) A is -CH2NHR' and B is -CH2NHR, or A = -NRR' and B = H; and (ii) Each of R and R' is independently selected from H, C1-C6 alkyl and C1-C6 heteroalkyl, provided that if B = H, then R and R' cannot both be H.

14. The method of claim 13, wherein the compound is Or its pharmaceutically acceptable salt.

15. The method of claim 13, wherein the condition is a neurodegenerative disorder, a neuropsychiatric disorder, diabetes, a metabolic disease, an eye disorder associated with mitochondrial dysfunction, an ischemia-related condition, aging, mitochondrial toxicity associated with therapeutic agents, or a migraine.

16. The method of claim 15, wherein the condition is selected from the following neurodegenerative disorders: Friedrich's ataxia; amyotrophic lateral sclerosis; mitochondrial myopathy, encephalopathy, lactic acidosis, stroke (MELAS); myoclonic epilepsy with broken red fibers (MERFF); epilepsy; Parkinson's disease; Alzheimer's disease and Huntington's disease.

17. The method of claim 15, wherein the condition is selected from the following neuropsychiatric disorders: bipolar disorder, schizophrenia, depression, addiction disorder, anxiety disorder, attention deficit disorder, personality disorder, autism, and Asperger's syndrome.

18. The method of claim 15, wherein the condition is selected from the following ocular disorders associated with mitochondrial dysfunction: glaucoma, diabetic retinopathy, and age-related macular degeneration.

19. The method of claim 15, wherein the condition is an ischemia-related condition caused by: vascular occlusion, arteriosclerosis, valvular heart disease, tachycardia, hypertension, and hypotension.

20. A method for treating a subject with chronic inflammatory conditions, the method comprising administering to the subject an effective amount of a compound of formula (I), Or a pharmaceutically acceptable acid addition salt thereof, in an amount sufficient to inhibit the aggregation of the protein or peptide, and wherein (i) A is -CH2NHR' and B is -CH2NHR, or A = -NRR' and B = H; and (ii) Each of R and R' is independently selected from H, C1-C6 alkyl, and C1-C6 heteroalkyl. The prerequisite is that if B = H, then R and R' cannot both be H.

21. The method of claim 20, wherein the compound is Or its pharmaceutically acceptable salt.

22. The method of claim 20, wherein the chronic inflammatory condition is selected from sinusitis, arthritis, dermatitis, vasculitis, acute malaria, sickle cell disease, gastrointestinal inflammatory disease, or inflammatory lung disease.

23. The method of claim 22, wherein the chronic inflammatory condition is selected from the following inflammatory lung conditions: asthma, chronic obstructive pulmonary disease (COPD), lung conditions caused by physical trauma, emphysema, bronchitis, sarcoidosis, histiocytosis, lymphangioleiomyomatosis, acute lung injury, chronic lung disease, bronchopulmonary dysplasia, pneumonia, airway deterioration, and acute respiratory distress syndrome (ARDS).

24. The method of claim 22, wherein the chronic inflammatory condition is selected from the following gastrointestinal inflammatory conditions: inflammatory bowel disease (IBD), ulcerative colitis (UC), Crohn's disease (CD), colitis, and chronic hepatitis, such as viral hepatitis, alcohol-related hepatitis, and non-alcoholic steatohepatitis.

25. The method of claim 20, wherein the chronic inflammatory condition is an autoimmune disease.

26. The method of claim 25, wherein the autoimmune disease is selected from multiple sclerosis, type 1 diabetes mellitus, rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, pancreatitis, psoriasis, plaque psoriasis, guttate psoriasis, inverted psoriasis, pustular psoriasis, erythrodermic psoriasis, paraneoplastic autoimmune diseases, autoimmune hepatitis, bullous pemphigoid, myasthenia gravis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, celiac disease, thyroiditis, Sjögren's syndrome, Guillain-Barré syndrome, Raynaud's phenomenon, Addison's disease, primary biliary cirrhosis, primary sclerosing cholangitis, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis, and diabetes mellitus.

27. The method of claim 20, wherein the chronic inflammatory condition is a neuroinflammatory disease.

28. The method of claim 27, wherein the neuroinflammatory disease is selected from multiple sclerosis, type 1 diabetes mellitus, neuromyelitis optica, anti-myelin oligodendrocyte glycoprotein antibody disease, autoimmune encephalitis, transverse myelitis, optic neuritis, and neurosarcoidosis, or from the following neurodegenerative disorders: Friedrich's ataxia; amyotrophic lateral sclerosis; mitochondrial myopathy, encephalopathy, lactic acidosis, stroke (MELAS); myoclonic epilepsy with broken red fibers (MERFF); epilepsy; Parkinson's disease; Alzheimer's disease, and Huntington's disease.

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