Oleanolic acid and plant extracts for treatment of diseases associated with dysregulated disorders of glucose-6-phosphate dehydrogenase, including Bag3path

Abstract

The present invention relates to a method of treating a glucose-6-phosphate dehydrogenase dysregulated disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of oleanolic acid, a conjugated salt thereof, or a prodrug thereof. The glucose 6-phosphate dehydrogenase dysregulated disorder can be myofibrillar myopathy, amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease and Alzheimer's disease caused by BCL2 associated immortal gene mutation.

Description

[0001] Reference to the Sequence Listing

[0002] The entire content of the sequence listing identified as Sequence_Listing_P24623PCT00.xml (size: 9048 bytes; creation date: September 12, 2023, submitted herewith with this application) is hereby incorporated by reference into this text. Technical Field

[0003] The present invention relates to oleanolic acid and plants containing oleanolic acid (such as Chaenomelis fructus) and their extracts for the treatment of glucose-6-phosphate dehydrogenase (G6PD) dysregulation disorders (such as myofibrillar myopathy (Bag3opathy), amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease, and Alzheimer's disease caused by mutations in the BCL2 associated athanogene 3 (BAG3)). Background Art

[0004] Myofibrillar myopathy (MFM) is a group of chronic neuromuscular diseases characterized by Z-disk disassembly, accumulation of myofibrillar degradation products, and mitochondrial abnormalities. MFM causes progressive muscle weakness in patients and is usually accompanied by cardiomyopathy and neuropathy. Due to its unique morphology, MFM has genetic heterogeneity and has been confirmed to be related to mutations in multiple genes, including desmin gene (DES), αB-crystallin gene (CRYAB), myotilin (MYOT), Z-band alternatively spliced PDZ motif protein (ZASP), filamin C (FLNC), BCL2 associated athanogene 3 (BAG3), plectin (PLEC), four and a half LIM domain 1 protein (FHL1), DnaJ heat shock protein family (Hsp40) member B6 (DNAJB6), and titin (TTN). In particular, Bag3opathy usually occurs in childhood and is accompanied by severe muscle weakness, cardiomyopathy, and respiratory failure.

[0005] BAG3 is a member of the BAG family of co-chaperone proteins and is highly expressed in skeletal and cardiac muscles. It interacts with heat shock protein 70 (Hsp70) through the BAG domain and with other binding partners through the WW domain. BAG3 is involved in many biological processes, including apoptosis, cytoskeleton organization, autophagy, and development, as well as the adaptive response to stress stimuli. Initially, knockout of BAG3 in mice was shown to result in fulminant myopathy and early lethality. In 2009, Selcen et al. first discovered Bag3opathy in three MFM patients. To date, fewer than twenty cases of Bag3opathy have been reported worldwide, and only one case is from a Chinese family.

[0006] Most cases of Bag3opathy are caused by the c.626C>T, p.Pro209Leu (P209L) mutation, which may disrupt the critical interaction between BAG3 and small heat shock proteins, including HspB8, HspB6, and CRYAB. Two mechanisms have been proposed for the pathogenesis of Bag3opathy: (1) the chaperone-assisted selective autophagy (CASA) pathway, in which BAG3 acts together with HspB8 and Hsc70 to promote the degradation of damaged components during muscle contraction; and (2) the maintenance of myofibril integrity under mechanical stress through the interaction of BAG3 and Hsc70 with the actin capping protein CapZ. Despite these findings, the pathogenesis of Bag3opathy remains poorly understood, and there is no treatment for the cause.

[0007] Therefore, more effective methods are needed to treat G6PD dysregulation disorders, such as Bag3opathy, amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease, and Alzheimer's disease. Summary of the Invention

[0008] The present invention relates to oleanolic acid and extracts of any plant containing oleanolic acid, such as papaya, for the treatment of G6PD dysregulation disorders, such as Bag3opathy, amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease, and Alzheimer's disease.

[0009] In a first aspect, the present invention provides a method for treating glucose-6-phosphate dehydrogenase (G6PD) dysregulation disorders, the method comprising administering to the subject a therapeutically effective amount of oleanolic acid, its conjugate salts, or its prodrugs.

[0010] In certain embodiments, the G6PD dysregulation disorders are the group consisting of BCL2-associated athanogene myofibrillar myopathy (Bag3opathy), amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease, and Alzheimer's disease.

[0011] In certain embodiments, the G6PD dysregulation disorder is BCL2-associated athanogene myofibrillar myopathy (Bag3opathy).

[0012] In certain embodiments, the subject has one or more of severe muscle weakness, cardiomyopathy, and respiratory failure.

[0013] In certain embodiments, a plant product or an extract containing oleanolic acid or its conjugate salts is administered to the subject.

[0014] In certain embodiments, the plant product comprises one or more of Crataegi fructus, Forsythiae fructus, Prunellae spica, Verbenae herba, Eriobotryae folium, Ligustri lucidi fructus, Kaki calyx, Chaenomelis fructus, Jujubae fructus, Corni fructus, or an extract thereof.

[0015] In certain embodiments, the plant product comprises papaya or an extract thereof.

[0016] In certain embodiments, the papaya extract is prepared by extracting at least a portion of oleanolic acid or a conjugate salt thereof present in papaya by mixing papaya with ethanol, and forming an ethanol extract comprising oleanolic acid or a conjugate salt thereof, and the ethanol may also be removed from the ethanol extract to form the papaya extract.

[0017] In certain embodiments, the subject has a BCL2 associated athanogene 3 (BAG3) gene comprising a c.626C>T mutation.

[0018] In certain embodiments, a prodrug of oleanolic acid has Chemical Formula 2:

[0019]

[0020] or a pharmaceutically acceptable salt thereof, wherein

[0021] R 1 is hydrogen, R(C=O)- or RO(C=O)-;

[0022] R 2 is hydrogen, alkyl, cycloalkyl, aralkyl or aryl; and

[0023] R is independently hydrogen, alkyl, aralkyl, aryl or heterocycloalkyl in each case, wherein R 1 and R 2 at least one of which is not hydrogen.

[0024] In certain embodiments, the subject is human.

[0025] In a second aspect, the present disclosure provides a method of treating BCL2 associated athanogene myofibrillar myopathy (Bag3opathy), the method comprising administering to the subject a therapeutically effective amount of oleanolic acid, a conjugate salt thereof, or a prodrug thereof.

[0026] In certain embodiments, oleanolic acid or a conjugate salt thereof is administered to a subject in the form of a plant product or an extract thereof.

[0027] In certain embodiments, the plant product comprises one or more of hawthorn, forsythia, selfheal, verbena, loquat leaf, glossy privet fruit, calyx kaki, papaya, Chinese date, dogwood or an extract thereof.

[0028] In certain embodiments, the plant product comprises papaya or an extract thereof.

[0029] In certain embodiments, the papaya extract is prepared by extracting at least a portion of the oleanolic acid or conjugate salt thereof present in papaya by mixing papaya with ethanol and forming an ethanol extract comprising oleanolic acid or a conjugate salt thereof, and the ethanol may be removed from the ethanol extract to form the papaya extract.

[0030] In certain embodiments, the subject has a BCL2 associated athanogene 3 (BAG3) gene comprising a c.626C> mutation.

[0031] In certain embodiments, the prodrug of oleanolic acid has Chemical Formula 2:

[0032]

[0033]

[0034] or a pharmaceutically acceptable salt thereof, wherein

[0035] R 1 is hydrogen, R(C=O)- or RO(C=O)-;

[0036] R 2 is hydrogen, alkyl, cycloalkyl, aralkyl or aryl; and

[0037] R is independently hydrogen, alkyl, aralkyl, aryl or heterocycloalkyl in each case, wherein at least one of R 1 and R 2 is not hydrogen.

[0038] In certain embodiments, the subject is human.

[0039] Other aspects and advantages of the invention will be apparent to those skilled in the art upon review of the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The above and other objects and features of the present invention will become apparent from the following description when taken in conjunction with the accompanying drawings, in which:

[0041] Figure 1Demonstrates the serum metabolomic analysis of Bag3opathy patients. The figure shows the metabolites with significant changes between Bag3opathy patients and healthy controls.

[0042] Figure 2 Demonstrates the metabolic pathways overrepresented by the significantly altered metabolites in the serum metabolomic analysis of Bag3opathy patients.

[0043] Figure 3 Demonstrates the levels of glucose-6-phosphate (G6P) and major converting enzymes, G6PD; GPI, glucose-6-phosphate isomerase (GPI), and phosphoglucomutase (PGM) in Bag3opathy patients and healthy controls measured by ELISA kits.

[0044] Figure 4 Demonstrates the identification of the mutation sites of the mouse Bag3 protein, which are equivalent to the mutation P209L (residues 199 to 219 of SEQ ID NO: 1; residues 205 to 225 of SEQ ID NO: 2; SEQ ID NO: 3; and SEQ ID NO: 4) that causes human Bag3opathy.

[0045] Figure 5 Demonstrates the genome editing performed at the mouse Bag3 locus by CRISPR / Cas9. The figure shows a schematic diagram of the mouse Bag3 locus. Exons are labeled in black, while introns are labeled in gray. The editing site is in the third exon. The cleavage sites of the guide RNA (gRNA) are indicated by thin arrows, while the mutated nucleotides are highlighted by thick arrows (SEQ ID NO: 5 and SEQ ID NO: 6).

[0046] Figure 6 Demonstrates the G6PD levels in the serum samples of P209-KI mice (Control: n = 4; P209L / +: n = 4; P209L / P209L: n = 3). *p < 0.05.

[0047] Figure 7 Demonstrates the G6PD levels in the myocardial samples of P209-KI mice (Control: n = 4; P209L / +: n = 4; P209L / P209L: n = 3).

[0048] Figure 8 Demonstrates the G6PD levels in the skeletal muscle samples of P209-KI mice (Control: n = 4; P209L / +: n = 4; P209L / P209L: n = 3). **p < 0.01.

[0049] Figure 9Shows the changes in G6PD protein in the serum of normal mice treated with oleanolic acid (50 mg / kg and 200 mg / kg) orally for 10 days. *p<0.05.

[0050] Figure 10 Shows the changes in G6PD protein in the myocardium of normal mice treated with oleanolic acid (50 mg / kg and 200 mg / kg) orally for 10 days. *p<0.05.

[0051] Figure 11 Shows the changes in G6PD protein in the skeletal muscle of normal mice treated with oleanolic acid (50 mg / kg and 200 mg / kg) orally for 10 days. *p<0.05.

[0052] Figure 12 Shows the changes in G6PD protein in the body of patients with Bag3opathy after three months of taking oleanolic acid capsules (180 mg / day) orally.

[0053] Figure 13 Shows the changes in CK-MB in the body of patients with Bag3opathy after three months of taking oleanolic acid capsules (180 mg / day) orally.

[0054] Figure 14 Shows the HPLC chromatograms before (A) and after (B) the purification of oleanolic acid from papaya extract using AB-8 resin column chromatography.

[0055] Figure 15 Shows the effect of papaya extract on muscle fatigue caused by tonic fatigue contractions. The forced fatigue tolerance of the plantaris muscles of BAG3P209L / + mice treated with saline (control group, A), 150 mg / kg / day of papaya extract (B), and 300 mg / kg / day of papaya extract (C) was evaluated by 80 times of low-frequency continuous stimulation. (n = 5 in each group), *p<0.05, **p<0.01.

[0056] Figure 16 Shows the changes in G6PD levels in the serum of BAG3P209L / + mice treated with papaya extract (150 mg / kg and 300 mg / kg) orally for 12 days. (n = 5 in each group), *p<0.05. Detailed implementation methods

[0057] Definition

[0058] Throughout the specification of this application, unless the context requires otherwise, the word "comprising" or variants thereof shall be understood to implicitly include the recited integer or group of integers, but not to exclude any other integer or group of integers. It should also be noted that in the present invention, particularly in the claims and / or each paragraph, terms such as "comprising" may have the meaning given to them by the United States Patent Law; for example, they may mean "including"; and terms such as "consisting essentially of" have the meaning given to them by the United States Patent Law, for example, they allow elements not expressly recited, but exclude elements found in the prior art or that would affect the basic or novel characteristics of the present invention.

[0059] Furthermore, throughout the specification and claims of this application, unless the context requires otherwise, variants of the words "include" and "including" shall be understood to implicitly include the recited integer or group of integers, but not to exclude any other integer or group of integers.

[0060] The term "alkyl" is well recognized in the art and includes saturated aliphatic groups, including straight-chain alkyls, branched-chain alkyls, cycloalkyls (alicyclic groups), alkyl-substituted cycloalkyls, and cycloalkyl-substituted alkyls. In certain embodiments, the straight-chain or branched-chain alkyl has about 30 or fewer carbon atoms in its main chain (e.g., C1-C in the straight chain 30 and C3-C in the branched chain 30 ), and optionally has about 20 or fewer carbon atoms.

[0061] Likewise, the cycloalkyl has about 3 to about 10 carbon atoms in its ring structure, and optionally has about 5, 6, or 7 carbon atoms in the ring structure.

[0062] The term "aralkyl" is well recognized in the art and refers to an aryl-substituted alkyl (e.g., aryl or heteroaryl).

[0063] The term "aryl" is well recognized in the art and refers to 5-membered monocyclic aryl, 6-membered monocyclic aryl, and 7-membered monocyclic aryl that may include from 0 to 4 heteroatoms. For example, benzene, naphthalene, anthracene, pyrene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, etc. Aryl groups having heteroatoms in the ring structure may also be referred to as "heterocyclic rings" or "heteroaromatic compounds". The aromatic ring may be substituted at one or more ring positions with the above-mentioned substituents (e.g., halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, alkoxy, amino, nitro, mercapto, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclic group, multiple aromatic or heteroaromatic moieties, -CF3, -CN, etc.). The term "aryl" also includes polycyclic ring systems having two or more rings, wherein two or more carbons are common to two adjacent rings (these rings are "fused rings"), and wherein at least one ring is an aromatic ring. For example, the other rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, and / or heterocyclic group.

[0064] The terms "heterocycloalkyl", "heteroaryl", or "heterocyclic group" are well recognized in the art and refer to 3- to about 10-membered ring structures, optionally 3- to about 7-membered rings, the ring structures of which include from 1 to 4 heteroatoms. The heterocycle may also be polycyclic. For example, heterocyclic groups include thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, benzoxanthene, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indene, isoindole, indole, indazole, purine, quinoline, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenopyrazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactone, lactam (such as azetidinone and pyrrolidone), sultam, sultone, etc. The heterocycle may be substituted at one or more positions with the above-mentioned substituents (e.g., halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, amino, nitro, mercapto, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclic group, aromatic or heteroaromatic moiety, -CF3, -CN, etc.).

[0065] The term "optionally substituted" refers to chemical groups (such as alkyl, cycloalkyl, aryl, etc.) in which one or more hydrogen atoms can be replaced by substituents described herein (e.g., halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, alkoxy, amino, nitro, mercapto, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclic group, multiple aromatic or heteroaromatic moieties, -CF3, -CN, etc.).

[0066] It should be noted that "substituted" or "substituted by" includes the following implicit restrictive conditions: such substitution conforms to the allowable valences of the atom being substituted and the substituent, and such substitution results in a stable compound, e.g., the compound does not undergo spontaneous transformation such as rearrangement, cyclization, elimination, or other reactions.

[0067] The term "substituted" is also considered to include all allowable substituents of organic compounds. Broadly speaking, allowable substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. For example, exemplary substituents include those described above. For suitable organic compounds, the allowable substituents can be one or more and can be the same or different. For the purposes of the present invention, a heteroatom (such as nitrogen) can have a hydrogen substituent and / or any allowable substituent of the organic compound that satisfies the valence of the heteroatom. The present invention is not intended to be limited in any way by the allowable substituents of organic compounds.

[0068] As used herein, the term "pharmaceutically acceptable salt" refers to those salts that are within the scope of sound medical judgment, suitable for contact with the tissues of a subject without undue toxicity, irritation, allergic response, etc., and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. described pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds provided herein include salts derived from suitable inorganic acids and bases and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are amino salts formed with inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid) or organic acids (such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid), or by using other methods employed in the art (such as ion exchange). Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartates, benzenesulfonates, besylates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecyl sulfates, ethanesulfonates, formates, fumarates, glucoheptanoates, glycerophosphates, gluconates, hemisulfates, heptanoates, hexanoates, hydroiodides, 2-hydroxyethanesulfonates, lactobionates, lactates, laurates, lauryl sulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, valerates, etc. In certain embodiments, for example, the organic acids from which the salts can be derived include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.

[0069] Pharmaceutically acceptable salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N + (C 1-4(alkyl)4 salts. Representative alkali metal salts or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, etc. Where appropriate, other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium salts, and amine cations formed using counterions (such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates). For example, organic bases from which salts can be derived include primary amines, secondary amines, tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, basic ion exchange resins, etc., such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In certain embodiments, the pharmaceutically acceptable base addition salts are selected from ammonium salts, potassium salts, sodium salts, calcium salts, and magnesium salts.

[0070] The term "composition" is intended to encompass a product containing a specified amount of specified ingredients and any product directly or indirectly resulting from the combination of the specified amounts of the specified ingredients.

[0071] As used herein, the term "isolated" in connection with the compound means that the compound is not present in a plant product and is separated from some or all of the components that are normally associated with it in the plant product.

[0072] As used herein, the term "substantially pure" in connection with a sample of the compound means that the sample contains at least 60 wt% of the compound. In certain embodiments, the sample contains at least 70 wt% of the compound; at least 75 wt% of the compound; at least 80 wt% of the compound; at least 85 wt% of the compound; at least 90 wt% of the compound; at least 95 wt% of the compound; or at least 98 wt% of the compound.

[0073] The term "treatment" as used herein refers to alleviating or improving a disorder / disease and / or its associated symptoms. It should be recognized that while not excluded, treating a disorder or condition does not require complete elimination of the disorder, condition, or its associated symptoms. In certain embodiments, treatment includes preventing a disorder or condition and / or its associated symptoms. The term "prevention" as used herein refers to any action that inhibits or at least delays the development of a disorder, condition, or its associated symptoms. Prevention can include primary prevention, secondary prevention, and tertiary prevention levels, where: a) primary prevention avoids the development of the disease; b) secondary prevention activities aim at early disease treatment, thereby increasing the opportunity for intervention to prevent disease progression and symptom appearance; and c) tertiary prevention reduces the negative impact of a diagnosed disease by restoring function and reducing disease-related complications.

[0074] The term "subject" as used herein refers to any animal (e.g., a mammal), including but not limited to humans, non-human primates, canines, felines, and rodents.

[0075] As used herein, the term "therapeutically effective amount" refers to the amount of an active compound or pharmaceutical preparation that elicits a biological or medical response (including alleviating the symptoms of a disease, disorder, or condition being treated) in a cell culture, tissue system, animal, or human that a researcher, veterinarian, clinician, or physician is seeking.

[0076] Other definitions of selected terms used herein can be found in the detailed description of the present disclosure and apply throughout the text. Unless otherwise defined, all other technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0077] Provided herein is a method for treating a subject in need of treatment for a G6PD dysregulation disorder, the method comprising administering to the subject a therapeutically effective amount of oleanolic acid, its conjugate salts, or its prodrugs.

[0078] Oleanolic acid can be represented by the compound of Formula 1:

[0079]

[0080] A prodrug refers to a derivative of oleanolic acid that is inactive or has significantly reduced biological activity and is metabolized in vivo to oleanolic acid and / or one or more active metabolites upon administration. Prodrugs can be formed by methods well known in the art and can thus be in substantially any form recognized by one of ordinary skill in the art. For example, prodrugs include oleanolic acid in which a hydroxyl or carboxyl group is bound to any group that cleaves to form a free hydroxyl or free carboxylic acid, respectively, when the prodrug is administered to a subject. Examples include, but are not limited to, alkyl acyl ester derivatives and aryl acyl ester derivatives of alcohols (such as acetate derivatives, formate derivatives, and benzoate derivatives), alkyl carbonate derivatives and aryl carbonate derivatives of alcohols, alkyl esters, carbocyclic esters, aryl esters, and alkylaryl esters (such as methyl ester, ethyl ester, propyl ester, isopropyl ester, butyl ester, isobutyl ester, sec-butyl ester, tert-butyl ester, cyclopropyl ester, phenyl ester, benzyl ester, and phenethyl ester, etc.).

[0081] In certain embodiments, the prodrug of oleanolic acid is represented by the compound of Formula 2:

[0082]

[0083] or a pharmaceutically acceptable salt thereof, wherein

[0084] R 1 is hydrogen, R(C═O)-, or RO(C═O)-;

[0085] R 2 is hydrogen, alkyl, cycloalkyl, aralkyl, or aryl; and

[0086] R is independently in each case hydrogen, alkyl, aralkyl, aryl or heterocycloalkyl, where R 1 and R 2 in at least one case is not hydrogen.

[0087] In certain embodiments, R is independently in each case hydrogen, C1-C4 alkyl, methyl, ethyl, isopropyl, n-propyl, tert-butyl, benzyl or phenyl.

[0088] Oleanolic acid or its conjugate salt can be administered in isolated and / or substantially pure form or in the form of a plant product containing oleanolic acid or its conjugate salt, an extract of a plant product containing oleanolic acid or conjugate salt, or a pharmaceutical composition containing any one or more of the foregoing substances.

[0089] The plant product can be any plant product containing oleanolic acid.

[0090] The plant product can include any component of a plant, higher plant, tree, alga, fungus or combination thereof that contains or is suspected of containing one or more polysaccharides. The plant product can include, but is not limited to, stems, leaves, bark, fruits, peels, vegetables, flowers, seeds, roots, rhizomes, beans or any organic component of a plant or tree.

[0091] In certain embodiments, the plant is one or more of hawthorn, forsythia, selfheal, verbena, loquat leaf, glossy privet fruit, calyx kaki, papaya, Chinese date, macrocarpium officinale or a mixture thereof. In certain embodiments, the plant is papaya.

[0092] Methods known in the art can be used to prepare an extract of the plant product. In certain embodiments, an extract of the plant product is prepared by contacting the plant product with a solvent so as to extract at least a portion of oleanolic acid or its conjugate salt and form an extraction solvent containing oleanolic acid or its conjugate salt; separating the plant product and the extraction solvent; and optionally removing the solvent from the extraction solvent so as to form an extract of the plant product.

[0093] The plant product can be directly extracted or optionally pretreated to remove impurities by, for example, mechanical pretreatment (such as covering, chopping, mincing, grinding, milling and / or sieving) and / or chemical pretreatment (such as acid or base extraction).

[0094] The solvent can include any solvent or solvent mixture in which oleanolic acid or its conjugate salt is at least partially soluble. In certain embodiments, the solvent is an alcohol, ester, ether, ketone, aromatic solvent, alkane, formamide, sulfoxide, alkyl halide, alkyl nitrile, nitroalkane, or a mixture thereof. In certain embodiments, the solvent is methanol, ethanol, isopropanol, n-propanol, acetone, ethyl acetate, diethyl ether, methyl tert-butyl ether, tetrahydrofuran (THF), 2-methyltetrahydrofuran, tetrahydropyran (THP), dioxane, 1,2-dimethoxyethane (DME), benzene, toluene, xylene, chlorobenzene, dichloromethane, dichloroethane, chloroform, acetonitrile, nitromethane, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), N-methylpyrrolidone (NMP), or a mixture thereof.

[0095] The solvent can include an alcohol solvent containing one or more C1-C6 fatty alcohols and an aqueous solvent containing at least one salt. In certain embodiments, the alcohol solvent contains one or more C1-C5, C1-C4, C1-C3, or C2-C3 fatty alcohols. Exemplary C1-C6 fatty alcohols include, but are not limited to, methanol, ethanol, 1-propanol, isopropanol, n-butanol, isobutanol, sec-butanol, tert-butanol, 1-pentanol, isopentanol, 2-methyl-1-butanol, neopentanol, 2-pentanol, 3-methyl-2-butanol, 3-pentanol, tert-pentanol, 1-hexanol, 2-hexanol, 3-hexanol, 2-methyl-1-pentanol, 3-methyl-1-pentanol, 4-methyl-1-pentanol, 2-methyl-2-pentanol, 3-methyl-2-pentanol, 4-methyl-2-pentanol, 2-methyl-3-pentanol, 3-methyl-3-pentanol, 2,2-dimethyl-1-butanol, 2,3-dimethyl-1-butanol, and 3,3-dimethyl-1-butanol. In certain embodiments, the alcohol solvent includes one or more of methanol, ethanol, 1-propanol, and isopropanol. In certain embodiments, the alcohol solvent is ethanol.

[0096] The step of removing the solvent from the extraction solvent can be accomplished by methods well known in the art (such as evaporation, distillation, e.g., distillation under reduced pressure and / or heating, lyophilization, spray drying, fluidized bed drying, direct oven heating drying, etc.).

[0097] In certain embodiments, the papaya extract is prepared by: extracting at least a portion of the oleanolic acid or its conjugate salt present in papaya by mixing papaya with ethanol, and forming an ethanol extract containing oleanolic acid or its conjugate salt, and optionally removing the ethanol from the ethanol extract to form the papaya extract.

[0098] The present invention also provides a pharmaceutical composition, which comprises oleanolic acid or its conjugate salts, or a herbal extract containing oleanolic acid or its conjugate salts, and at least one pharmaceutically acceptable excipient and / or pharmaceutically acceptable carrier.

[0099] According to standard pharmaceutical practice, oleanolic acid or a herbal extract containing oleanolic acid and its pharmaceutically acceptable salts can be administered to a subject alone or in combination with a pharmaceutically acceptable carrier or diluent in a pharmaceutical composition. Oleanolic acid or a herbal extract containing oleanolic acid can be administered orally or parenterally. Parenteral administration includes intravenous administration, intramuscular administration, peritoneal administration, subcutaneous administration, and topical administration, and the preferred method is intravenous administration.

[0100] Accordingly, the present invention provides pharmaceutically acceptable compositions, which comprise a therapeutically effective amount of oleanolic acid or a herbal extract containing oleanolic acid or its pharmaceutically acceptable salts, formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents. The pharmaceutical compositions of the present invention can be specifically formulated for administration in solid or liquid form, including the following administration methods: (1) parenteral administration, such as subcutaneous injection, intramuscular injection, intravenous injection, or epidural injection in the form of a sterile solution or suspension or a sustained-release preparation; and (2) oral administration, such as a liquid medicine (aqueous or non-aqueous solution or suspension), tablets (e.g., those for oral absorption, sublingual absorption, and systemic absorption), pills, powders, granules, and pastes applied to the tongue).

[0101] Oleanolic acid contains acidic functional groups and can thus form pharmaceutically acceptable salts with pharmaceutically acceptable bases. In these cases, the term "pharmaceutically acceptable salts" refers to relatively non-toxic inorganic base and organic base addition salts of oleanolic acid. Similarly, such salts can be prepared by in-situ synthesis in the production process of the administration carrier or dosage form, or by reacting the purified oleanolic acid in free acid form with a suitable base alone, and the suitable base is, for example, hydroxides, carbonates, or bicarbonates of pharmaceutically acceptable metal cations, ammonia, or pharmaceutically acceptable organic primary amines, secondary amines, or tertiary amines. Representative alkali metal salts or alkaline earth metal salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts, etc. Representative organic amines used to form base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, etc.

[0102] Wetting agents, emulsifiers, and lubricants (such as sodium lauryl sulfate and magnesium stearate), as well as coloring agents, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, solubilizers, buffering agents, and antioxidants can also be present in the composition.

[0103] A method for preparing a medicament comprising oleanolic acid or a herbal extract containing oleanolic acid comprises the following steps: combining oleanolic acid or a herbal extract containing oleanolic acid with a carrier and one or more auxiliary components (optional). Generally, the preparation is carried out by uniformly mixing oleanolic acid or a herbal extract containing oleanolic acid with a liquid carrier for preparing a liquid dosage form; or subsequently subjecting the liquid carrier to a lyophilization process to form a sterile water-reconstitutable powder preparation; or uniformly mixing oleanolic acid or a herbal extract containing oleanolic acid with a finely divided solid carrier for constructing a solid dosage form; or a combined system of the above carriers can be employed; subsequently, according to the requirements of the preparation, necessary shaping and packaging processes are carried out on the product.

[0104] The pharmaceutical composition suitable for parenteral administration of the present invention comprises a combination of oleanolic acid or a herbal extract containing oleanolic acid with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions or sterile powders that can be reconstituted into a sterile injectable solution or dispersion before use, and the one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions or sterile powders may contain sugars (such as sucrose), alcohols, non-ionic surfactants (such as Tween 20), antioxidants, buffers, bacteriostatic agents, chelating agents, solutes that render the preparation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0105] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical composition of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and their suitable mixtures, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Appropriate fluidity can be maintained, for example, by using coating materials (such as lecithin), by maintaining the required particle size in the case of dispersants, and by using surfactants.

[0106] These compositions may also contain adjuvants (such as preservatives, wetting agents, emulsifying agents and dispersing agents). By including various antibacterial and antifungal agents (for example, parabens, chlorobutanol, phenol, sorbic acid, etc.), it is possible to ensure protection against the action of microorganisms on the compounds of the present disclosure. It may also be desirable to include isotonic agents (such as sugars, sodium chloride, etc.) in the composition. In addition, the absorption of injectable drug forms can be prolonged by including agents that delay absorption (such as aluminum monostearate and gelatin).

[0107] The G6PD dysregulation disorder can be selected from the group consisting of Bag3opathy, amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease and Alzheimer's disease. In certain embodiments, the G6PD dysregulation disorder is Bag3opathy.

[0108] Viral infections, cardiovascular diseases (Meng et al., Frontiers in Pharmacology, 2022)

[0109] The subject may have one or more of severe muscle weakness, cardiomyopathy, and respiratory insufficiency. In certain embodiments, the subject has a BAG3 gene containing the c.626C>T mutation.

[0110] The specific route of administration and dosage regimen will be determined by a skilled clinician based on factors such as the specific nature of the disorder being treated, the severity of the disorder, and the age and general physical condition of the patient. In certain embodiments, oleanolic acid or its conjugate salt is administered to the subject orally or intravenously.

[0111] Those skilled in the art can readily determine the optimal dose and dosage regimen to be administered, and the optimal dose and dosage regimen will vary with the mode of administration, formulation strength, and progression of the disease condition. In addition, factors related to the particular patient being treated, including the patient's sex, age, weight, diet, physical activity, time of administration, and concomitant diseases, can result in the need for adjustment of the dose and / or dosage regimen. In certain embodiments, the compounds described herein are administered to the subject once daily, once weekly, twice weekly, three times weekly, once monthly, or twice monthly.

[0112] Although the dose will vary depending on the age, weight, symptoms to be treated, desired therapeutic effect, route of administration, duration of treatment, etc. of the subject, satisfactory results can be obtained by systemic administration in divided doses 1 to 5 times a day or in a sustained-release form at a dose of 0.001 - 1000 mg / kg.

[0113] The present disclosure reports a new role of BAG3 in the metabolic network, which has been rarely reported before. Previous studies in human hepatocellular carcinoma (HCC) cell lines found that BAG3 was shown to directly interact with G6PD and inhibit the activity of G6PD. However, in patients and mouse models carrying the BAG3 P209L mutation, the G6PD protein was reduced, indicating a new role of BAG3 as a positive regulator of G6PD. G6PD is the rate-limiting enzyme of the pentose phosphate pathway (PPP), which is responsible for maintaining the coenzyme nicotinamide adenine dinucleotide phosphate (NADPH). PPP is also important for cellular defense against oxidative stress. Without being bound by theory, these findings suggest that BAG3 can protect cells from oxidative stress by regulating G6PD. The underlying molecular mechanism deserves further investigation.

[0114] As is well known, X-linked genetic defects in G6PD cause non-immune hemolytic anemia and affect over 400 million people globally. Recent studies have shown that G6PD is associated with many neurological and neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), Parkinson's disease (PD), and Alzheimer's disease (AD). Based on the results disclosed herein, dysregulated G6PD is also associated with Bag3opathy. Without wishing to be bound by theory, it is believed that G6PD can play a neuroprotective role in all of these disorders. A decrease in G6PD levels and impaired activity may lead to elevated reactive oxygen species (ROS) and oxidative damage. These findings also open up a path for new targeted G6PD therapies for these disorders.

[0115] Oleanolic acid is known to be an activator of Nrf2. It was first demonstrated that oleanolic acid can increase G6PD by activating Nrf2 in mice and humans. Oleanolic acid can be developed into a therapy for G6PD dysregulated disorders such as Bag3opathy, ALS, HD, PD, and AD.

[0116] To confirm that plant extracts containing oleanolic acid can also increase G6PD and improve muscle dysfunction in Bag3opathy, in vivo studies were conducted in BAG3 P209L / + mice using papaya extract containing 27.18% oleanolic acid. The results showed that the papaya extract could alleviate the decline in contractile force of the posterior tibial muscle and improve contractile force recovery ability, indicating that the papaya extract has the potential to be developed into a new therapy for G6PD dysregulated disorders.

[0117] Experiment

[0118] Bag3opathy patients and healthy controls

[0119] A 16-year-old Chinese girl patient (in 2014) was diagnosed with MFM since the age of 6 and gradually developed bilateral Achilles tendon contracture. She underwent genetic testing and mutation analysis using genomic DNA from the proband at Princess Margaret Hospital on August 9, 2010. In light microscopy and ultrastructural studies, her muscle biopsy results indicated MFM. Thirty healthy female subjects were recruited as controls in accordance with the ethical principles of the Declaration of Helsinki, and these subjects gave informed consent. Before the subjects participated in the study, complete blood picture (CBP), fasting blood glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, creatinine, fasting triglycerides, and total cholesterol were tested. The mean age of the study participants was 15.5 years (SD: 8.80).

[0120] Metabolomics analysis of serum

[0121] Serum collection and preparation

[0122] Serum was collected from patients and healthy volunteers and immediately stored at -80 °C for analysis. The sample preparation procedure obtained the metabolic profiles of liquid biological fluids using standard methods. Briefly, for metabolite extraction, four volumes of methanol / water (1:1 v / v) were added to the samples, vortexed for 30 s, and centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was then transferred to a new tube containing 10 μL of internal standard (IS, 4-chlorophenylalanine solution at 0.1 mg mL -1 for metabolomics analysis. Meanwhile, quality control samples for the analysis platform were prepared from a pooled mixture equally sourced from all samples and pretreated according to the above steps.

[0123] UPLC / QTOF-MS analysis

[0124] Endogenous metabolites were separated by an ultra-high performance liquid chromatography system (UHPLC, Agilent 1290 Infinity, USA) connected to a 1.7 μm BEH Amide column (2.1 mm × 100 mm, Waters, USA). The mobile phase consisted of water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). The gradient program started from 90% B and went to 30% B in 12 min, held at 30% B for 1 min, then returned to the starting conditions in 0.1 min and re-equilibrated until 18 min. The flow rate was 0.25 mL / min and the column temperature was 40 °C. The separated components were then fragmented and these components were analyzed using a mass spectrometer. A quadrupole time-of-flight mass spectrometer (QTOF-MS, Agilent 6543, USA) was coupled with an electrospray ionization source for fragment collection. For full-scan MS analysis, the QTOF-MS conditions were set as follows: the temperature of the desolvation gas was 300 °C; the gas flow rate was 8 L / min. The capillary voltage and cone voltage for ESI+ were set to 3.2 kV and 35 V respectively, and the capillary voltage and cone voltage for ESI- were set to 3 kV and 50 V respectively. The mass range was set to 80 to 1000 m / z. The scan time was set to 0.3 s and the inter-scan delay was set to 0.02 s. The raw data were extracted and preprocessed by the XCMS software package (version 2.13.2) based on the R language for baseline correction, smoothing, noise reduction, deconvolution, peak alignment, and area calculation. The intensity of each metabolic signal was normalized by using the internal standard. The p-values of each fragment were calculated by a t-test, and these p-values were corrected using the Benjamini-Hochberg false discovery rate (FDR) control procedure.

[0125] Metabolite identification

[0126] Fragments with adjusted p-values less than 0.1 were considered metabolic features. The targeted MS / MS acquisition mode was performed with the MS / MS range set to 30 to 800 m / z and the collision energy set to 10 eV, 20 eV, and 40 eV for comparison with chemical standards obtained from Sigma-Aldrich (St. Louis, MO, USA) or the METLIN database. Other parameters in the targeted MS / MS acquisition mode were the same as those set in the full scan mode. Pathway analysis was performed using MetaboAnalyst 3.0.

[0127] Determination of serum metabolites and proteins using ELISA kits

[0128] The concentration of G6P in serum was measured using a high-sensitivity G6P assay kit (Catalog No.: K687-100, BioVision, Inc., USA) according to the manufacturer's instructions. Briefly, before the reaction, 200 μL of the sample was deproteinized using a 10 kDa MWCO spin filter. After adding 50 μL of the appropriate reaction mixture to each 50 μL of the filtrate, the reaction mixture was incubated at 37 °C for 5 minutes in the dark. Finally, the concentration of G6P was calculated based on the fluorescence intensity measured by a fluorometer (EnVision 2104 Multilabel Reader, PerkinElmer, USA) (λ ex = 535 / λ em = 587). The serum levels of G6PD (SEA716Hu, Cloud-Clone Corp), GPI, catalase (CAT), thioredoxin (TXN), and superoxide dismutase 1 (SOD1) were measured using ELISA kits purchased from Cloud-Clone Crop Inc. (USA). Serum samples were diluted 100-fold in PBS for the measurement of CAT and TXN and diluted 800-fold for the SOD1 test. The activity of PGM was measured using a colorimetric assay kit purchased from BioVision (Inc., USA). Samples were prepared and measured at 450 nm using an ELISA reader (Benchmark Plus, BioRad, USA) according to the manufacturer's instructions.

[0129] Establishment of BAG3 P209L gene knock-in mouse model using CRISPR-Cas9

[0130] Using CRISPR / Cas9-mediated genome engineering, a C57BL / 6 mouse model with a point mutation (P215L in the mouse BAG3 gene) was established by Cyagen Biosciences (Guangzhou, China). Through sequence alignment between human (UniProt ID: O95817) and mouse (UniProt ID: Q9JLV1) BAG3 protein sequences, the equivalent mouse BAG3 protein mutation site was identified. Exon 3 of the mouse BAG3 locus was selected as the target site. Two gRNA targeting vectors and donor oligonucleotide sequences were designed. The vectors containing Cas9 mRNA, gRNA, and donor oligonucleotide were co-injected into fertilized eggs for the production of knock-in mice. The mutation site (P215L, CCC mutated to CTC) in the donor oligonucleotide was introduced into exon 3 of the BAG3 locus by homologous directed repair. Subsequently, the mouse pups were genotyped by sequence analysis using PCR.

[0131] Determination of G6PD (GZ) in mouse serum, myocardium and skeletal muscle

[0132] BAG3 P209-KI mice were bred by mating P209L / + male mice with female mice. All mice had free access to food and water in plastic cages at 22 ± 2 °C and were maintained on a 12-hour light / dark cycle. Animal welfare and experimental procedures were carried out in accordance with the relevant ethical regulations of Yunnan Minzu University. Eleven 16-month-old male BAG3 P209L-KI mice (4 WT, 4 P209L / +, and 3 P209L / P209L) were sacrificed, and blood was collected by eye enucleation. The blood was kept at room temperature for 2 - 3 hours and then centrifuged at 1000g for 20 minutes for serum separation. Myocardial and skeletal muscle samples were obtained after cardiac perfusion with cold PBS. Muscle tissues were lysed into homogenates in ice-cold lysis buffer (150 mM NaCl, 50 mM Tris, pH 7.5, 1% NP-40, 0.1% sodium deoxycholate) and 1 mM PMSF. Then the samples were sonicated on ice and centrifuged at 10000g for 5 minutes. The concentration of G6PD in serum, myocardium, and skeletal muscle was measured by a G6PD Elisa kit (Catalog No.: SEA716Mu, Cloud-Clone Corp. USA) according to the manufacturer's instructions. The plate was read at 450 nm by a microplate reader (iMark, Biorad, USA). The concentration of total protein in tissue samples was measured by a spectrophotometer (NanoDrop One, Thermo Scientific, USA).

[0133] Treatment of mice with oleanolic acid

[0134] Seventeen normal male C57BL / 6 mice at 6 - 8 weeks of age were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China). These mice were allowed free access to food and water in plastic cages at 22 ± 2 °C and maintained on a 12-hour light / dark cycle. Animal welfare and experimental procedures were carried out in accordance with the relevant ethical regulations of Yunnan Minzu University. The mice were randomly divided into a control group (n = 6) and two drug treatment groups (n = 5, 6). Oleanolic acid was dispersed in a 0.5% sodium carboxymethylcellulose (CMC-Na) solution to prepare solutions of 10 mg / mL and 2.5 mg / mL. The control group and the drug treatment groups were orally administered 0.5% CMC-Na and oleanolic acid (50 mg / kg / day; 200 mg / kg / day) for 10 days. On the 11th day, the mice were sacrificed, and serum samples and tissue samples were prepared, and the serum samples and tissue samples were detected according to the aforementioned method.

[0135] Treatment of Bag3opathy patients with oleanolic acid

[0136] Patients with Bag3opathy (patient weight: 40 kg) orally took a traditional Chinese medicine supplement containing oleanolic acid (180 mg per day) to evaluate the therapeutic effect on BAG3-related MFM for 3 months. 3 mL of serum was collected for analysis before and after treatment. This trial was approved by the Ethics Committee of Hong Kong Baptist University (HASC / 15-16 / 0268) according to the principles of the Declaration of Helsinki, and informed consent was obtained.

[0137] Preparation of papaya extract and determination of oleanolic acid content

[0138] Extraction and purification of oleanolic acid from papaya

[0139] Dry papaya powder was extracted with an aqueous ethanol solution under reflux conditions. Then, the ethanol extract was centrifuged, and ethanol was removed by concentrating the supernatant under reduced pressure. The viscous residue was thoroughly mixed with AB-8 resin and dried, and then AB-8 resin column chromatography was performed. First, the column loaded with the sample was eluted with 5 bed volumes (BV) of deionized water, 5 BV of 50% ethanol, and 5 BV of 65% ethanol to remove impurities, and then the column loaded with the sample was eluted with 10 BV of 80% ethanol to obtain a fraction rich in oleanolic acid, and this fraction was further concentrated under reduced pressure to produce a product rich in oleanolic acid.

[0140] HPLC analysis of oleanolic acid in papaya

[0141] The papaya extract was analyzed using an Agilent 1290 Infinity II LC system (Agilent, Pal Alto, CA, USA) connected to an Alltima C18 column (250 mm × 4.6 mm, 5 μm). The mobile phase consisted of solvent A (acetic acid:triethylamine:H2O = 0.30:0.15:900) and solvent B (acetic acid:triethylamine:methanol = 0.30:0.15:900). The elution program was isocratic elution for 0 to 40 min with 90% B. The total flow rate was 0.8 mL / min. The column temperature was set at room temperature, and the detection wavelength was set at 210 nm.

[0142] Treatment of mice with papaya extract

[0143] BAG3 P209L / + mice (32 weeks old) were randomly divided into 3 groups of 5 mice each (control group, 150 mg papaya extract / kg / day, 300 mg papaya extract / kg / day). The mice were allowed free access to food and water in plastic cages at 22 ± 2 °C and maintained on a 12-hour light / dark cycle.

[0144] To study the contractility of the skeletal muscle of the hindlimbs of BAG3 P209L / + mice under physiological conditions, the mice were anesthetized temporarily by respiratory anesthesia and their body temperature was maintained using a warming lamp. The hindlimbs to be tested were fixed with a cloth bandage at the knees and feet. The gastrocnemius muscle was stimulated by needle electrodes inserted subcutaneously to cause contraction.

[0145] During a 12-day skeletal muscle exercise training protocol, four sub-groups of multi-cycle low-frequency tetanic contractions were performed, with each sub-group separated by 4 days, and the fatigue trends of the peaks of the four groups of low-frequency tetanic contractions were analyzed comparatively. The protocol used a stimulation frequency of 40 Hz to repetitively contract the skeletal muscle 80 times, with each stimulation lasting 1000 ms and a rest interval of 2000 ms.

[0146] Statistical methods

[0147] The comparison of two groups of biological samples is common in biomedical research. For measurements that follow a normal distribution, the t-statistic is commonly used to test for differences between two groups (Equation 1).

[0148]

[0149] where is the mean of the measurements of the first group, and is the mean of the measurements of the second group. is the sample variance of the first group, and is the sample variance of the second group. And n1 and n2 are the numbers of biological replicates in the first and second groups, respectively. Bag3opathy cases are very rare, and we could only find one Chinese patient. Therefore, the disease group (the first group) has one biological replicate (n1 = 1), while the healthy control group (the second group) has 30 biological replicates (n2 = 30). The key issue is that, without biological replicates, the sample variance of the disease group cannot be directly calculated. Even so, an alternative method to estimate is to "borrow" the sample variance from the healthy control group, making equal to This yields the following statistic.

[0150]

[0151] The T′ statistic follows an exact t - distribution with n2 - 1 degrees of freedom. This method (i.e., the t - type test) is used to compare a single patient with multiple healthy controls.

[0152] Results

[0153] Clinical manifestations of Bag3opathy patients

[0154] The girl has a history of restrictive lung disease (previously diagnosed as asthma). Her condition progressed rapidly, presenting with proximal myopathy, spinal ankylosis, and bilateral tendo - Achilles tightness, requiring surgical elongation. Echocardiogram showed hypertrophic cardiomyopathy with restrictive physiology. In addition, the patient was found to have a prolonged QT interval. The family history was not remarkable, but incidentally, her father was found to have a prolonged QT interval. She was found to have mild proximal muscle weakness in her legs and arms successively, with a Medical Research Council grade of 4 in both. Nerve conduction studies showed reduced motor (tibial motor and peroneal motor) amplitudes (0.3 / 0.7 mV; 0.4 / 0.5 mV) and delays (29 / 33 m / s; 28.7 / 34.3 m / s). She has been using a wheelchair for about one and a half years (using the wheelchair 70 - 80% of the time) and was found to have weakened lower limb strength. Her pulmonary function tests showed a moderate restrictive pattern and she requires BiPAP use every night. She has a good appetite and normal menstrual cycles.

[0155] Previously, mutation analysis showed that the girl was a heterozygous carrier of the BAG3 gene with a primary mutation c.626C>T (p.Pro209Leu, P209L) and a germline variant c.772C>T (p.Arg258Trp). The girl had lower levels of eosinophils and platelets than normal. Using biochemical assays, it was found that creatine kinase (CK) and normal blood sugar were elevated six-fold. Total cholesterol, including HDL cholesterol, LDL cholesterol, and triglycerides, was also lower than normal (Table 1). To further investigate the global perturbations induced by the mutated BAG3 in the girl, a serum metabolomics study was conducted to systematically analyze her serum samples and compare them with 30 healthy, matched girls.

[0156] Table 1. Patient characteristics

[0157]

[0158]

[0159] Serum metabolomics analysis identified abnormally high levels of G6P and low levels of G6PD

[0160] Ultra-high performance liquid chromatography-high resolution mass spectrometry was used to characterize small molecule metabolites in the serum of Bag3opathy patients. Principal component analysis (PCA) was used to completely separate patients and healthy controls in negative mode features or positive mode features. More than 100 metabolites were identified in both modes. A t-test combined with FDR control was used to explore the significance of differences between individual patients and a group of healthy controls. Therefore, a total of 64 metabolites with significant changes (adjusted p-value < 0.3) were selected ( Figure 1 and Table 2). Pathway analysis showed pathways overrepresented in amino acid metabolism and glucose metabolism ( Figure 2 and Table 3).

[0161] Table 2. Metabolites with significant changes in Bag3opathy patients

[0162]

[0163]

[0164]

[0165] Table 3. Metabolic pathways overrepresented by metabolites with significant changes in Bag3opathy patients

[0166]

[0167] The most significant finding was that glucose-6-phosphate (G6P) was significantly elevated in Bag3opathy patients (log2 fc = 4.88, adjusted p-value < 0.01). Consistently, the serum G6P level in Bag3opathy patients measured by ELISA kit was 1.19 μM, significantly higher than (p-value = 5.56E-8) the serum G6P level (0.15 ± 0.13 μM) of the healthy control group( Figure 3 ). Since the abnormally elevated G6P might be caused by the dysregulation of several G6P-related enzymes, the inventors also measured the serum levels / activities of three major G6P-converting enzymes: G6PD, GPI, and PGM. As expected, the serum G6PD level in Bag3opathy patients was substantially decreased (0.07 ng / mL), and was significantly lower (-99.0%, p-value = 9.72E-6) than the normal level (7.25 ± 9.38 ng / mL)( Figure 3 ). Meanwhile, no differences in the serum level of GPI and the activity of PGM were shown between Bag3opathy patients and healthy controls( Figure 3 ). These findings indicate that the level of G6PD enzyme in Bag3opathy patients is abnormally low. However, whether the mutation of BAG3 leads to the decrease of G6PD remains unclear. To test this hypothesis, we set out to establish a Bag3opathy mouse model.

[0168] P209L gene knock-in mice showed muscle weakness and reduced G6PD

[0169] Previous work has reported that knocking out (KO) Bag3 in mice leads to severe muscle dysfunction and early lethality, or exacerbates diabetic nephropathy. Recently, a transgenic (TG) mouse model with cardiomyocyte-specific expression of human BAG3P209L was reported. To some extent, these KO or TG mouse models are similar to the situation of Bag3opathy patients, but not close. Therefore, a knock-in (Kl) mouse model carrying an equivalent disease mutation in the mouse BAG3 gene was established..

[0170] To identify the mutation site, the human BAG3 protein sequence (UniProt ID: O95817) was aligned with the mouse BAG3 protein sequence (UniProt ID: Q9JLV1). A conserved "IPV" motif was found in the mouse BAG3 protein( Figure 4 ), indicating that the conserved biological function can be mediated by the "IPV" motif in both humans and mice. From the sequence comparison, it can be inferred that the pathogenic mutation in mouse BAG3 would be P215L( Figure 4 ), which is encoded by exon 3 in the mouse BAG3 locus( Figure 5, Table 4). The guide RNA (gRNA) and donor oligonucleotide sequences were carefully designed ( Figure 5 ), and a single mutation (CCC to CTC) was introduced into the mouse Bag3 gene by CRISPR-Cas9-mediated genome editing and homology-directed repair ( Figure 5 ). The newborn mouse pups were genotyped by PCR and DNA sequencing (Table 5).

[0171] Table 4. Structure of the mouse Bag3 locus (chromosome 7)

[0172] Exon / intron Start position End position Length Exon 1 128523616 128524044 429 Intron 1-2 128524045 128539972 15928 Exon 2 128539973 128540311 339 Intron 2-3 128540312 128541806 1495 Exon 3 128541807 128542208 402 Intron 3-4 128542209 128545589 3381 Exon 4 128545590 128546981 1392

[0173] Table 5. Primers for PCR genotyping of the Bag3opathy mouse model by DNA sequencing

[0174]

[0175] Hindlimb weakness was observed in two female heterozygous P209L-KI mice (P209L / +), indicating that the KI mouse model could develop a muscle weakness-like phenotype observed in human Bag3opathy patients. Meanwhile, the mouse G6PD levels were measured by ELISA kits. Consistently, compared with wild type (6.57 ± 0.73 ng / mL and 1.61 ± 0.08 ng / mg in serum and skeletal muscle respectively, n = 4), the G6PD protein in the serum (4.29 ± 0.87 ng / mL, n = 4) and skeletal muscle (1.09 ± 0.17 ng / mg, n = 4) samples of P209L KI mice was significantly reduced in the heterozygous rather than the homozygous condition ( Figure 6 and Figure 8 ). No difference in G6PD was detected in the myocardium ( Figure 7 ). These results indicate that in the mouse model, the BAG3 P209L mutation leads to muscle weakness and reduced G6PD.

[0176] Oral administration of oleanolic acid increased G6PD in the serum, myocardium and skeletal muscle of mice

[0177] Normal mice were orally administered oleanolic acid (50 mg / kg and 200 mg / kg) daily for 10 days, which significantly increased the G6PD protein level in the serum samples ( Figure 9 ): control group (n = 6), 1.79 ± 0.99 ng / mL; 50 mg oleanolic acid / kg group (n = 6), 3.74 ± 1.91 ng / mL (p = 0.140); 200 mg oleanolic acid / kg group (n = 6), 4.47 ± 3.07 ng / mL (p = 0.049). The G6PD protein in the myocardium also increased ( Figure 10):Control group (n = 6), 0.36 ± 0.26 ng / mg; 50 mg oleanolic acid / kg group (n = 6), 0.54 ± 0.32 ng / mg (p = 0.36); 200 mg oleanolic acid / kg group (n = 6), 0.93 ± 0.43 ng / mg (p = 0.011). Similar changes in G6PD were also observed in skeletal muscle ( Figure 11 ):Control group (n = 6), 0.11 ± 0.04 ng / mg; 50 mg oleanolic acid / kg group (n = 6), 0.19 ± 0.04 ng / mg (p = 0.035); 200 mg oleanolic acid / kg group (n = 6), 0.19 ± 0.10 ng / mL (p = 0.035). Overall, oral administration of oleanolic acid increased the G6PD protein concentration in the serum, myocardium, and skeletal muscle of normal mice.

[0178] Treatment with oleanolic acid increased serum G6PD and improved cardiac function in Bag3opathy patients

[0179] Patients with Bag3opathy may benefit from oleanolic acid treatment due to its effect of increasing G6PD. Through a well-designed protocol, this 18-year-old girl took oleanolic acid capsules (180 mg / day) for three months. Consistently, the serum G6PD protein increased significantly, from 0.09 ng / mL to 2.43 ng / mL, which is closer to the average level of normal girls (7.25 ± 9.38 ng / mL) ( Figure 12 )。Surprisingly, the cardiac marker creatine kinase-MB (CK-MB) in the subject's blood decreased significantly: 24 U / L before treatment; 16 U / L after treatment ( Figure 13 )。These results indicate that oral administration of oleanolic acid can increase G6PD in this Bag3opathy patient and improve their cardiac function.

[0180] Oral administration of papaya extract improved muscle dysfunction in P209L-KI mouse model

[0181] Figure 14 The HPLC chromatograms of oleanolic acid in papaya extract before and after purification are shown. The oleanolic acid content in papaya is 0.33%, and after purification using AB-8 resin column chromatography, the oleanolic acid content in the extract increased from 1.87% to 27.18%.

[0182] In vivo experiments were performed on BAG3P209L / + mice by administering papaya extract and applying short-interval continuous low-frequency stimulation to the posterior tibial muscle. The control group of BAG3 P209L / + mice showed equal peak contractile force during the training cycle on day 0 and day 12, and it decreased significantly on day 8 and day 12. ( Figure 15A). In the group of BAG3P209L / + mice treated with 150 mg / kg papaya extract, the peak contractile force of the posterior tibial muscle did not change during all 4 training cycles ( Figure 15 B). In the group of BAG3 P209L / + mice treated with 300 mg / kg papaya extract, a slight decrease in the peak contractile force of the posterior tibial muscle was shown on the 4th day, while there were no significant changes compared to the 0th day on the 8th and 12th days ( Figure 15 C). The results showed that papaya extract could alleviate the attenuation of the contractile force of the posterior tibial muscle caused by the accumulation of exercise injuries and improve the contractile force recovery ability. As expected, papaya extract significantly increased the serum G6PD level in BAG3 P209L / + mice ( Figure 16 ).

Claims

1. A method for treating glucose-6-phosphate dehydrogenase (G6PD) dysregulation disorders, the method comprising administering to the subject a therapeutically effective amount of oleanolic acid, its conjugate salts, or its prodrugs.

2. The method according to claim 1, wherein the G6PD dysregulation disorders are selected from, but not limited to, Bag3opathy caused by mutations in Bcl2 associated athanogene 3 (BCL2), amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease, and Alzheimer's disease.

3. The method according to claim 1, wherein the G6PD dysregulation disorder is Bag3opathy caused by mutations in BCL2.

4. The method according to claim 3, wherein the subject has one or more of severe muscle weakness, cardiomyopathy, and respiratory insufficiency.

5. The method according to claim 1, wherein oleanolic acid or its conjugate salts are administered to the subject in the form of a plant product or its extract.

6. The method according to claim 5, wherein the plant product comprises one or more of Crataegi fructus, Forsythiae fructus, Prunellae spica, Verbenae herba, Eriobotryae folium, Ligustri lucidi fructus, Kaki calyx, Chaenomelis fructus, Jujubae fructus, Corni fructus, or their extracts.

7. The method according to claim 5, wherein the plant product comprises Chaenomelis fructus or its extract.

8. The method according to claim 7, wherein the Chaenomelis fructus extract is prepared by mixing Chaenomelis fructus with ethanol to extract at least a portion of the oleanolic acid or its conjugate salts present in Chaenomelis fructus and forming an ethanol extract containing oleanolic acid or its conjugate salts, and the ethanol can also be removed from the ethanol extract to form the Chaenomelis fructus extract.

9. The method according to claim 1, wherein the subject has a Bcl2 associated athanogene 3 (BAG3) gene containing the c.626C>T mutation.

10. The method according to claim 1, wherein the prodrug of oleanolic acid has Chemical Formula 2: or a pharmaceutically acceptable salt thereof, wherein R 1 is hydrogen, R(C=O)- or RO(C=O)-; R 2 is hydrogen, alkyl, cycloalkyl, aralkyl or aryl; and R is independently hydrogen, alkyl, aralkyl, aryl or heterocycloalkyl in each case, where R 1 and R 2 at least one of which is not hydrogen.

11. The method according to claim 1, wherein the subject is human.

12. A method for treating Bag3opathy caused by mutations in BCL2 in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of oleanolic acid, its conjugate salts, or its prodrugs.

13. The method according to claim 12, wherein oleanolic acid or its conjugate salt is administered to the subject in the form of a plant product or an extract thereof.

14. The method according to claim 13, wherein the plant product comprises one or more of hawthorn, forsythia, selfheal fruit-spike, verbena, loquat leaf, glossy privet fruit, calyx kaki, papaya, Chinese date, dogwood or an extract thereof.

15. The method according to claim 13, wherein the plant product comprises papaya or an extract thereof.

16. The method according to claim 15, wherein the papaya extract is prepared by: extracting at least a portion of oleanolic acid or its conjugate salt present in papaya by mixing papaya with ethanol and forming an ethanol extract containing oleanolic acid or its conjugate salt, and optionally removing the ethanol from the ethanol extract to form the papaya extract.

17. The method according to claim 12, wherein the subject has a BCL2 associated athanogene 3 (BAG3) gene comprising a c.626C> mutation.

18. The method according to claim 12, wherein the prodrug of oleanolic acid has Chemical Formula 2: or a pharmaceutically acceptable salt thereof, wherein R 1 is hydrogen, R(C=O)- or RO(C=O)-; R 2 is hydrogen, alkyl, cycloalkyl, aralkyl or aryl; and R is independently in each case hydrogen, alkyl, aralkyl, aryl or heterocycloalkyl, where R 1 and R 2 at least one of which is not hydrogen.

19. The method according to claim 12, wherein the subject is human.

Images