Methods, compositions and uses relating to treatment and prevention of liritic syndrome

Abstract

The present disclosure provides methods for treating, preventing, alleviating, inhibiting, or delaying the onset of Like's syndrome in a subject in need thereof, and methods for alleviating, inhibiting, alleviating, inhibiting, or delaying the onset of related signs and / or symptoms of Like's syndrome, the methods comprise administering to the subject various compounds, mixtures of compounds, or compositions, formulations, or medicaments derived therefrom. The disclosure further provides compositions, formulations, or medicaments and related uses for treating, preventing, alleviating, inhibiting, or delaying the onset of Like's syndrome in a subject and addressing signs and / or symptoms associated therewith.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 528,186, filed July 21, 2023, and U.S. Provisional Patent Application No. 63 / 606,826, filed December 6, 2023, the entire contents of each of which are incorporated herein by reference for any and all purposes. Technical Field

[0003] This application generally relates to methods, compositions / formulations / medications, and related uses for treating, preventing, alleviating, suppressing, or delaying the onset of Leigh syndrome and / or related signs and / or symptoms in a subject in need. The methods, compositions / formulations / medications, and related uses all relate to the administration of one or more of the four compounds specifically specified herein. Background Technology

[0004] The following description is provided to aid the reader's understanding. It is not acknowledged that the information provided herein or the references cited herein are prior art to the compounds, compositions, products, and / or methods disclosed herein.

[0005] Lewynn syndrome (also known as Lewynn disease) is a rare neurodegenerative disorder associated with mitochondrial dysfunction, typically beginning in infancy between three months and two years of age. In rare cases, the onset of the disease or its signs and symptoms may be delayed into adolescence or even adulthood. For children with Lewynn syndrome, the disease usually progresses rapidly. Children with early-onset Lewynn syndrome typically die before the age of three, and 90% of deaths usually occur before the age of six. Children with Lewynn syndrome often die from respiratory failure. People with adult-onset Lewynn syndrome can live into their fifties.

[0006] Early signs and symptoms of Leydig syndrome include loss of appetite, poor sucking ability, vomiting, irritability, persistent crying, and seizures. The disease then typically progresses to general weakness, hypotonia (including loss of head control and motor skills), and episodes of lactic acidosis, which can lead to respiratory and renal impairment. Clinical symptoms include neurodevelopmental regression, often accompanied by brainstem dysfunction, including abnormal tone, strength, responsiveness, ataxia, dysphagia, and seizures. Other signs or symptoms include dysphagia, dyspnea, difficulty eating, hypotonia, developmental delay, ataxia, dysarthria, dystonia, and paralysis. While clinical presentation may vary from person to person, abnormalities in the brainstem, cerebellum, basal ganglia, oculomotor nerves, and cranial nerves significantly influence the symptoms of Leydig syndrome. Because Leydig syndrome involves mitochondrial dysfunction, and the heart is rich in mitochondria, cardiomyopathy (e.g., hypertrophic cardiomyopathy and dilated cardiomyopathy) is also frequently observed in patients with Leydig syndrome. Patients may also experience visual impairment (e.g., color blindness or vision loss).

[0007] Vatiquinone (also known as EPI743) has been clinically tested as a potential treatment for Leishmaniasis, but recent results appear to be disappointing. It is believed that in treated subjects, vatiquinone targets ferroptosis (see: Kahn-Kirby et al., Targeting ferroptosis: A novel therapeutic strategy for treatment of mitochondrial disease-related epilepsy). Treatment typically involves addressing the signs and symptoms of the disease. Better treatments are needed to address this devastating rare disease. Summary of the Invention

[0008] In one aspect, this technology provides a method for treating, preventing, alleviating, suppressing, or delaying the onset of Lechner's syndrome or related signs and / or symptoms in a subject in need, the method comprising administering to the subject a therapeutically effective amount of compound 1, compound 2, compound 3, compound 4, or a mixture of any two or more compounds 1 to 4, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, tautomer, hydrate, and / or solvate thereof, wherein compounds 1 to 4 have the following structures: .

[0009] In some embodiments, signs or symptoms of Leydig syndrome include dysphagia, dyspnea, dysphagia, hypotonia, lactic acidosis, developmental delay, cardiomyopathy, seizures, ataxia, dysarthria, dystonia, paralysis, respiratory and renal impairment, color blindness and / or vision loss.

[0010] In some embodiments, the subject is diagnosed with Leydig syndrome. In some embodiments, the subject is a person.

[0011] In some embodiments, the compound is administered daily for 6 weeks or longer.

[0012] In some embodiments, the compound is administered orally, topically, intranasally, systemically, intravenously, subcutaneously, intraperitoneally, intradermally, intraocularly, intraocularly, intrathecally, intrathecally, intravaginally, via iontophoresis, through mucous membranes, intravitreal, or intramuscularly.

[0013] In some embodiments, the compound is administered to the subject to treat, prevent, alleviate, inhibit, or delay the onset of cardiomyopathy in the subject.

[0014] In some embodiments, cardiomyopathy is hypertrophic cardiomyopathy, left ventricular hypertrophy, dilated cardiomyopathy, pericardial effusion, and / or arrhythmia / conduction abnormality. In some embodiments, cardiomyopathy is hypertrophic cardiomyopathy.

[0015] In some embodiments, administering the compound to a subject reduces lactate levels in the subject's blood, urine, or cerebrospinal fluid (CSF).

[0016] In some embodiments, the compound is administered to the subject to treat, prevent, reduce, inhibit, or delay the onset of multi-organ and / or central nervous system (CNS) injury, which includes lesions of the brainstem, basal ganglia, and spinal cord.

[0017] In some embodiments, the compound is administered to the subject to treat, prevent, alleviate, suppress, or delay the onset of color blindness and / or vision loss.

[0018] In some embodiments, administering a compound to a subject prolongs the subject's lifespan.

[0019] In some embodiments, the compound is administered to the subject to treat, prevent, reduce, suppress, or delay the occurrence of seizures, ataxia, dysarthria, dystonia, and / or paralysis.

[0020] In one aspect, this technology provides a composition, medicament, or formulation comprising any one of compounds 1 to 4 or a mixture of any two or more compounds 1 to 4, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, tautomer, hydrate, and / or solvate thereof, for the treatment, prevention, relief, inhibition, or delay of the onset of Leishman's syndrome or related signs and / or symptoms in a subject of need, wherein compounds 1 to 4 have the following structures: .

[0021] In some embodiments, signs or symptoms of Leydig syndrome include dysphagia, dyspnea, dysphagia, hypotonia, lactic acidosis, developmental delay, cardiomyopathy, seizures, ataxia, dysarthria, dystonia, paralysis, respiratory and renal impairment, color blindness, and vision loss.

[0022] In some embodiments, the subject is diagnosed with Leydig syndrome. In some embodiments, the subject is a person.

[0023] In some embodiments, the composition, drug, or formulation is administered daily for 6 weeks or longer.

[0024] In some embodiments, the composition, drug, or formulation is administered orally, topically, intranasally, systemically, intravenously, subcutaneously, intraperitoneally, intradermally, intraocularly, intraocularly, intrathecally, intrathecally, intracranially, via iontophoresis, through mucous membranes, intravitreally, or intramuscularly.

[0025] In some embodiments, the composition, drug, or formulation is administered to a subject to treat, prevent, alleviate, inhibit, or delay the onset of cardiomyopathy in the subject.

[0026] In some embodiments, cardiomyopathy is hypertrophic cardiomyopathy, left ventricular hypertrophy, dilated cardiomyopathy, pericardial effusion, and / or arrhythmia / conduction abnormality. In some embodiments, cardiomyopathy is hypertrophic cardiomyopathy.

[0027] In some embodiments, administering a composition, drug, or formulation to a subject reduces lactate levels in the subject's blood, urine, or cerebrospinal fluid (CSF).

[0028] In some embodiments, a composition, drug, or formulation is administered to a subject to treat, prevent, reduce, inhibit, or delay the onset of multi-organ and / or central nervous system (CNS) injury, said multi-organ and / or CNS injury including lesions of the brainstem, basal ganglia, and spinal cord.

[0029] In some embodiments, the composition, drug, or formulation is administered to the subject to treat, prevent, alleviate, suppress, or delay the onset of color blindness and / or vision loss in the subject.

[0030] In some embodiments, administering a composition, drug, or formulation to a subject may prolong the subject's lifespan.

[0031] In some embodiments, the composition, drug, or formulation is administered to a subject to treat, prevent, reduce, suppress, or delay seizures of the subject’s epileptic seizures, ataxia, dysarthria, dystonia, and / or paralysis.

[0032] In one aspect, this technology provides the use of compounds 1 to 4, or any two or more of compounds 1 to 4, or pharmaceutically acceptable salts, stereoisomers, mixtures of stereoisomers, tautomers, hydrates, and / or solvates thereof, in the preparation of compositions, pharmaceuticals, or formulations comprising any one of compounds 1 to 4, or any two or more of compounds 1 to 4, or pharmaceutically acceptable salts, stereoisomers, mixtures of stereoisomers, tautomers, hydrates, and / or solvates thereof, suitable for treating, preventing, alleviating, inhibiting, or delaying the onset of Lechner's syndrome or signs and / or symptoms of Lechner's syndrome in a subject of need, wherein compounds 1 to 4 have the following structures: .

[0033] In some embodiments, signs or symptoms of Leydig syndrome include dysphagia, dyspnea, dysphagia, hypotonia, lactic acidosis, developmental delay, cardiomyopathy, seizures, ataxia, dysarthria, dystonia, paralysis, respiratory and renal impairment, color blindness, and vision loss.

[0034] In some embodiments, the subject is diagnosed with Leydig syndrome. In some embodiments, the subject is a person.

[0035] In some embodiments, the compound, composition, drug, or formulation is administered daily for six weeks or longer.

[0036] In some embodiments, the compound, composition, drug, or formulation is administered orally, topically, intranasally, systemically, intravenously, subcutaneously, intraperitoneally, intradermally, intraocularly, intraocularly, intrathecally, intrathecally, intravaginally, via iontophoresis, through mucous membranes, intravitreal, or intramuscularly.

[0037] In some embodiments, a compound, composition, drug, or formulation is administered to a subject to treat, prevent, alleviate, inhibit, or delay the onset of cardiomyopathy in the subject.

[0038] In some embodiments, cardiomyopathy is hypertrophic cardiomyopathy, left ventricular hypertrophy, dilated cardiomyopathy, pericardial effusion, and / or arrhythmia / conduction abnormality. In some embodiments, cardiomyopathy is hypertrophic cardiomyopathy.

[0039] In some embodiments, administration of a compound, composition, drug, or formulation to a subject reduces lactate levels in the subject's blood, urine, or cerebrospinal fluid (CSF).

[0040] In some embodiments, a compound, composition, drug, or formulation is administered to a subject to treat, prevent, reduce, inhibit, or delay the onset of multi-organ and / or central nervous system (CNS) injury, said multi-organ and / or CNS injury including lesions of the brainstem, basal ganglia, and spinal cord.

[0041] In some embodiments, a compound, composition, drug, or formulation is administered to a subject to treat, prevent, reduce, suppress, or delay the onset of color blindness and / or vision loss in the subject.

[0042] In some embodiments, administering a compound, composition, drug, or formulation to a subject prolongs the subject's lifespan.

[0043] In some embodiments, a compound, composition, drug, or formulation is administered to a subject to treat, prevent, reduce, suppress, or delay seizures of the subject’s epileptic seizures, ataxia, dysarthria, dystonia, and / or paralysis. Attached Figure Description

[0044] Figure 1 It is a graphical representation of data obtained from in vitro assays designed to examine and quantify (i.e., by determining EC) 50 (Quantitative) Compound 1 exhibits the bypass activity of Complex I.

[0045] Figure 2 It is a graphical representation of data obtained from in vitro assays designed to examine and quantify (i.e., by determining EC) 50 (Quantitative) Compound 2 exhibits the bypass activity of Complex I.

[0046] Figure 3 It is a graphical representation of data obtained from in vitro assays designed to examine and quantify (i.e., by determining EC) 50 (Quantitative) Compound 3 exhibits the ability of complex I to bypass activity.

[0047] Figure 4It is a graphical representation of data obtained from in vitro assays designed to examine and quantify (i.e., by determining EC) 50 (Quantitative) Compound 4 exhibits the ability of complex I to bypass activity.

[0048] Figure 5 It is a graphical representation of data obtained from in vitro assays designed to evaluate the effect of compound 3 on ROS generated by reverse electron transfer (RET) from complex II to complex I.

[0049] Figure 6 It is a graphical representation of data obtained from in vitro assays designed to evaluate the effect of compound 4 on ROS generated by reverse electron transfer (RET) from complex II to complex I.

[0050] Figure 7 This is a graphical representation of data from in vitro assays used to demonstrate the comparative effect of RSL3 on healthy and diseased cells (i.e., fibroblasts derived from patients with Leydig syndrome) to determine the concentration of RSL3 for subsequent studies in each cell line.

[0051] Figure 8 The image is a graphical representation of data from in vitro assays used to examine and compare the ability of compounds 1 to 4 and vatiquidon to reduce RSL3-induced ferroptosis in patient-derived fibroblasts (cell line GM08402) from healthy individuals.

[0052] Figure 9 The image is a graphical representation of data from in vitro assays used to examine and compare the ability of compounds 1 to 4 and vatiquidon to reduce RSL3-induced ferroptosis in patient-derived fibroblasts (cell line GM03672) from individuals diagnosed with Leishman's syndrome.

[0053] Figure 10 The image is a graphical representation of data from in vitro assays used to examine and compare the ability of compounds 2 to 4 and vatiquidon to act as inhibitors of RSL3-induced ferroptosis 15-lipoxygenase (15-LO) in patient-derived fibroblasts from healthy individuals.

[0054] Figure 11 The image is a graphical representation of data from in vitro assays used to examine and compare the ability of compounds 2 to 4 and vatiquidone to act as 15-lipoxygenase inhibitors of RSL3-induced ferroptosis in patient-derived fibroblasts from individuals diagnosed with Leydig syndrome.

[0055] Figure 12It is a graphical representation of data from in vitro assays used to examine and compare the ability of compounds 1 to 4 and vatiquidon to alleviate damage to patient-derived fibroblasts from healthy individuals caused by erastin.

[0056] Figure 13 It is a graphical representation of data from in vitro assays used to examine and compare the ability of compounds 1 to 4 and vatiquidone to alleviate damage to patient-derived fibroblasts from individuals diagnosed with Leydig syndrome caused by alastine.

[0057] Figure 14 The image is a graphical representation of data from in vitro assays used to examine and compare the ability of compounds 2, 3, 4 and vatiquidone to act as inhibitors of 15-lipoxygenase in fibroblasts with ageratin-damaged cells from healthy individuals.

[0058] Figure 15 The image is a graphical representation of data from in vitro assays used to examine and compare the ability of compounds 1 to 4 and vatiquidon to act as inhibitors of 15-lipoxygenase in fibroblasts with agestin damage from individuals diagnosed with Leishman's syndrome.

[0059] Figure 16 This is a graphical representation of comparative data obtained from a mouse plasma pharmacokinetic (PK) characterization study, in which compound 3 was studied at 20 mg / kg and 60 mg / kg SC, and compound 4 was studied at 60 mg / kg SC.

[0060] Figure 17 This is a graphical representation of comparative data obtained from plasma pharmacokinetic studies, in which compound 3 was studied in mice at a 20 mg / kg SC dose and in rats at a 10 mg / kg SC dose (regardless of the species examined, 60 mg / m²). 2 All are valid.

[0061] Figure 18 This is a graphical representation of comparative data obtained in a PK characterization study that examined drug uptake in mouse heart tissue resulting from subcutaneous administration of 20 mg / kg and 60 mg / kg doses of compound 3 and 60 mg / kg dose of compound 4.

[0062] Figure 19 This is a graphical representation of comparative data obtained in a PK characterization study that examined drug uptake in mouse brain tissue resulting from subcutaneous administration of 20 mg / kg and 60 mg / kg doses of compound 3 and 60 mg / kg dose of compound 4.

[0063] Figure 20 This is a bar graph presenting data obtained using fibroblast cell lines derived from patients with Leydig syndrome. It compares the membrane potential of untreated cells with that of cells treated with RSL3 alone, and the membrane potential of cells treated with both RSL3 and compound 3 (1 μM).

[0064] Figure 21 This is a bar graph presenting data obtained using fibroblast cell lines derived from patients with Leydig syndrome. It compares the membrane potential of untreated cells with that of cells treated with RSL3 alone, and the membrane potential of cells treated with both RSL3 and compound 4 (1 μM).

[0065] Figure 22A The graph shows the plasma PK characteristics of mice after treatment with the following doses of compound 4 for 5 days: (i) 60 mg / kg in PBS containing 5% Kolliphor ELP (K-ELP); (ii) 60 mg / kg in PBS containing 15% K-ELP; and (iii) 180 mg / kg in PBS containing 15% K-ELP.

[0066] Figures 22B to 22C This shows the results of treating mouse tissues (heart, etc.) with the following dosage of compound 4 for 5 days. Figure 22B ) and brain ( Figure 22C Charts of exposure characteristics: (i) 60 mg / kg PBS containing 5% Kolliphor ELP (K-ELP); (ii) 60 mg / kg PBS containing 15% K-ELP; and (iii) 180 mg / kg PBS containing 15% K-ELP. Detailed Implementation

[0067] It should be understood that certain aspects, patterns, embodiments, variations, and features of this disclosure are described below in varying degrees of detail to provide a substantial understanding of the technology. Definitions of certain terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology pertains.

[0068] In practicing this technique, many routine techniques from molecular biology, protein biochemistry, cell biology, immunology, microbiology, and recombinant DNA were used. These techniques are well-known and explained in the following literature: for example, *A Guide to Experimental Molecular Biology* (…). Current Protocols in Molecular Biology), Volumes I-III, edited by Ausubel, (1997); Sambrook et al., Molecular Cloning: A Laboratory Manual ( Molecular Cloning: A Laboratory Manual ), Second Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989); DNA Cloning: Practical Methods DNA Cloning: A Practical Approach ), Volume I and Volume II, edited by Glover (1985); Oligonucleotide Synthesis ( Oligonucleotide Synthesis )》, edited by Gait (1984); Nucleic Acid Hybridization ( Nucleic Acid Hybridization ), Hames and Higgins (eds.) (1985); Transcription and Translation ( Transcription and Translation ), Hames and Higgins (eds.) (1984); Animal Cell Culture ( Animal Cell Culture "Immobilized Cells and Enzymes", edited by Freshney (1986); "Immobilized Cells and Enzymes" Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, *A Practical Guide to Molecular Cloning* (…). A Practical Guide to Molecular Cloning )》; Series, "Meth. Enzymol" (Academic Press, Inc., 1984); "Mammalian Cell Gene Transfer Vectors" Gene Transfer Vectors for Mammalian Cells ), edited by Miller and Calos (Cold Spring Harbor Laboratory, New York, 1987); and Enzymatic Methods, Volumes 154 and 155, edited by Wu and Grossman and Wu, respectively.

[0069] I. Definition

[0070] The definitions of specific functional groups and chemical terms are described in more detail below. Chemical elements are identified according to the Periodic Table of the Elements, GAS edition, and the Handbook of Chemistry and Physics, 7Sh edition, inner pages. In addition, the general principles of organic chemistry, as well as specific functional components and reactivity, are described in the following literature: Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5th ed., John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd ed., Cambridge University Press, Cambridge, 1987.

[0071] It should be understood that certain aspects, modes, embodiments, variations, and features of this technology are described below in varying degrees of detail to provide a substantial understanding of this disclosure. Definitions of certain terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology pertains.

[0072] As used in this specification and the accompanying embodiments, unless the content expressly indicates otherwise, the singular forms “a,” “an,” and “the” include plural referents. For example, references to “cell” include combinations of two or more types of cells, etc.

[0073] As used herein, “administering” or “administration” of a drug agent (i.e., a therapeutic agent) or compound / pharmaceutical product (including compositions (i.e., formulations or drugs)) to a subject includes any route of introduction or delivery of the therapeutic agent / compound / pharmaceutical product to the subject to exert its intended function. Administration may be performed via any suitable route, such as oral administration. Administration may be performed subcutaneously. Administration may be performed intravenously. Administration may be performed intraocularly. Administration may be performed systemically. Alternatively, administration may be performed topically, intranasally, intraperitoneally, intradermally, intraocularly, intrathecally, intraventricularly, iontophoretally, transmucosally, intravitreally, or intramuscularly. Administration includes self-administration, administration by another person, or administration using a device (e.g., an infusion pump).

[0074] As used in this article, “ameliorate” or “ameliorating” a disease, condition, or symptom means, in a statistical sample or specific subject, an outcome that makes the occurrence of a disease, condition, or symptom (or its signs or symptoms) better or more tolerable compared to a control sample, control subject, or control subject group.

[0075] As used herein, the term "carrier" or "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or mediator used with or in combination with a therapeutic agent / compound / pharmaceutical product / composition (including formulations or drugs) for administration. Non-limiting examples of such pharmaceutically acceptable carriers include liquids (e.g., water, saline, oil) and solids (e.g., gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, silica particles (nanoparticles or microparticles), urea, etc.). Additionally, adjuvants, stabilizers, thickeners, lubricants, flavoring agents, and coloring agents may be used. Other examples of suitable drug carriers are described in EW Martin's *Remington Pharmaceutical Sciences*. Remington's Pharmaceutical Sciences The reference mentioned above is incorporated herein by reference in its entirety.

[0076] As used herein, the phrase “delay the onset of…” means, in a statistical sample, to postpone or prevent the onset of a disease, condition, or symptom in a sample or subject who has been given one or more therapeutic agents, or to cause one or more signs or symptoms of a disease, condition, or symptom to occur more slowly than normal, relative to a control sample, control subject, or control subject group.

[0077] As used herein, the term "effective amount" refers to an amount of therapeutic agent / compound / composition / pharmaceutical product sufficient to achieve the desired therapeutic and / or preventive effect, such as the amount used to treat, prevent, suppress, alleviate, or delay the onset of a disease, condition, or symptom, or the physiological signs or symptoms of a disease, condition, or symptom. In the context of therapeutic or preventive application, in some embodiments, the amount of therapeutic agent / compound / composition / pharmaceutical product administered to the subject will depend on the type and severity of the disease and on individual characteristics such as general health status, age, sex, weight, and tolerance to the drug. In some embodiments, it also depends on the extent, severity, and type of the disease. Those skilled in the art will be able to determine the appropriate dosage based on these and other factors. The compound / composition / pharmaceutical product may also be administered in combination with one or more other therapeutic compounds / pharmaceutical agents (so-called "co-administration," where, for example, additional or other therapeutic agents may be administered simultaneously, sequentially, or by means of individual administration).

[0078] As used herein, the term "hydrate" refers to a compound that is associated (e.g., complexed) with water. The number of water molecules in a hydrate of a compound may (or may not) be in a proportional relationship with the number of compound molecules in the hydrate.

[0079] As used herein, “inhibit” means a reduction in signs, symptoms, or symptom associated with Lechner’s syndrome (e.g., risk factors). In one embodiment, inhibition means a reduction of at least a statistically significant amount compared to a control, control subject, or control subject cohort. In one embodiment, inhibition means a reduction of at least 5% compared to a control, control subject, or control subject cohort. In various individual embodiments, inhibition means a reduction of at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 33%, 40%, 50%, 60%, 67%, 70%, 75%, 80%, 90%, 95%, or 99% compared to a control, control subject, or control subject cohort.

[0080] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a therapeutic compound that can be prepared using a relatively non-toxic acid or base, depending on the specific substituent present on the compound described herein. When the compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting such a compound in its neutral form with a sufficient amount of the desired base, either purely or in a suitable inert solvent. Examples of pharmaceutically acceptable salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts, or similar salts. When the compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting such a compound in its neutral form with a sufficient amount of the desired acid, either purely or in a suitable inert solvent. Salts derived from pharmaceutically acceptable inorganic bases include ammonium, calcium, copper, iron, ferrous, lithium, magnesium, manganese, manganese, potassium, sodium, and zinc salts, etc. Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary, and tertiary amines, including substituted amines, cyclic amines, and naturally occurring amines, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-methylmorpholine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, heparin, isopropylamine, lysine, methylglucosamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine (NEt3), trimethylamine, tripropylamine, tromethamine, etc., such as salts containing the protonated form of the organic base (e.g., [HNEt3]+). Salts derived from pharmaceutically acceptable inorganic acids include salts of the following acids: boric acid, carbonic acid, hydrohalic acids (hydrobromic acid, hydrochloric acid, hydrofluoric acid, or hydroiodic acid), nitric acid, phosphoric acid, aminosulfonic acid, and sulfuric acid. Salts derived from pharmaceutically acceptable organic acids include salts of the following acids: aliphatic hydroxy acids (e.g., citric acid, gluconic acid, glycolic acid, lactic acid, lactobionic acid, malic acid, and tartaric acid), aliphatic monocarboxylic acids (e.g., acetic acid, butyric acid, formic acid, propionic acid, and trifluoroacetic acid), amino acids (e.g., aspartic acid and glutamic acid), aromatic carboxylic acids (e.g., benzoic acid, p-chlorobenzoic acid, diphenylacetic acid, gentianic acid, hippuric acid, and triphenylacetic acid), and aromatic hydroxy acids (e.g., o-hydroxybenzoic acid, p ... Hydroxybenzoic acid, 1-hydroxynaphth-2-carboxylic acid and 3-hydroxynaphth-2-carboxylic acid), ascorbic acid, dicarboxylic acids (e.g. fumaric acid, maleic acid, oxalic acid and succinic acid), glucuronic acid, mandelic acid, mucoic acid, nicotinic acid, orotic acid, dihydroxy acids, pantothenic acid, sulfonic acids (e.g. benzenesulfonic acid, camphorsulfonic acid, ethanedisulfonic acid, ethanesulfonic acid, hydroxyethylsulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, naphthalene-1,5-disulfonic acid, naphthalene-2,6-disulfonic acid and p-toluenesulfonic acid (PTSA)), hydroxynaphthic acid, etc.In some embodiments, pharmaceutically acceptable counterions are selected from the group consisting of: acetates, benzoates, benzenesulfonates, bromides, camphorsulfonates, chlorides, theophylline, citrates, ethanedisulfonates, fumarates, gluconate, gluconate, glucuronide, hippurate, iodides, hydroxyethanesulfonates, lactates, lactobionates, lauryl sulfate, malates, maleates, methanesulfonates, methyl sulfates, naphthates, sapsylates, nitrates, octadecanoates, oleates, oxalates, dihydroxynaphthyl salts, phosphates, polygalacturonates, succinates, sulfates, sulfosalicylates, tartrates, toluenesulfonates, and trifluoroacetates. In some embodiments, the salt is a tartrate, fumarate, citrate, benzoate, succinate, octanoate, lactate, oxalate, phthalate, methanesulfonate, benzenesulfonate, maleate, trifluoroacetate, hydrochloride, or toluenesulfonate. It also includes salts of amino acids such as arginine, and salts of organic acids such as glucuronic acid or galacturonic acid (see, for example, Berge et al., Journal of Pharmaceutical Science, 66: 1-19 (1977)). Certain specific compounds may contain both basic and acidic functional groups that allow the compound to be converted into a base addition salt or an acid addition salt, or to exist in zwitterionic form. These salts can be prepared by methods known to those skilled in the art. Any other pharmaceutically acceptable carrier known to those skilled in the art is suitable for use with this technique.

[0081] As used herein, “prevention” or “preventing” of a disease, condition, or symptom refers to a reduction in the incidence of the disease, condition, or symptom in a sample or subject that has been administered one or more therapeutic agents, relative to a control sample, control subject, or control subject group. Such prevention is sometimes referred to as preventive treatment.

[0082] As used herein, the term “alone” means the simultaneous or substantially simultaneous administration of at least two active ingredients (e.g., therapeutic agents) via different routes.

[0083] As used herein, the term "sequentially" means the application of at least two active ingredients (e.g., a therapeutic agent) at different times, via the same or different routes of administration. More specifically, sequential application means the complete application of one active ingredient (e.g., a therapeutic agent) followed by the application of another or other active ingredients. Thus, an active ingredient may be applied minutes, hours, or days before the application of one or more other active ingredients. Simultaneous treatment does not exist under this definition.

[0084] As used herein, the term “simultaneous” means the application of at least two active ingredients (e.g., therapeutic agents) via the same route and simultaneously or substantially simultaneously.

[0085] As used herein, the term "solvent" refers to a compound form that may associate with a solvent through a solvent decomposition reaction. This physical association can include hydrogen bonding. Common solvents include water, methanol, ethanol, isopropanol, acetic acid, ethyl acetate, acetone, hexane, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), diethyl ether, etc.

[0086] As used herein, "subject" refers to a live animal. In various embodiments, the subject is a mammal. In various embodiments, the subject is a non-human mammal, including but not limited to mice, rats, hamsters, guinea pigs, rabbits, sheep, goats, cats, dogs, pigs, miniature pigs, horses, cattle, or non-human primates. In some embodiments, the subject is a human.

[0087] It should also be understood that, in some embodiments, the various treatment or prevention modalities of medical conditions as described herein are intended to represent “basic”, which includes complete treatment, prevention, mitigation, or suppression, but also includes less complete treatment, prevention, mitigation, or suppression, in which some biological or medically relevant outcome is achieved.

[0088] As used herein, "synergistic therapeutic effect" means a therapeutic effect greater than the sum of its parts produced by the combination of at least two agents, and the effect exceeding the effect produced by the agents being applied alone in other ways.

[0089] As used herein, the term "tautomer" refers to a compound that is an interchangeable form of a particular compound structure, with variations in the shifts of hydrogen atoms and electrons. Thus, two structures can reach equilibrium through the movement of π electrons and one atom (typically H). For example, enols and ketones are tautomers because they can be rapidly interconverted by treatment with acids or bases. The tautomer form can be related to the attainment of optimal chemical reactivity and biological activity of the compound of interest.

[0090] As used herein, the term "treating" refers to therapeutic treatment in which the aim is to reduce, alleviate, or slow down (relieve) a pre-existing disease, condition, or symptom, or its associated signs or symptoms. For example, but not limited to, a subject's disease is successfully "treated" if, following administration of an effective amount of a therapeutic agent / compound / composition / pharmaceutical product or its pharmaceutically acceptable salt, stereoisomer, tautomer, hydrate, and / or solvate, the subject exhibits an observable and / or measurable reduction or absence of one or more signs, symptoms, or symptoms associated with the disease or condition. It should also be understood that the various modes of treatment for medical conditions described are intended to represent "fundamental," which includes complete remission of the signs or symptoms of the disease, condition, or symptom, as well as "partial" remission in which some biologically or medically relevant outcome is achieved.

[0091] II. Pharmaceutical Composition, Route of Administration and Dosage

[0092] The methods, uses, and compositions of this application utilize therapeutically effective amounts of compound 1, compound 2, compound 3, compound 4, or mixtures of any two or more compounds 1 to 4, or pharmaceutically acceptable salts, stereoisomers, mixtures of stereoisomers, tautomers, hydrates, and / or solvates thereof, wherein compounds 1 to 4 have the following structures: .

[0093] Compounds 1 to 4, or mixtures of any two or more of compounds 1 to 4, or their pharmaceutically acceptable salts, stereoisomers, mixtures of stereoisomers, tautomers, hydrates, and / or solvates, may be formulated into pharmaceutical products suitable for administration to a subject in need. Such pharmaceutical products may be referred to as compositions, formulations, or drugs, depending on their intended use. Any mixture prepared by mixing any one or more of compounds 1 to 4, or their pharmaceutically acceptable salts, stereoisomers, mixtures of stereoisomers, tautomers, hydrates, and / or solvates, with solvents and / or other compounds is a composition or formulation and may or may not be intended for administration to a subject. A drug is generally considered to be a composition or formulation specifically prepared for administration to a subject to address a disease, symptom, or condition (e.g., Lechner's syndrome). For the sake of brevity, whenever this document refers to “compounds 1 to 4 or a mixture of two or more of compounds 1 to 4”, such reference is intended to imply any or pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, tautomer, hydrate and / or solvate of compounds 1 to 4.

[0094] Compounds 1 to 4 or any two or more of compounds 1 to 4 disclosed herein may be used alone or in combination with other therapeutic agents to address the needs of subjects with Leydig syndrome. For administration to subjects in need, compounds 1 to 4 or any two or more of compounds 1 to 4 will generally need to be formulated for the intended route of administration (for single administration or in combination). In some embodiments, the same route of administration may be used to deliver compounds 1 to 4 or any two or more of compounds 1 to 4. In some embodiments, two or more of compounds 1 to 4 or any two or more of compounds 1 to 4 may be administered via different routes. The formulated product may be considered a composition or medicine comprising compounds 1 to 4 or any two or more of compounds 1 to 4 and optionally one or more other (i.e., additional) therapeutic agents.

[0095] In some embodiments, the therapeutic agent may be formulated to have very little or no excipients or carriers. In some embodiments, the therapeutic agent may be formulated such that the majority of the formulation is an excipient or carrier. In short, those skilled in the art will tailor the formulation to have appropriate amounts of excipients or carriers based on the needs / symptoms of the subject, the type and severity of the disease to be treated, the nature of one or more therapeutic agents to be delivered, and the selected administration method of one or more particular therapeutic agents.

[0096] In some embodiments, the pharmaceutical composition may further comprise at least one therapeutic agent other than compounds 1 to 4 or a mixture of any two or more compounds 1 to 4 (e.g., another (or additional) therapeutic agent used in combination with compounds 1 to 4 or a mixture of any two or more compounds 1 to 4). The at least one additional / alternative therapeutic agent may be an agent for treating Leishman's syndrome, or may be administered, for example, to alleviate the side effects of administering compounds 1 to 4 or a mixture of any two or more compounds 1 to 4 (e.g., to resolve adverse reactions, or because combination therapy is the preferred treatment for Leishman's syndrome). Therefore, in some embodiments, the pharmaceutical composition may be prepared, for example, by combining compounds 1 to 4 or a mixture of any two or more compounds 1 to 4 with a pharmaceutically acceptable carrier and optionally one or more additional therapeutic agents, or otherwise, by combining the other / alternative therapeutic agent only when administering compounds 1 to 4 or a mixture of any two or more compounds 1 to 4.

[0097] The pharmaceutical composition may contain an effective amount of one or more therapeutic agents as described herein, and may optionally be distributed (e.g., dissolved, suspended, or otherwise distributed) in a pharmaceutically acceptable carrier. The components of the pharmaceutical composition may also be able to mix with the compounds of this application and in a manner that does not significantly impair the desired pharmaceutical efficiency.

[0098] As stated above, "effective amount" refers to any amount of a specific therapeutic agent sufficient to achieve the desired biological effect. In conjunction with the teachings provided herein, an effective prophylactic (i.e., preventative) or therapeutic treatment regimen can be planned by selecting from a variety of therapeutic compounds and weighing factors such as potency, relative bioavailability, patient weight, severity of adverse side effects, and administration mode. This regimen will not cause significant undesirable toxicity and will be effective in addressing a specific symptom, condition, or disease in a particular subject in a therapeutic manner. The effective amount of a therapeutic agent for any particular indication can vary depending on factors such as the disease, condition, or symptom being treated, one or more specific compounds administered, the subject's body type, the subject's age, the subject's overall health condition, and / or the severity of the disease, condition, or symptom. The effective amount can be determined during preclinical and / or clinical trials using methods familiar to physicians and clinicians. Those skilled in the art can determine the effective amount of one or more specific therapeutic agents empirically without the need for excessive experimentation. A maximum dose, i.e., the highest safe dose based on some medical judgment, can be used. Multiple daily doses can be envisioned to achieve appropriate systemic levels of the compound. Appropriate systemic levels can be determined, for example, by measuring the patient's peak or sustained plasma levels. The terms "dose" and "dosage" are used interchangeably herein. Dosage can be administered by oneself, by another person, or via a device (e.g., a pump).

[0099] For any therapeutic compound described herein, the therapeutically effective dose can be determined, for example, initially from animal models. The therapeutically effective dose can also be determined based on human data from compounds already tested in humans and compounds known to exhibit similar pharmacological activity (such as other relevant active agents). Parenteral administration may require higher doses. The applied dose can be adjusted based on relative bioavailability and the potency of the compound being administered. Adjusting the dose to achieve maximum efficacy based on the methods described above and other methods well known in the art is entirely within the capabilities of a person skilled in the art.

[0100] Therapeutic compounds (alone or formulated in a pharmaceutical composition / drug) for therapeutic or preventative purposes can be tested in suitable animal model systems. Suitable animal model systems include, but are not limited to, rats, mice, chickens, cattle, monkeys, rabbits, pigs, miniature pigs, etc., prior to testing in human subjects. In vivo testing can be performed using any animal model system known in the art prior to administration to human subjects. In some embodiments, administration can be tested directly in humans.

[0101] The dosage, toxicity, and therapeutic efficacy of any therapeutic agent or composition (e.g., a formulation or drug comprising compounds 1 to 4 or a mixture of any two or more compounds 1 to 4), other / additional therapeutic agents, or mixtures thereof can be determined by standard pharmaceutical procedures in cell cultures or laboratory animals, for example, by determining the LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic effects and therapeutic effects is the therapeutic index, and said therapeutic index can be expressed as the ratio LD50 / ED50. Compounds exhibiting a high therapeutic index are advantageous. Although compounds exhibiting toxic side effects may be used, in such cases, delivery systems targeting such compounds to the site of the affected tissue can be carefully designed to minimize potential damage to uninfected cells and thereby reduce side effects.

[0102] Exemplary treatment regimens may require administration once daily, twice daily, three times daily, weekly, or monthly. In therapeutic applications, relatively high doses are sometimes required at relatively short intervals until disease progression is slowed, reduced, or terminated, or until the subject shows partial or complete relief of disease symptoms. Afterward, a prophylactic regimen may be administered to the patient.

[0103] For use in therapy, an effective amount of the therapeutic compound (alone or formulated) may be administered to a subject by any mode of delivery of the compound to the desired surface. Administration of the pharmaceutical composition may be performed in any manner known to those skilled in the art. Routes of administration include, but are not limited to, oral, topical, intranasal, systemic, intravenous, subcutaneous, intraperitoneal, intradermal, intraocular, ocular, intrathecal, intraventricular, via iontophoresis, transmucosal, intravitreal, or intramuscular administration. Administration includes self-administration, administration by another person, or administration via a device (e.g., a pump).

[0104] The therapeutic compounds / pharmaceuticals disclosed herein (e.g., compounds 1 to 4 or mixtures of any two or more compounds 1 to 4) can be delivered to a subject in the form of a formulation or a drug (i.e., a pharmaceutical composition). Formulations and drugs can be prepared, for example, by dissolving or suspending the therapeutic compounds / pharmaceuticals disclosed herein (e.g., compounds 1 to 4 or mixtures of any two or more compounds 1 to 4) in water, a solvent, a pharmaceutically acceptable carrier, a salt (e.g., NaCl or sodium phosphate), a buffer, a preservative, a compatible carrier, an adjuvant, and optionally other therapeutically acceptable ingredients.

[0105] Pharmaceutical compositions (e.g., formulations or drugs) may include a carrier (e.g., a pharmaceutically acceptable carrier), which may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using coatings such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. Glutathione and other antioxidants may be included to prevent oxidation. In many cases, it will be advantageous to include isotonic agents in the composition, such as sugars (e.g., trehalose), polyols (e.g., mannitol, sorbitol), or sodium chloride. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption (e.g., aluminum monostearate or gelatin) in the composition.

[0106] Solutions or suspensions (e.g., formulations or drugs) intended for parenteral, intradermal, subcutaneous, or intraocular application may include the following components: sterile diluents, such as water for injection, saline solution, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates; and agents for adjusting tension, such as sodium chloride or dextrose. pH may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be sealed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. For the convenience of patients or treating physicians, the medication preparation can be provided alone or in a kit containing all the necessary equipment (e.g., vials, diluent vials, syringes, and needles) for the course of treatment (e.g., treatment for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or longer).

[0107] When systemic delivery of a therapeutic compound / pharmaceutical agent or pharmaceutical composition is desired, the therapeutic compound / pharmaceutical agent or pharmaceutical composition can be formulated for parenteral administration by injection, such as by bolus injection or continuous infusion (e.g., by IV injection or by pump administration over a defined time). The formulation for injection may be present in unit dosage forms, such as in ampoules or multi-dose containers, with added preservatives. The pharmaceutical composition may be in the form of a suspension, solution, or emulsion in an oily or aqueous medium and may contain formulation agents such as suspending agents, stabilizers, and / or dispersants. Additionally, suspensions of therapeutic compounds (e.g., compounds 1 to 4 or any two or more mixtures of compounds 1 to 4) can be prepared as suitable oily injectable suspensions. Suitable lipophilic solvents or mediums include fatty oils (such as sesame oil) or synthetic fatty acid esters (such as ethyl oleate or triglycerides) or liposomes. Aqueous injectable suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents to increase the solubility of the compound, in order to allow for the preparation of high-concentration solutions.

[0108] Systemic formulations include formulations designed for administration by injection (e.g., subcutaneous, intravenous, intramuscular, intrathecal, or intraperitoneal injection), as well as formulations designed for oral administration via the skin, mucosa, or lungs.

[0109] For intravenous and other parenteral routes of administration, compounds (e.g., compounds 1 to 4 or any two or more of compounds 1 to 4) may be formulated as lyophilized formulations, lyophilized formulations of therapeutic compounds with liposome embedding or lipid encapsulation, lipid complexes in aqueous suspensions, or salt complexes. Lyophilized formulations are typically reconstituted in a suitable aqueous solution, such as sterile water or saline, shortly before administration.

[0110] Suitable pharmaceutical compositions for injection (e.g., formulations or drugs) may include sterile aqueous solutions (in the case of water solubility) or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial aqueous solutions, Cremophor EL... TM (BASF, Parsippony, New Jersey) or phosphate-buffered saline (PBS). Compositions intended for injection will generally be sterile and should have a degree of fluidity sufficient for easy injection. They should be stable under manufacturing and storage conditions and be preserved against contamination by microorganisms such as bacteria and fungi.

[0111] As needed, sterile injectable solutions (e.g., formulations or pharmaceuticals) can be prepared by incorporating a therapeutic compound (e.g., compounds 1 to 4 or a mixture of any two or more compounds 1 to 4) in a desired amount into a suitable solvent having one or a combination of the components listed above, followed by filtration sterilization. Typically, dispersions are prepared by incorporating the therapeutic compound into a sterile mediator containing a basic dispersion medium and other desired components from the components listed above. In the case of sterile powders used to prepare sterile injectable solutions, typical preparation methods include vacuum drying and freeze-drying, which can produce powders containing the active ingredient plus any additional desired components from its previously sterile filtered solution.

[0112] For oral administration, the compound can be readily formulated by combining a therapeutic compound (e.g., compounds 1 to 4 or a mixture of any two or more compounds 1 to 4) with a pharmaceutically acceptable carrier well known in the art. Such carriers enable the therapeutic compound to be formulated into tablets, pills, sugar-coated pills, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by a subject to be treated. Tablets, pills, capsules, lozenges, etc., may contain any of the following components or compounds with similar properties: binders, such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients, such as starch or lactose; dispersants, such as alginate, Primogel®, or corn starch; lubricants, such as magnesium stearate or stearates; flow aids, such as colloidal silica; sweeteners, such as sucrose or saccharin; or flavorings, such as peppermint, methyl salicylate, or orange flavoring.

[0113] Pharmaceutical formulations for oral use can be obtained as solid excipients, and optionally, after the addition of suitable adjuvants (if desired), the resulting mixture is ground and processed into granules to obtain tablets or sugar-coated cores. Suitable excipients are specifically fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose formulations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrants such as crospyvinylpyrrolidone, agar, or alginate or its salts, such as sodium alginate, can be added. Optionally, oral formulations can also be prepared in saline or buffer solutions, such as EDTA, to neutralize internal acidic conditions, or can be administered without any carrier.

[0114] Specifically, chemically modified oral dosage forms of the above are also envisioned to enable effective oral delivery of the derivatives. Typically, the envisioned chemical modification involves attaching at least one portion to a therapeutic agent, ingredient, and / or excipient, wherein said portion allows for (a) inhibition of acid hydrolysis; and (b) uptake from the stomach or intestine into the bloodstream. Increased overall stability of the therapeutic agent, ingredient, and / or excipient, as well as increased circulation time in vivo, are also desired. Examples of such portions include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, and polyproline. Abuchowski and Davis, “Soluble Polymer-Enzyme Adducts,” in *Enzymes as Drugs*, edited by Hocenberg and Roberts, Wiley-Interscience, New York, NY, pp. 367-383 (1981); Newmark et al., *Journal of Applied Biochemistry* (… J Appl Biochem (4:185-9 (1982)). Other polymers that can be used are poly-1,3-dioxolane and poly-1,3,6-trioxane. As mentioned above, polyethylene glycol (PEG) moieties of various molecular weights are suitable for pharmaceutical applications.

[0115] For formulations of therapeutic agents, ingredients, and / or excipients, the release site can be the stomach, small intestine (duodenum, jejunum, or ileum), or large intestine. Those skilled in the art have available formulations that do not dissolve in the stomach but release the material in the duodenum or other parts of the intestine. Preferably, release will avoid harmful effects on the gastric environment by protecting the therapeutic compound / pharmaceutical or by releasing the bioactive material outside the gastric environment (e.g., in the intestine).

[0116] Coatings or coating mixtures can also be used on tablets, not necessarily to protect the stomach. This may include sugar coatings or coatings that make the tablets easier to swallow. Capsules can consist of a hard shell (such as gelatin) for delivering dry therapeutic agents (e.g., powders); for liquid forms, a soft gelatin shell can be used. The shell material for flat capsules can be thick starch or other edible paper. For pills, lozenges, molded tablets, or tablet grinders, a wet massing technique can be used.

[0117] Therapeutic compounds / pharmaceuticals (as used herein) are intended to refer to compounds 1 to 4, or mixtures of any two or more compounds 1 to 4, and any other active pharmaceutical ingredient (e.g., other therapeutic agents) that can be administered in combination with compounds 1 to 4 or mixtures of any two or more compounds 1 to 4. Formulations may comprise fine multiply particles in the form of granules or clumps with a particle size of about 1-2 mm. Formulations for materials used in capsule administration may also be powders, slightly compressed suppositories, or even tablets. Therapeutic compounds / pharmaceuticals or pharmaceutical compositions may be prepared by compression.

[0118] Colorants and flavorings can both be included. For example, therapeutic compounds / pharmaceuticals or pharmaceutical compositions can be formulated and then further incorporated into edible products, such as chilled beverages containing colorants and flavorings.

[0119] The volume of therapeutic compounds / pharmaceuticals or pharmaceutical compositions can be diluted or increased using inert materials. These diluents may include carbohydrates, particularly mannitol, lactose, anhydrous lactose, cellulose, sucrose, modified dextran, and starch. Certain inorganic salts may also be used as fillers, including calcium triphosphate, magnesium carbonate, and sodium chloride. Some commercially available diluents include Fast-Flo®, Emdex®, STARCH 1500®, Emcompress®, and Avicel®.

[0120] Disintegrants can be included in formulations of therapeutic compounds / pharmaceuticals or pharmaceutical compositions to form solid dosage forms. Materials used as disintegrants include, but are not limited to, starch, including the commercially available starch-based disintegrant Explotab. Sodium glycolate starch, Amberlite®, sodium carboxymethyl cellulose, hyperbranched starch, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponges, and bentonite can also be used. Another form of disintegrant is an insoluble cation exchange resin. Powdered gums can be used as both disintegrants and binders, and these can include powdered gums such as agar, karaya gum, or astragalus gum. Alginic acid and its sodium salts can also be used as disintegrants.

[0121] Binders can be used to hold therapeutic agents together to form rigid tablets and include materials derived from natural products such as gum arabic, astragalus gum, starch, and gelatin. Other binders include methylcellulose (MC), ethylcellulose (EC), and carboxymethylcellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropyl methylcellulose (HPMC) can both be used in alcoholic solutions to granulate therapeutic agents.

[0122] Anti-friction agents may be included in formulations of therapeutic compounds / pharmaceuticals or pharmaceutical compositions to prevent adhesion during the compounding process. Lubricants may be used as a layer between the therapeutic agent and the mold wall, and these may include, but are not limited to: stearic acid, including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils, and waxes. Water-soluble lubricants such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol (PEG) of various molecular weights, and Carbowax may also be used. TM 4000 and 6000.

[0123] A flow aid may be added, which can improve the flowability of the drug during dispensing and facilitate rearrangement during compression. Flow aids may include starch, talc, pyrolytic silica, and hydrated aluminosilicates.

[0124] To aid in the dissolution of therapeutic compounds / pharmaceuticals or pharmaceutical compositions in an aqueous environment, surfactants may be added as wetting agents. Surfactants may include anionic detergents such as sodium lauryl sulfate, sodium dioctyl sulfosuccinate, and sodium dioctyl sulfonate. Cationic detergents may be used, and said cationic detergents may include benzalkonium chloride and benzyl chloride. Potential nonionic detergents that may be included as surfactants in formulations or pharmaceuticals include polidocanol 400, polyethylene glycol 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glyceryl monostearate, polysorbate 40, 60, 65, and 80, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. These surfactants may be present alone or as mixtures in varying proportions in the formulations, pharmaceuticals, or derivatives disclosed herein.

[0125] Orally applicable pharmaceutical compositions include push-fit capsules made of gelatin and soft-sealable capsules made of gelatin and plasticizers such as glycerin or sorbitol. Such push-fit capsules may contain an active ingredient mixed with a filler such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the therapeutic compound may be dissolved or suspended in a suitable liquid, such as fatty oil, liquid paraffin, or liquid polyethylene glycol. Additionally, a stabilizer may be added. Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be at doses suitable for such administration.

[0126] For buccal administration, the composition can be in the form of tablets or lozenges prepared in a conventional manner.

[0127] For topical application, therapeutic compounds / pharmaceuticals or pharmaceutical compositions can be formulated as solutions, gels, ointments, creams, suspensions, etc., as is well known in the art. Solutions, gels, ointments, creams, or suspensions can be applied topically. Compounds can also be formulated into rectal or vaginal compositions (such as suppositories or retention enemas) containing, for example, a conventional suppository base (such as cocoa butter or other glycerides).

[0128] For inhalation administration, the therapeutic compound / pharmaceutical agent or pharmaceutical composition used according to this application can be conveniently delivered from a pressurized package or nebulizer in the form of an aerosol spray using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas). In some embodiments, the formulation, drug, and / or therapeutic compound / pharmaceutical agent can be delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, such as a gas (e.g., carbon dioxide). Such methods include those described in U.S. Patent No. 6,468,798. In the case of pressurized aerosols, the dosage unit can be determined by providing a valve for delivering the amount of the dose. For example, capsules and cartridges (e.g., gelatin) for inhalers or blowpipes can be formulated into a powder mixture containing a therapeutic compound / pharmaceutical agent and a suitable powder matrix (e.g., lactose or starch). Alternatively, therapeutic compounds / pharmaceuticals or pharmaceutical compositions may be in powder form so that they can be prepared together with a suitable medium (e.g., sterile, pyrogen-free water) prior to use.

[0129] Nasal delivery of therapeutic compounds / pharmaceuticals or pharmaceutical compositions is also envisioned. Nasal delivery allows the therapeutic compound / pharmaceutical or pharmaceutical composition to be delivered directly into the bloodstream after administration to the nose, without the need for product deposition in the lungs. Formulations for nasal delivery include formulations containing dextran or cyclodextrin.

[0130] For nasal administration, a useful device is a small, rigid bottle to which a metered-dose nebulizer is attached. In some embodiments, the metered dose is delivered by inhaling a pharmaceutical composition (in solution form) into a volume-defined chamber having an orifice sized to atomize the aerosol formulation by forming a spray when the liquid within the chamber is compressed. The chamber is compressed to administer the therapeutic compound / agent or pharmaceutical composition. In a particular embodiment, the chamber is arranged as a piston. Such devices are commercially available.

[0131] Alternatively, a plastic squeeze bottle with an orifice or opening sized to atomize the aerosol formulation by forming a spray upon squeezing. The opening is typically located at the top of the bottle, and the top is usually tapered to partially align with the nasal passage for efficient application of the aerosol formulation. Preferably, the nasal inhaler will deliver a metered amount of the aerosol formulation to administer a measured dose of a therapeutic compound / pharmaceutical or pharmaceutical composition.

[0132] This paper also envisions pulmonary delivery of the compounds disclosed herein. Therapeutic compounds / pharmaceuticals or pharmaceutical compositions are delivered to the lungs of mammals upon inhalation and cross the pulmonary epithelial lining to reach the bloodstream. Other reports of inhaled molecules include Adjei et al., *Pharmaceutical Research*. Pharm Res )》 7:565-569 (1990); Adjei et al., International Journal of Pharmacy ( Int J Pharmaceutics Leuprorelin acetate; Braquet et al., Journal of Cardiovascular Pharmacology (63:135-144 (1990)); J Cardiovasc Pharmacol Endothelin-1; Hubbard et al., Annals of Internal Medicine (1989) 13(Supplement 5):143-146; Hubbard et al., Annals of Internal Medicine (1989) 13(Supplement 5):143-146; Annal Int Med )》 3:206-212 (1989) (α1-antitrypsin); Smith et al., 1989, Journal of Clinical Research ( J Clin Invest )》 84:1145-1146 (a-1-protease); Oswein et al., 1990, “Aerosolization of Proteins”, Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, (recombinant human growth hormone); Debs et al., 1988, Journal of Immunology ( J Immunol (Interferon-γ and tumor necrosis factor α) and Platz et al., U.S. Patent No. 5,284,656 (Granocyte colony-stimulating factor; incorporated by reference). Methods and compositions for pulmonary delivery of drugs to exert systemic effects are described in U.S. Patent No. 5,451,569 (incorporated by reference), issued September 19, 1995, by Wong et al.

[0133] It is envisioned that the practice of this technology will use mechanical devices designed for the transpulmonary delivery of therapeutic products, including but not limited to nebulizers, metered-dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.

[0134] Some specific examples of commercially available devices suitable for practicing this technology are the Ultravent manufactured by Mallinckrodt, Inc., St. Louis, Mo., Missouri. TM Inhalers; Acorn II® inhalers manufactured by Marquest Medical Products, Englewood, Colo.; Ventolin® metered-dose inhalers manufactured by Glaxo Inc., Research Triangle Park, North Carolina; and Spinhaler® powder inhalers manufactured by Fisons Corp., Bedford, Mass.

[0135] All such devices require the use of formulations suitable for dispensing therapeutic compounds / pharmaceuticals or pharmaceutical compositions. Typically, each formulation is specific to the type of device employed, and may involve the use of appropriate propellant materials in addition to the usual diluents, adjuvants, and / or carriers used in therapy. Furthermore, the use of liposomes, microcapsules, microspheres, nanoparticles, nanospheres, inclusion complexes, or other types of carriers is envisioned.

[0136] Formulations suitable for use with nebulizers (jet or ultrasonic) may, for example, contain a therapeutic compound / pharmaceutical agent or pharmaceutical composition dissolved in water at a concentration of about 0.01 mg to 50 mg of a bioactive compound per mL of solution. The formulation may also include buffers and, optionally, monosaccharides (e.g., for inhibitor stabilization and osmotic adjustment). Nebulizer formulations may also contain surfactants to reduce or prevent surface-induced aggregation of the therapeutic compounds / pharmaceutical agents or pharmaceutical compositions disclosed herein caused by solution atomization during aerosol formation.

[0137] Formulations used with metered-dose inhaler devices may typically comprise a fine powder containing a therapeutic compound / pharmaceutical or pharmaceutical composition disclosed herein, suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material used for this purpose, such as chlorofluorocarbons, chlorofluorocarbons, hydrofluorocarbons, or hydrocarbons, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soy lecithin. Oleic acid may also be used as a surfactant.

[0138] The formulation for dispensing from a powder inhaler device may comprise a finely divided dry powder containing a therapeutic compound / agent or pharmaceutical composition, and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol, in an amount that facilitates dispersion of the powder from the device, for example, 50% to 90% by weight of the formulation. The compound / therapeutic agent / pharmaceutical composition may advantageously be prepared in the form of microparticles or nanoparticles with an average particle size of less than 10 micrometers (μm), most preferably 0.5 to 5 μm, for most efficient delivery to deep lungs.

[0139] For ocular or intraocular indications, any suitable mode of delivery of therapeutic compounds / pharmaceuticals or pharmaceutical compositions to the eye or surrounding area may be used. Generally, for ophthalmic formulations, see Mitra (edited), Ophthalmic Drug Delivery Systems (…). Ophthalmic Drug Delivery Systems Marcel Dekker, Inc., New York, NY, (1993) and see also Havener, WH, Ocular Pharmacology ( Ocular Pharmacology , CV Mosby Co., St. Louis, (1983). Non-limiting examples of pharmaceutical compositions suitable for intraocular or periocular application include, but are not limited to, ocular inserts, microtablets, and topical formulations such as eye drops, ointments, and in-situ gels. In one embodiment, a contact lens is coated with a pharmaceutical composition comprising (or containing a pharmaceutical composition encapsulated therein). In some embodiments, a single dose may comprise 0.1 ng to 5000 μg, 1 ng to 500 μg, or 10 ng to 100 μg of a therapeutic compound / agent or pharmaceutical composition for application to the eye.

[0140] Eye drops may contain a sterile liquid formulation that can be applied directly to the eye. In some embodiments, the eye drops contain at least one therapeutic agent (and possibly several), and may further contain one or more preservatives. In some embodiments, the optimal pH of the eye drops is equal to the pH of the tear film and is about 7.4, and the pH may be within any range that is harmless to the subject's eyes. For eye drops, the therapeutic compound / agent may be present in the drop solution at about 0.1% to about 5% (w / v or v / v, depending on the physical properties of the active ingredient (i.e., solid or liquid)). In some embodiments, the therapeutic compound / agent may be present in the drop solution at about 1% to about 3% (w / v or v / v, as applicable). In some embodiments, the therapeutic compound / agent may be present in the drop solution at about 0.2% to about 1.5% (w / v or v / v, as applicable). In some embodiments, the therapeutic compound / agent may be present in the drop solution at about 0.1% to about 1.0% (w / v or v / v, as applicable).

[0141] In-situ gels are viscous liquids that, when influenced by external factors such as appropriate pH, temperature, pressure, and / or the presence of electrolytes, exhibit the ability to undergo a sol-gel transition. This property results in a slower rate of drug excretion from the ocular surface and an increased bioavailability of the active ingredient. Commonly used polymers in in-situ gel formulations include, but are not limited to, gellan gum, poloxamer, silicone-containing formulations, silica-based formulations, and cellulose acetate phthalate. In some embodiments, a therapeutic compound / pharmaceutical or pharmaceutical composition is formulated into an in-situ gel (as a formulation / pharmaceutical).

[0142] For topical ocular application, therapeutic compounds / pharmaceuticals or pharmaceutical compositions can be formulated as solutions, gels, ointments, creams, suspensions, etc., as is well known in the art. Ointments are semi-solid dosage forms for external use, such as for topical application to the eyes or skin. In some embodiments, ointments comprise a solid or semi-solid hydrocarbon matrix with a melting or softening point close to the core temperature of the human body. In some embodiments, ointments applied to the eyes break down into small droplets, which remain in the conjunctival sac for a longer period, thereby improving bioavailability.

[0143] Ocular inserts are solid or semi-solid dosage forms, avoiding the disadvantages of traditional ocular drug dosage forms. They are less susceptible to defense mechanisms (such as outflow through the nasolacrimal duct), can remain in the conjunctival sac for a longer period, and can be more stable than conventional dosage forms. They also offer advantages such as precise administration of one or more therapeutic compounds / agents or drug compositions, slow release of one or more therapeutic compounds / agents at a constant rate, and limitation of systemic absorption of one or more therapeutic compounds / agents. In some embodiments, the ocular insert comprises one or more therapeutic compounds / agents and one or more polymeric materials. The polymeric materials may include, but are not limited to, methylcellulose and its derivatives (e.g., hydroxypropyl methylcellulose (HPMC)), ethylcellulose, polyvinylpyrrolidone (PVP K-90), polyvinyl alcohol, chitosan, carboxymethyl chitosan, gelatin, and various mixtures of the above polymers. The ocular insert may contain silica. The ocular insert may contain liposomes, nanoparticles, or microparticles of a degradable or biodegradable polymer (as described in more detail below).

[0144] Microtablets are biodegradable solid drug dosage forms that transform into a gel upon application to the conjunctival sac, thereby prolonging the contact time between the active ingredient (i.e., the therapeutic compound / agent) and the ocular surface, which in turn improves the bioavailability of the therapeutic compound / agent. Advantages of microtablets include ease of application to the conjunctival sac, immunity to defense mechanisms such as tearing or drainage through the nasolacrimal duct, longer contact with the cornea due to the presence of mucosal adhesion polymers, and gradual release of the active ingredient from the formulation at the application site due to swelling of the outer carrier layer. Microtablets may contain one or more therapeutic compounds / agents and one or more polymers. Non-limiting examples of polymers suitable for use in microtablet formulations include cellulose derivatives such as hydroxypropyl methylcellulose (HPMC), hydroxyethyl cellulose (HEC), sodium carboxymethyl cellulose, ethyl cellulose, acrylates (e.g., polyacrylic acid and its cross-linked forms), carbopol® or carbomer, chitosan, and starch (e.g., drum-dried waxy corn starch). In some embodiments, microtablets further contain one or more excipients. Non-limiting examples of excipients include mannitol and magnesium stearate.

[0145] Ocular or intraocular preparations and medications may contain non-toxic excipients, such as antibacterial components that are generally harmless when used, for example, thimerosal, benzalkonium chloride, methylparaben and propylparaben, phenylchlorobenzyl bromide, benzyl alcohol or phenylethanol; buffering components, such as sodium chloride, sodium borate, sodium acetate, sodium citrate or gluconate buffer; and other conventional components, such as sorbitol monolaurate, triethanolamine, polyoxyethylene sorbitol monopalmitate, ethylenediaminetetraacetic acid (EDTA), etc.

[0146] In some embodiments, the viscosity of ocular formulations containing one or more therapeutic compounds / agents is increased to improve contact with the cornea and bioavailability in the eye. Viscosity can be increased by adding high molecular weight hydrophilic polymers that do not diffuse through biological membranes and form a three-dimensional network in water. Non-limiting examples of such polymers include polyvinyl alcohol, poloxamer, hyaluronic acid, carbomer and polysaccharides, cellulose derivatives, gellan gum, and xanthan gum.

[0147] In addition to the formulations described above, therapeutic compounds / pharmaceuticals or pharmaceutical compositions may also be formulated into reservoir formulations. Such long-acting formulations may be formulated with suitable polymeric or hydrophobic materials (e.g., emulsions in acceptable oils) or ion exchange resins, or formulated as sparingly soluble derivatives, such as sparingly soluble salts.

[0148] In some embodiments, a therapeutic compound / pharmaceutical agent or pharmaceutical composition is administered as a reservoir formulation, wherein the active therapeutic agent is encapsulated or disposed within silica-based microparticles. Such formulations may be controlled-release, delayed-release, or extended-release formulations (terms defined below). Such controlled-release, delayed-release, or extended-release formulations may comprise particles, such as microparticles or nanoparticles.

[0149] The pharmaceutical composition may also contain a suitable solid or gel phase carrier or excipient. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starch, cellulose derivatives, gelatin, silica / silicone, and polymers (such as polyethylene glycol).

[0150] Suitable liquid or solid pharmaceutical formulations (e.g., formulations or drugs) may be, for example, aqueous or saline solutions for inhalation; microencapsulated, embedded, coated on microscopic gold particles, contained in liposomes, atomized, aerosolized, granules for implantation in the skin, or granules dried on a sharp object for scraping into the skin. Pharmaceutical compositions / formulations may also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops, or sustained-release formulations of therapeutic compounds, in which excipients and additives and / or adjuvants (such as disintegrants, binders, coating agents, swelling agents, lubricants, flavoring agents, sweeteners, or solubilizers) are typically used as described above. The pharmaceutical compositions may be suitable for a variety of drug delivery systems. For a brief overview of drug delivery methods, see Langer R, *Science*. Science )》 249:1527-33 (1990).

[0151] Therapeutic compounds / pharmaceuticals or pharmaceutical compositions may be provided in particulate form. As used herein, "particulate" refers to nanoparticles or microparticles (or, in some cases, larger particles) that may consist wholly or partially of the therapeutic compound / pharmaceutical as described herein. The particles may contain the therapeutic compound / pharmaceutical within a coated core, said coating including, but not limited to, enteric coating. Therapeutic compounds / pharmaceuticals may also be dispersed throughout the particle. Therapeutic compounds / pharmaceuticals may also be adsorbed into the particle. The particles may have release kinetics of any order, including zero-order release, first-order release, second-order release, delayed release, sustained release, immediate release, and any combination thereof. In addition to any therapeutic compound / pharmaceutical, the particles may comprise any of those materials conventionally used in the pharmaceutical and medical fields, including, but not limited to, erosive, non-erosive, biodegradable, or non-biodegradable materials, or combinations thereof. The particles may be microcapsules containing the therapeutic compound / pharmaceutical in a solution or semi-solid state. The particles may virtually have any shape.

[0152] Both non-biodegradable and biodegradable polymeric materials can be used to manufacture particles for delivering therapeutic compounds / pharmaceuticals. These polymers can be natural or synthetic. The polymer can be selected based on the desired release time period. Bioadhesive polymers of particular interest include those by Sawhney HS et al. (1993), "Macromolecules ( Macromolecules Biodegradable hydrogels described in 26:581-7 of the Standard for the Prophecy of the Informatics of the People's Republic of China (SAPPRFT) are incorporated herein by reference. These include polyhyaluronic acid, casein, gelatin, gelatin protein, polyanhydride, polyacrylic acid, alginate, chitosan, polyethylene glycol (PEG), polyvinyl alcohol (PVA), poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), polylactic acid (PLA), poly(lactic-co-ethanol) acid (PLGA), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), and poly(ε-caprolactone), or mixtures of two or more of the foregoing. Biodegradable polymeric materials can be substantially pure single polymers or mixtures of two or more polymers, wherein said materials comprise mixtures of single monomers, block copolymers, or mixtures thereof.

[0153] Therapeutic compounds / pharmaceuticals or mixtures of two or more therapeutic compounds / pharmaceuticals can be formulated into a carrier system. The carrier can be a colloidal system. The carrier or colloidal system can be liposomes or phospholipid bilayers. In one embodiment, the therapeutic compound / pharmaceutical or mixture of two or more therapeutic compounds / pharmaceuticals can be encapsulated in liposomes while maintaining the integrity of the therapeutic compound / pharmaceutical. Those skilled in the art will understand that various methods exist for preparing liposomes. (See Lichtenberg et al., *Biochemical Analysis Methods*) Methods Biochem. Anal .)》, 33:337-462 (1988); Anselem et al., Liposome Technology ( Liposome Technology ), CRC Press (1993). Liposome formulations can delay clearance and increase cellular uptake (see Reddy, *Annals of Drug Therapy*). Ann. Pharmacother ( . )》, 34(7-8):915-923 (2000)). For example, therapeutic agents can also be loaded into particles prepared from pharmaceutically acceptable ingredients, including but not limited to soluble, insoluble, permeable, impermeable, biodegradable, or gastric-retention polymers or liposomes. Such particles include, but are not limited to, nanoparticles, biodegradable nanoparticles, microparticles, biodegradable microparticles, nanospheres, biodegradable nanospheres, microspheres, biodegradable microspheres, capsules, emulsions, liposomes, micelles, and viral vector systems.

[0154] The carrier can also be a polymer, such as a biodegradable, biocompatible polymer matrix. In one embodiment, a therapeutic compound / pharmaceutical or a mixture of two or more therapeutic compounds / pharmaceuticals can be embedded in a polymer matrix while maintaining the integrity of the composition. The polymer can be microparticles or nanoparticles encapsulating the therapeutic compound / pharmaceutical. The polymer can be natural, such as peptides, proteins, or polysaccharides, or synthetic, such as polyalphahydroxy acids. Examples include carriers made from, for example, collagen, fibronectin, elastin, cellulose acetate, cellulose nitrate, polysaccharides, fibroin, gelatin, and combinations thereof. In one embodiment, the polymer is polylactic acid (PLA), polylactic acid / glycolic acid (PLGA), or a mixture thereof. The polymer matrix can be prepared and separated in a variety of forms and sizes, including microspheres and nanospheres. Polymer formulations can prolong the duration of therapeutic effects. (See Reddy, Annals of Drug Therapy, 34(7-8):915-923 (2000)). Polymer formulations for human growth hormone (hGH) have been used in clinical trials. (See Kozarich and Rich, Chemical Biology) Chemical Biology )》, 2:548-552 (1998)).

[0155] Examples of sustained-release formulations of polymeric microspheres are described in the following: PCT Publication WO 99 / 15154 (Tracy et al.), U.S. Patents 5,674,534 and 5,716,644 (both by Zale et al.), PCT Publication WO 96 / 40073 (Zale et al.), and PCT Publication WO 00 / 38651 (Shah et al.). U.S. Patents 5,674,534 and 5,716,644, and PCT Publication WO 96 / 40073 describe polymer matrices containing erythropoietin particles stabilized with salt to prevent aggregation.

[0156] In some embodiments, nanoparticles or microparticles may be based on silica or silane (see, for example, WO2002 / 080977, entitled “Biodegradable carrier and method for preparation thereof”).

[0157] In some embodiments, a therapeutic compound / pharmaceutical agent or a mixture of two or more therapeutic compounds / pharmaceutical agents is prepared together with a carrier (such as a controlled-release formulation, including implants and microencapsulated delivery systems) that protects the therapeutic compound / pharmaceutical agent or other therapeutic agent or mixture thereof from rapid elimination from the body. Biodegradable polymers and biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such formulations can be prepared using known techniques. The materials can also be commercially available, for example, from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (including liposomes targeting monoclonal antibodies against cell-specific antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0158] Therapeutic compounds / pharmaceuticals or mixtures of two or more therapeutic compounds / pharmaceuticals may be included in controlled-release systems. The term "controlled release" is intended to refer to any formulation containing a drug in which the manner and profile of drug release from the formulation are controlled. This refers to both immediate-release and non-immediate-release formulations, with non-immediate-release formulations including, but not limited to, sustained-release and delayed-release formulations. The term "sustained-release" (also known as "extended-release") is used in its conventional sense to refer to a drug formulation that provides a gradual release of the drug over an extended period of time and preferably, but not necessarily, results in a substantially constant blood level of the drug over the extended period of time. The term "delayed-release" is used in its conventional sense to refer to a drug formulation in which there is a time delay between administration of the formulation and release of the drug from it, thereby making it available to a subject. "Delayed-release" may or may not involve a gradual release of the drug over an extended period of time and therefore may or may not be "continuous release."

[0159] The use of long-term controlled-release or sustained-release implants or depot formulations may be particularly suitable for the treatment of chronic conditions. The terms "implant" and "depot formulation" are intended to include a single composition (such as a mesh) or a composition comprising multiple components (e.g., a fibrous mesh composed of several individual mesh materials) or multiple individual compositions, wherein said multiple compositions remain localized and provide long-term sustained release of an active pharmaceutical ingredient arising from an aggregate of one or more compositions. As used herein, "long-term" release means that the implant or depot formulation is constructed and arranged to deliver a therapeutic or preventative level of active pharmaceutical ingredient for at least 2 days. In some embodiments, the implant or depot formulation is constructed and arranged to deliver a therapeutic or preventative level of active pharmaceutical ingredient for at least 7 days. In some embodiments, the implant or depot formulation is constructed and arranged to deliver a therapeutic or preventative level of active pharmaceutical ingredient for at least 14 days. In some embodiments, the implant or depot formulation is constructed and arranged to deliver a therapeutic or preventative level of active pharmaceutical ingredient for at least 30 days. In some embodiments, the implant or depot preparation is configured and arranged to deliver a therapeutic or preventative level of active pharmaceutical ingredient for at least 60 days. In some embodiments, the implant or depot preparation is configured and arranged to deliver a therapeutic or preventative level of active pharmaceutical ingredient for at least 90 days. In some embodiments, the implant or depot preparation is configured and arranged to deliver a therapeutic or preventative level of active pharmaceutical ingredient for at least 180 days. In some embodiments, the implant or depot preparation is configured and arranged to deliver a therapeutic or preventative level of active pharmaceutical ingredient for at least one year. In some embodiments, the implant or depot preparation is configured and arranged to deliver a therapeutic or preventative level of active pharmaceutical ingredient for 15-30 days. In some embodiments, the implant or depot preparation is configured and arranged to deliver a therapeutic or preventative level of active pharmaceutical ingredient for 30-60 days. In some embodiments, the implant or depot preparation is configured and arranged to deliver a therapeutic or preventative level of active pharmaceutical ingredient for 60-90 days. In some embodiments, the implant or reservoir preparation is configured and arranged to deliver a therapeutic or prophylactic level of active pharmaceutical ingredient for 90-120 days. In some embodiments, the implant or reservoir preparation is configured and arranged to deliver a therapeutic or prophylactic level of active pharmaceutical ingredient for 120-180 days. In some embodiments, the implant or reservoir preparation is configured and arranged to deliver a therapeutic or prophylactic level of active pharmaceutical ingredient for up to one year. In some embodiments, long-release implants or reservoir preparations are well known to those skilled in the art and include some of the release systems described above. In some embodiments, such implants or reservoir preparations can be administered surgically. In some embodiments, such implants or reservoir preparations can be administered topically or by injection.

[0160] III. Preparations and medications: Compounds 1 to 4, or mixtures of any two or more of compounds 1 to 4, may be used alone or in combination with one or more other therapeutic agents to address the needs of subjects with Leydig syndrome. For administration to subjects in need, compounds 1 to 4, or mixtures of any two or more of compounds 1 to 4, and / or other therapeutic agents will typically need to be formulated for a suitable route of administration. For example, if compounds 1 to 4, or mixtures of any two or more of compounds 1 to 4, and / or other therapeutic agents are to be administered to a subject by injection, they will typically be formulated as an injectable liquid or liquid suspension. This can be achieved, for example, by dissolving or suspending the therapeutic agent in a suitable diluent, adjuvant, excipient, mediator, or pharmaceutically acceptable carrier, as previously described herein (see the section titled: Pharmaceutical Compositions, Routes of Administration, and Dosing). In some embodiments, the diluent, adjuvant, excipient, mediator, or pharmaceutically acceptable carrier may be water, saline, or a buffered aqueous solution. The foregoing has discussed suitable methods, reagents, and compositions for formulating compounds 1 to 4 or mixtures of any two or more of compounds 1 to 4 and / or other therapeutic agents into suitable medicaments.

[0161] Similarly, if compounds 1 to 4, or mixtures of any two or more compounds 1 to 4, and / or other therapeutic agents can be administered to a subject orally, the selected active ingredient may be formulated into pills, tablets, capsules, or other media for such administration as discussed in the section entitled “Pharmaceutical Compositions, Routes of Administration, and Dosing” above, or other administrations as known to those skilled in the art. Suitable methods, reagents, and compositions for formulating compounds 1 to 4, or mixtures of any two or more compounds 1 to 4, and / or other therapeutic agents into suitable orally administerable medicaments have been discussed above.

[0162] Similarly, compounds 1 to 4, or mixtures of any two or more compounds 1 to 4, and / or other therapeutic agents, may be formulated for ocular, buccal, topical, nasal, or any other mode of administration previously discussed herein or known to those skilled in the art. Suitable methods, reagents, and compositions for formulating compounds 1 to 4, or mixtures of any two or more compounds 1 to 4, and / or other therapeutic agents, into suitable medicaments for ocular, buccal, topical, or nasal application have been discussed above.

[0163] In short, any formulation described in the section titled "Pharmaceutical Compositions, Routes of Administration, and Dosing" above (which may also be referred to as a drug or composition when formulated for administration to a subject suffering from a disease or symptom requiring medical care) can be used to produce a composition (i.e., a formulation or a drug) suitable for administration to a subject in need. Therefore, in some embodiments, this application relates to compositions, formulations, and drugs suitable for administration to a subject suffering from or believed to suffer from Lechner's syndrome.

[0164] In some embodiments, the composition, formulation, or drug is administered subcutaneously. In some embodiments, the composition, formulation, or drug is administered orally, topically, intranasally, systemically, intravenously, intraperitoneally, intradermally, intraocularly, intrathecally, intraocularly, intrathecally, intraventricularly, via iontophoresis, transmucosally, intravitreal, or intramuscularly.

[0165] In some embodiments, compounds 1 to 4 and / or other therapeutic agents may be administered in the form of formulations prepared using pharmaceutically acceptable salts. In some embodiments, compounds 1 to 4 and / or other therapeutic agents may be administered in the form of formulations prepared using hydrates. In some embodiments, compounds 1 to 4 and / or other therapeutic agents may be administered in the form of formulations prepared using solvated forms. In some embodiments, compounds 1 to 4 and / or other therapeutic agents may be administered in the form of formulations prepared using tautomeric forms. In some embodiments, compounds 1 to 4 and / or other therapeutic agents may be administered in the form of formulations prepared using stereoisomers of compounds 1 to 4 and / or other therapeutic agents.

[0166] IV. Treatment methods and related uses of the disclosed compounds in addressing Leishman's syndrome.

[0167] In one aspect, this disclosure provides a treatment for, prevention of, reduction of, inhibition of or delay of the onset of Lechner's syndrome or related signs or symptoms in a subject in need, comprising administering to the subject a therapeutically effective amount of compound 1, compound 2, compound 3, compound 4, or a mixture of any two or more of compounds 1 to 4, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, tautomer, hydrate and / or solvate thereof, wherein compounds 1 to 4 have the following structures: .

[0168] In some embodiments of the method, signs or symptoms of Lechner's syndrome include dysphagia, dyspnea, dysphagia, hypotonia, lactic acidosis, developmental delay, cardiomyopathy, seizures, ataxia, dysarthria, dystonia, paralysis, respiratory and renal impairment, color blindness and / or vision loss. In some embodiments of the method, the subject is diagnosed with Lechner's syndrome. In some embodiments of the method, the subject is a human being.

[0169] In some embodiments of the method, the compound (i.e., compound 1, compound 2, compound 3, compound 4, or any two or more compounds 1 to 4, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, tautomer, hydrate, and / or solvate thereof) is administered orally, topically, intranasally, systemically, intravenously, subcutaneously, intraperitoneally, intradermally, intraocularly, intraocularly, intrathecally, intraventricularly, via iontophoresis, transmucosally, intravitreally, or intramuscularly. In some embodiments of the method, the compound is administered daily for six weeks or longer.

[0170] In some embodiments of the method, administration of a compound (i.e., compound 1, compound 2, compound 3, compound 4, or any two or more of compounds 1 to 4, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, tautomer, hydrate, and / or solvate thereof) to a subject treats, prevents, alleviates, inhibits, or delays the onset of cardiomyopathy. In some embodiments of the method, the cardiomyopathy is hypertrophic cardiomyopathy, left ventricular hypertrophy, dilated cardiomyopathy, pericardial effusion, and / or arrhythmia / conduction abnormality. In some embodiments of the method, the cardiomyopathy is hypertrophic cardiomyopathy.

[0171] In some embodiments of the method, administration of a compound (i.e., compound 1, compound 2, compound 3, compound 4, or any two or more of compounds 1 to 4, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, tautomer, hydrate, and / or solvate thereof) to a subject reduces lactate levels in the subject's blood, urine, or cerebrospinal fluid (CSF).

[0172] In some embodiments of the method, administering a compound (i.e., compound 1, compound 2, compound 3, compound 4, or any two or more of compounds 1 to 4, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, tautomer, hydrate, and / or solvate thereof) to a subject to treat, prevent, alleviate, inhibit, or delay the onset of multi-organ and / or central nervous system (CNS) injury in the subject, said multi-organ and / or CNS injury including lesions of the brainstem, basal ganglia, and spinal cord.

[0173] In some embodiments of the method, administering a compound (i.e., compound 1, compound 2, compound 3, compound 4, or any two or more of compounds 1 to 4, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, tautomer, hydrate, and / or solvate thereof) to a subject to treat, prevent, alleviate, inhibit, or delay the onset of color blindness and / or vision loss.

[0174] In some embodiments of the method, administering a compound (i.e., compound 1, compound 2, compound 3, compound 4, or any two or more of compounds 1 to 4, or their pharmaceutically acceptable salts, stereoisomers, mixtures of stereoisomers, tautomers, hydrates, and / or solvates) to a subject prolongs the subject's lifespan.

[0175] In some embodiments of the method, administering a compound (i.e., compound 1, compound 2, compound 3, compound 4, or any two or more of compounds 1 to 4, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, tautomer, hydrate, and / or solvate) to a subject treats, prevents, alleviates, inhibits, or delays seizures, ataxia, dysarthria, dystonia, and / or paralysis.

[0176] In another aspect, this disclosure provides a composition, medicament, or formulation comprising any one of compounds 1 to 4 or a mixture of any two or more compounds 1 to 4, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, tautomer, hydrate, and / or solvate thereof, for the treatment, prevention, relief, inhibition, or delay of the onset of Leishman's syndrome or related signs or symptoms in a subject of need, wherein compounds 1 to 4 have the following structure: .

[0177] In some embodiments of the compositions, drugs, or formulations, signs or symptoms of Lechner's syndrome include dysphagia, dyspnea, dysphagia, hypotonia, lactic acidosis, developmental delay, cardiomyopathy, seizures, ataxia, dysarthria, dystonia, paralysis, respiratory and renal impairment, color blindness, and / or vision loss. In some embodiments of the compositions, drugs, or formulations, the subject is diagnosed with Lechner's syndrome. In some embodiments of the compositions, drugs, or formulations, the subject is a human being.

[0178] In some embodiments, a composition, drug, or formulation comprising a compound (i.e., compound 1, compound 2, compound 3, compound 4, or any two or more of compounds 1 to 4, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, tautomer, hydrate, and / or solvate thereof) is administered orally, topically, intranasally, systemically, intravenously, subcutaneously, intraperitoneally, intradermally, intraocularly, intraocularly, intrathecally, intraventricularly, via iontophoresis, transmucosal, intravitreal, or intramuscularly. In some embodiments, the composition, drug, or formulation comprising the compound is administered daily for six weeks or longer.

[0179] In some embodiments, a composition, drug, or formulation comprising a compound (i.e., compound 1, compound 2, compound 3, compound 4, or any two or more compounds of compounds 1 to 4, or their pharmaceutically acceptable salts, stereoisomers, mixtures of stereoisomers, tautomers, hydrates, and / or solvates) is administered to a subject to treat, prevent, alleviate, inhibit, or delay the onset of cardiomyopathy in the subject. In some embodiments, cardiomyopathy is hypertrophic cardiomyopathy, left ventricular hypertrophy, dilated cardiomyopathy, pericardial effusion, and / or arrhythmia / conduction abnormality. In some embodiments, cardiomyopathy is hypertrophic cardiomyopathy.

[0180] In some embodiments, administering to a subject a composition, drug, or formulation comprising a compound (i.e., compound 1, compound 2, compound 3, compound 4, or a mixture of any two or more of compounds 1 to 4, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, tautomer, hydrate, and / or solvate thereof) reduces lactate levels in the subject's blood, urine, or cerebrospinal fluid (CSF).

[0181] In some embodiments, a composition, drug, or formulation comprising a compound (i.e., compound 1, compound 2, compound 3, compound 4, or any two or more of compounds 1 to 4, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, tautomer, hydrate, and / or solvate thereof) is administered to a subject to treat, prevent, alleviate, inhibit, or delay the onset of multi-organ and / or central nervous system (CNS) injury in the subject, said multi-organ and / or CNS injury including lesions of the brainstem, basal ganglia, and spinal cord.

[0182] In some embodiments, a composition, drug, or formulation comprising a compound (i.e., compound 1, compound 2, compound 3, compound 4, or a mixture of any two or more of compounds 1 to 4, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, tautomer, hydrate, and / or solvate thereof) is administered to a subject to treat, prevent, alleviate, inhibit, or delay the onset of color blindness and / or vision loss in the subject.

[0183] In some embodiments, administering to a subject a composition, drug, or formulation comprising a compound (i.e., compound 1, compound 2, compound 3, compound 4, or a mixture of any two or more of compounds 1 to 4, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, tautomer, hydrate, and / or solvate thereof) prolongs the subject's life.

[0184] In some embodiments, a composition, drug, or formulation comprising a compound (i.e., compound 1, compound 2, compound 3, compound 4, or a mixture of any two or more of compounds 1 to 4, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, tautomer, hydrate, and / or solvate thereof) is administered to a subject to treat, prevent, reduce, suppress, or delay seizures of the subject’s epileptic seizures, ataxia, dysarthria, dystonia, and / or paralysis.

[0185] On the other hand, this disclosure provides the use of compounds 1 to 4, or mixtures of any two or more of compounds 1 to 4, or pharmaceutically acceptable salts, stereoisomers, mixtures of stereoisomers, tautomers, hydrates, and / or solvates thereof, in the preparation of compositions, formulations, or medicaments comprising any one of compounds 1 to 4, or mixtures of any two or more of compounds 1 to 4, or pharmaceutically acceptable salts, stereoisomers, mixtures of stereoisomers, tautomers, hydrates, and / or solvates thereof, for the treatment, prevention, relief, inhibition, or delay of the onset of Leishman's syndrome or related signs or symptoms in a subject of need, wherein compounds 1 to 4 have the following structure: .

[0186] In some embodiments of the described use, signs or symptoms of Lechner's syndrome include dysphagia, dyspnea, dysphagia, hypotonia, lactic acidosis, developmental delay, cardiomyopathy, seizures, ataxia, dysarthria, dystonia, paralysis, respiratory and renal impairment, color blindness and / or visual loss. In some embodiments of the described use, the subject is diagnosed with Lechner's syndrome. In some embodiments of the described use, the subject is a human being.

[0187] In some embodiments of the described use, the compound (i.e., compound 1, compound 2, compound 3, compound 4, or any two or more of compounds 1 to 4, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, tautomer, hydrate, and / or solvate thereof, including compositions, formulations, or pharmaceuticals derived therefrom) is administered orally, topically, intranasally, systemically, intravenously, subcutaneously, intraperitoneally, intradermally, intraocularly, intraocularly, intrathecally, intrathecally, intravaginally, via iontophoresis, transmucosally, intravitreal, or intramuscularly. In some embodiments of the described use, the compound is administered daily for six weeks or longer.

[0188] In some embodiments of the described use, the composition, formulation, or drug is administered to a subject to treat, prevent, alleviate, inhibit, or delay the onset of cardiomyopathy in the subject. In some embodiments of the described use, the cardiomyopathy is hypertrophic cardiomyopathy, left ventricular hypertrophy, dilated cardiomyopathy, pericardial effusion, and / or arrhythmia / conduction abnormality. In some embodiments of the described use, the cardiomyopathy is hypertrophic cardiomyopathy.

[0189] In some embodiments of the described use, administration of the composition, formulation, or drug to a subject reduces lactate levels in the subject's blood, urine, or cerebrospinal fluid (CSF).

[0190] In some embodiments of the described use, the composition, formulation, or drug is administered to a subject to treat, prevent, reduce, inhibit, or delay the onset of multi-organ and / or central nervous system (CNS) injury, including lesions of the brainstem, basal ganglia, and spinal cord.

[0191] In some embodiments of the described use, the composition, formulation, or drug is administered to a subject to treat, prevent, alleviate, suppress, or delay the onset of color blindness and / or vision loss in the subject.

[0192] In some embodiments of the described use, administering the composition, formulation, or drug to a subject prolongs the subject's lifespan.

[0193] In some embodiments, the composition, drug, or formulation is administered to a subject to treat, prevent, reduce, suppress, or delay seizures of the subject’s epileptic seizures, ataxia, dysarthria, dystonia, and / or paralysis.

[0194] Example

[0195] The following examples further illustrate the technology, and these examples should not be construed as limiting it in any way.

[0196] Example 1: In vitro assay

[0197] I. Materials and methods

[0198] Cell lines: Fibroblasts were obtained from patients at the Coriell Institute for Medical Research (Camden, New Jersey, USA). GM03672 is an untransformed fibroblast line from a patient with Lechner's syndrome carrying an unknown mutation. GM08402 is an untransformed fibroblast line from a seemingly healthy individual. Cells were cultured at 37°C in Eagle's Minimal Basis Medium (MEM, Corning) supplemented with Earl's salts and non-essential amino acids, 15% qualified fetal bovine serum, and 1% penicillin / streptomycin (100 U / 100 g) to produce "complete MEM".

[0199] Mitochondrial isolation: For mitochondrial isolation, brains of C57BL / 6 mice supplied by Jackson Laboratories, Inc. (Maine, USA) were dissected at a local animal facility (Explora, Massachusetts, USA). The brains were collected on ice in ice-cold mitochondrial isolation buffer (225 mM mannitol, 75 mM sucrose, 5 mM HEPES, 1 mM EGTA, pH 7.4). In a Piper glass dish, the brains were mixed with a few drops of buffer and cut into small, paste-like pieces with scissors. Mitochondrial isolation buffer was added at a ratio of 1:10–1:20 (w / v), and the tissue paste was transferred to a glass Dunns tissue homogenizer and homogenized further for 10–15 strokes. The paste was centrifuged at 1,000 xg for 10 minutes to form clumps and remove cell debris, etc. The supernatant was transferred to a 2.0 mL tube and centrifuged at 10,000 xg. Resuspend the mitochondrial clumps in the desired amount of miR05 buffer (Oroboros, Innsbruck, Austria) and add directly to Oroboros oxygraph-2K (Oroboros, Innsbruck, Austria) for experiments, or incubate on ice for up to 4 hours.

[0200] High-resolution breath measurement (HRR): All high-resolution breath measurement experiments were performed using the Oroboros O2k-fluorescence breath meter (Oroboros GmbH, Innsbruck, Austria). The Oroboros O2k-fluorescence breath meter was calibrated daily using the air calibration protocol on DatLab 7.4 (Oroboros GmbH, Innsbruck, Austria). The fluorescence signal was calibrated using the AmpR calibration protocol from DatLab 7.4 before each experiment requiring the use of the fluorescence module. Preliminary data analysis, including label setup, was performed using DatLab 7.4.

[0201] Complex I-bypass assay ((CI)-bypass assay): O2 consumption was assessed using HRR measurements to evaluate the activity of the I-bypass complex. Briefly, Riesler (GM03672) fibroblasts were harvested with trypsin, centrifuged at 300 xg to form clumps, and resuspended in pre-warmed intact MEM. Cells were added to each chamber via buffer replacement at a concentration of 1 million to 2 million cells per mL. The chambers were sealed, and O2 consumption measurements began. The negative slope signal, the derivative of O2 consumption, stabilized within minutes. O2 consumption was measured continuously using a Hamilton syringe, with glutamate (10 mM final concentration) and malate (0.1 mM final concentration) added to the sealed chambers through the central capillary of the stopper. The negative slope signal was allowed to stabilize for several minutes, and rotenone was added at a final concentration of 0.5 μM. One of compounds 1 to 4 or vatiquione was added at progressively increasing concentrations, allowing the negative slope signal to stabilize for several minutes between titrations. A marker was set at the signal peak between titrations. Values ​​were output and analyzed using a GraphPad Prism 9. Data for vatiquitone were not presented because no CI bypass activity was observed in the concentration range used for compounds 1 to 4 (i.e., titrations between 125 nM and 8 μM). Cellular responsiveness was confirmed using idebenone.

[0202] The structure of vatiquidon for reference purposes: .

[0203] Reverse electron transfer measurement (RET measurement): Reverse electron transfer (RET) was measured using HRR with Oroboros O2k and DatLab 7.4. To configure the Amp channel, the light or "fluorescence intensity" was set to 1000, the signal amplification or "fluorescence sensor gain" to 500, and the illumination in the chamber was turned off. To calibrate the fluorescence sensor, the instrument protocol "AmR Calibration" was used. In short, the chambers were loaded with miR05 buffer and the following reagents were added at the listed final concentrations: 15 μM DTPA, 5 U / mL superoxide dismutase (SOD), 1 U / mL horseradish peroxidase (HRP), and 10 μM Amplex Ultra Red. A three-point calibration curve was obtained by two 5 μL injections of 40 μM H2O2 before the experiment, and each chamber was calibrated using this curve. Mitochondria were suspended in preheated whole miR05 buffer at a concentration of approximately 1,500 μg per mL. 100 μL of the mitochondrial suspension was added to each chamber using a Hamilton syringe through the central capillary of the chamber stopper. Regular respiration was established for several minutes. Add sufficient amounts of one of compounds 1 through 4 from the stock vial to produce a final concentration of 1 μM or 2 μM, as indicated. The “no treatment” control did not receive any of compounds 1 through 4. Previous studies have shown that the drug mediator has no effect on mitochondrial respiration. Before adding succinate (10 mM final concentration), allow the negative slope signal to stabilize for several minutes. Succinate is a substrate of complex II, which generates reactive oxygen species (ROS) through the reverse transfer of electrons to complex I. After adding succinate (10 mM final concentration), allow the negative slope signal to stabilize for several minutes. Mark the trajectory of the negative slope of H2O2 as needed to determine H2O2 production (a form of ROS). Export the values ​​to GraphPad Prism 9 (GraphPad Software, Boston, Massachusetts) for analysis.

[0204] RSL3 cell protection assay (cell protection assay): To initiate the RSL3 cell protection assay, healthy fibroblasts (GM08402) and Lechner's syndrome fibroblasts (GM03672) were resuspended in intact MEM. Cells were seeded at 10,000 cells per well in clear, tissue-culture-treated 96-well plates and incubated overnight at 37°C. After incubation, the supernatant was manually aspirated, taking care not to damage the cells. Healthy fibroblasts were then inoculated with 1000 nM (1... S ,3 R )-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1-[4-(methoxycarbonyl)phenyl]-1 H -pyrido[3,4- bMethyl indole-3-carboxylate (RSL3) and compounds 1 to 4, or vatiquidon, in titration ranges from 10 nM to 1000 nM (Cayman Chemical, Ann Arbor, Michigan), were co-treated. Riesler fibroblasts were co-treated in a final volume of 100 μL with 375 nM or 700 nM RSL3 and one of compounds 1 to 4, or vatiquidon, in titration ranges from 10 nM to 1000 nM. Cells were incubated at 37°C for 4 hours. Cell viability was measured using the CellTiter-Glo luminescent cell viability kit (Promega) according to the manufacturer's instructions. The luminescent units were quantified using GEN5 software on a BioTek SYNERGY neo2 reader (LUM filter).

[0205] 15(S)-HETE ELISA after RSL3 treatment (HETE ELISA / RSL3 assay): Samples for the 15(S)-HETE ELISA were generated using the RSL3 cell protection assay described above. Healthy fibroblasts or Rijksbauer fibroblasts were resuspended in assay medium and seeded at 200,000 cells per well in clear, tissue-culture-treated 24-well plates. Cells were incubated at 37°C for 24 hours. After incubation, the supernatant was aspirated from each well. Fibroblasts were co-treated with 700 nM RSL3 and 1 μM of one of compounds 1 to 4 or vatiquidon in a final volume of 0.4 mL and incubated at 37°C for 24 hours. The concentration of 15(S)-HETE was measured in the supernatant using the Abcam 15(S) HETE-ELISA kit (catalog #: ab133035) according to the manufacturer's instructions. The absorbance at 405 nM was measured using GEN5 software on a BioTek SYNERGY neo2 plate reader.

[0206] Erasin Cell Protection Assay (Erastin Assay)

[0207] In short, healthy fibroblasts (GM08402) or Riesler fibroblasts (GM03672) were resuspended in assay medium and seeded at 10,000 cells per well in clear, tissue-culture-treated 96-well plates and incubated overnight at 37°C. After incubation, the supernatant was manually aspirated from the wells, taking care not to damage the cells. The fibroblasts were co-treated in a final volume of 100 μL with 2 μM ellastatin (Millipore Corp., catalog #), 250 μM ferric ammonium citrate (FAC), 50 μM ascorbic acid (AA), and each of compounds 1 to 4, or vatiquidon, in titrations ranging from 10 nM to 1000 nM. After an additional 24 hours of incubation at 37°C, cell viability was measured using the CellTiter-Glo luminescent cell viability kit (Promega) according to the manufacturer's instructions. The light-emitting units were quantized using GEN5 software on the BioTek SYNERGY neo2 reader (LUM filter).

[0208] 15(S)-HETE ELISA after ellastatin treatment (HETE ELISA / ellastatin assay): Samples for the 15(S)-HETE ELISA were generated using the ellastatin assay described above. Healthy fibroblasts or Rijksbauer fibroblasts were resuspended in assay medium and seeded at 200,000 cells / well in clear, tissue-culture-treated 24-well plates and incubated at 37°C for 24 hours. The supernatant was aspirated from each well. Fibroblasts were co-treated with 1 μM ellastatin (Millipore, catalog #), 250 μM ferric ammonium citrate (FAC), 50 μM ascorbic acid (AA), and 1 μM of one of compounds 1 to 4 in a final volume of 0.4 mL and incubated at 37°C for 24 hours. The concentration of 15(S)-HETE in the supernatant was measured using the Abogen 15(S)-HETE-ELISA kit (catalog #: ab133035). The absorbance at 405 nM was measured using GEN5 software on a BioTek SYNERGY neo2 plate reader.

[0209] Statistical analysis

[0210] Statistical analysis was performed using GraphPad Prism 9. Nonlinear sigmoid 4P fitting was used to fit the I-bypass data of the complex. RET determination data were analyzed using two-way ANOVA with a p-value of 0.05.

[0211] II. result: A. (CI-bypass assay) The Complex 1 bypass assay (CI-Borrow assay) assesses the ability of a compound to donate electrons to the electron transport chain (ETC) when Complex 1 is impaired or otherwise deficient. Normally, electrons are fed into the ETC via Complex 1. In cases of Complex 1 impairment, electrons must enter the ETC downstream of Complex 1 for oxidative phosphorylation (OXPHOS) to occur. Complex 1 consists of 46 subunits encoded by mitochondrial and nuclear genes. Due to its large size, mutations in Complex 1 are a common cause of ETC dysfunction in mitochondrial diseases. In fact, mutations in the genes encoding the subunits of Complex 1 are a significant cause of Lechner's syndrome and Lechner-like syndromes.

[0212] Using this assay, the ability of compounds 1 through 4 and vatiquidon to bypass complex I, which inhibits rotenone, was determined. Rotenone is a widely used inhibitor of complex I and blocks the transfer of electrons from the iron-sulfur complex on complex I to ubiquinone. Therefore, when rotenone was introduced into cells undergoing normal respiration, a decrease in oxygen consumption was observed, indicating disruption of OXPHOS. The ability of compounds 1 through 4 and vatiquidon to “bypass” complex I and restore OXPHOS was measured by increasing the oxygen consumption rate after adding the compounds to the chamber.

[0213] In performing this assay, fibroblasts were added to each chamber of the Oroboros Oxygraph-2K (“O2k”) in each case. O2k produces two trajectories: 1) total oxygen consumption over a period of time, and 2) the rate of oxygen consumption over a period of time (negative slope). Baseline respiration was established for several minutes by stabilizing the negative slope. Rotenone was introduced into each chamber at a final concentration of 0.5 μM. The addition of rotenone was expected to significantly reduce OXPHOS, as measured by the reduction in the rate of oxygen consumption. The test preparation (i.e., in this case, one of compounds 1 to 4 or vatiquidone) was then added to the chamber. If oxygen consumption recovered after the addition of the test preparation, it was assumed that the test preparation allowed OXPHOS to continue bypassing through complex I, which was still inhibited by rotenone.

[0214] refer to Figure 1This study demonstrates the ability of compound 1 to “bypass” rotenone inhibition by complex I. The graph represents a set of six experiments (n=6) in which compound 1 was titrated into the chambers of rotenone-inhibited Ridley fibroblasts. The points on the graph correspond to “markers” made on the negative slope trajectory produced by O2k. Markers are set on stable or “flat” regions of the trajectory after the addition of a given treatment agent (such as rotenone or a defined amount of the test compound). Compound 1 restores oxygen consumption in a dose-dependent manner. Data from the six runs (i.e., n=6) were fitted using a GraphPad Prism 9 with a four-parameter sigmoid curve. The EC50 value of compound 1 in the assay was determined to be approximately 13.7 μM.

[0215] Similarly, refer to Figure 2 This demonstrated the ability of compound 2 to "bypass" the inhibition of rotenone by complex I. The assays were performed and data collected as described above, except that each assay was run three times (i.e., n=3). The EC50 value of compound 2 in the assays was determined to be approximately 4.09 μM.

[0216] refer to Figure 3 This demonstrated the ability of compound 3 to "bypass" the rotenone-inhibiting complex I. The assays were performed and data collected as described above, except that the individual runs were performed four times (i.e., n=4). The EC50 value of compound 3 in the assays was determined to be approximately 3.41 μM.

[0217] refer to Figure 4 This demonstrated the ability of compound 4 to "bypass" the inhibition of rotenone by complex I. The assays were performed and data collected as described above, except that each assay was run twice (i.e., n=2). The EC50 value of compound 4 in the assays was determined to be approximately 8.80 μM.

[0218] At concentrations ranging from 125 nM to 8 μM, vatiquidon was inactive in the complex I bypass assay.

[0219] B. RET measurement

[0220] Reverse electron transfer assays are a common measurement in the HRR for the generation of reactive oxygen species (ROS). In this case, isolated mouse brain mitochondria are used for the assay. In the absence of a complex I inhibitor or substrate, the introduction of succinate as a substrate of complex II leads to a reverse electron transfer to complex I. The reverse electron transfer produces ROS, which is measured by HRR using O2k.

[0221] In the following experiments, RET measurements were used to determine whether compounds 1 through 4 reduced ROS production. ROS reduction may occur through several mechanisms, which require further experimental elucidation. A reduction in H₂O₂ production (which represents ROS) indicates that the test compound reduced ROS generated by electron backtransfer. This reduction in ROS may indicate that the test compound acts as a scavenger or enhances electron flow through the electron transport chain (ETC).

[0222] refer to Figure 5 Treatment of isolated mitochondria with 2 μM compound 3 before the addition of succinic acid in the RET assay resulted in a statistically significant reduction in H2O2 production.

[0223] refer to Figure 6 Treatment of isolated mitochondria with 1 μM or 2 μM compound 4 before the addition of succinic acid in RET assays resulted in a statistically significant reduction in H2O2 production.

[0224] Compounds 1 and 2 did not produce results that could be interpreted in this assay. Valtiquidone was not tested in the RET assay.

[0225] C. Cell protection assay

[0226] RSL3 is an inhibitor of glutathione peroxidase 4 (GPX4), which reduces lipid peroxides. RSL3 inhibition of GPX4 induces ferroptosis through iron-dependent accumulation of lipid ROS. In this assay, healthy fibroblasts or Rieger's fibroblasts were treated with RSL3 for 4 hours to induce ferroptosis, or left untreated. Endogenous ATP levels were measured as an indicator of cell viability using a commercially available kit (CellTiter-Glo® 2.0 Cell Viability Assay (Promag)).

[0227] In the field of mitochondrial research, ferroptosis and 15-LO have been proposed as plausible targets for Lechmann syndrome. Modeling of Lechmann syndrome in mice has shown that impaired iron homeostasis contributes to the disease pathogenesis, highlighting the importance of ferroptosis. Ferroptosis is also considered a trigger for epilepsy. The 15-LO inhibitor vatiquidone has been shown to reduce the incidence of seizures in patients with pontine dysplasia type 6, a mitochondrial disorder that, like Lechmann syndrome, is associated with epilepsy. Therefore, interfering with ferroptosis may help reduce the incidence and pathogenesis of Lechmann syndrome.

[0228] In this assay, the ability of compounds 1 to 4 and vatiquidon to inhibit / alleviate / delay iron-mediated cell death induced by ferroptosis was analyzed. Increased cell viability after addition of the test compound indicated a protective effect against ferroptosis. In short, ferroptosis was induced in healthy fibroblasts with 1 μM RSL3 and in fibroblasts from patients with Leydig syndrome with 375 nM RSL3. Cells were co-treated with dimethyl sulfoxide (DMSO) or ethanol (EtOH; each as a mediator control), each of compounds 1 to 4, or vatiquidon. Each of compounds 1 to 4 or vatiquidon was administered at a range of concentrations to obtain titration curves. Cell viability was determined using a commercially available kit for measuring ATP. On the graph, the X-axis shows the range of drug concentrations used, while the Y-axis shows cell viability (measured by ATP production), as measured by relative luminescent units.

[0229] Preliminary experiments were conducted to determine the effects of RSL3 damage on fibroblasts from healthy individuals (cell line: GM08402) and fibroblasts from patients diagnosed with Leydig syndrome (cell line: GM03672). The data obtained indicated that the ferroptosis inducer (RSL3) elicited different sensitivities in fibroblasts from healthy individuals and those from patients with Leydig syndrome. Figure 7 The data presented indicate that treatment with 1 μM RSL3 from healthy individuals and treatment with 700 nM RSL3 from patients with Riemann's syndrome resulted in roughly equivalent damage. These data suggest that 1 μM RSL3 is sufficient for healthy fibroblasts, while 700 nM is sufficient for Riemann's fibroblasts to achieve the experimental window of killing 25-30% of cells.

[0230] refer to Figure 8 The assay was performed on healthy cells. The test products were compounds 1 to 4 and vatiquidon. Each of compounds 1 to 4 and vatiquidon produced a protective effect on the cells, indicating that each of compounds 1 to 4 and vatiquidon possesses antiferroptosis properties. Additionally, data showed that compounds 2 and 3 had substantially equivalent antiferroptosis properties and, for the treatment of these fibroblasts from healthy individuals in this particular assay, their antiferroptosis properties were superior to those of compounds 1, 4, and vatiquidon (where the antiferroptosis properties of compounds 1, 4, and vatiquidon are substantially equivalent).

[0231] refer to Figure 9The assay was performed on fibroblasts derived from patients with Leydig's syndrome. The test products were compounds 1 to 4 and vatiquidon. Each of compounds 1 to 4 and vatiquidon exhibited a protective effect on the cells, indicating that each of compounds 1 to 4 and vatiquidon possesses antiferroptotic properties. Similarly (as observed in fibroblasts derived from healthy individuals), data indicated that compounds 2 and 3 had substantially equivalent antiferroptotic properties and were superior to compounds 1, 4, and vatiquidon in terms of their antiferroptotic properties for treating these fibroblasts derived from patients with Leydig's syndrome in this assay (where the antiferroptotic properties of compounds 1, 4, and vatiquidon are substantially equivalent). Notably, the relative effects of each of compounds 1 to 4 and vatiquidon in this assay were substantially similar regardless of whether the cell type was derived from a healthy individual or a patient diagnosed with Leydig's syndrome.

[0232] In summary, compounds 1 through 4 and vatiquidon produced similar results regardless of the cell type examined. Compounds 2 and 3 appeared to be superior to compounds 1, 4, and vatiquidon in their ability to protect (healthy or diseased) cells from RSL3-induced ferroptosis, but all compounds 1 through 4 and vatiquidon were found to be protective against RSL3-induced ferroptosis in both cells from healthy individuals and cells from patients diagnosed with Leydig's syndrome.

[0233] D. HETE ELISA / RSL3 assay

[0234] This assay was used as an indirect method to determine the ability of each of compounds 1 to 4 and vatiquidon to inhibit 15-lipoxygenase (“15-LO”) using a commercially available ELISA assay format. 15(S)-HETE is a metabolite of arachidonic acid. A reduction in 15(S)-HETE in the supernatant (as determined by the ELISA assay) indicates that 15-LO is inhibited by the addition of the test product compared to the untreated control. Therefore, all data were normalized against the “untreated” (i.e., untreated control) control group. The abilities of compounds 1 to 4 and vatiquidon were examined again. In these assays, ferroptosis was induced by the addition of RSL3, and the inhibition of 15-lipoxygenase (“15-LO”) attributable to each test product was indirectly measured by measuring the output of 15(S)-HETE. Fibroblasts from both healthy individuals and patients diagnosed with Lechner's syndrome were examined (but not all compounds were examined in each cell type). Activation of ferroptosis is considered a trigger for seizures commonly associated with Lechner's syndrome. In type 6 pontine dysplasia, the 15-LO inhibitor vatiquidone has been shown to reduce the frequency and incidence of seizures. Kahn-Kirby et al., PLoS One 2019;14(3): e0214250. Published online on March 28, 2019. doi:10.1371 / journal.pone.0214250. As a key regulator of ferroptosis, 15-LO serves as a rational therapeutic target for Lechner's syndrome.

[0235] refer to Figure 10 The data presented in graphs are derived from fibroblasts from healthy individuals subjected to 700 nM RSL3 to induce ferroptosis. The data indicate that compounds 2, 3, and 4 (compound 1 was not tested) and vatiquidon have protective effects because they inhibit 15-lipoxygenase. In this experiment, compound 3 appeared to be superior to compounds 2, 4, and vatiquidon; each produced roughly equivalent protective effects in this assay.

[0236] refer to Figure 11 The data presented graphically are from fibroblasts diagnosed with Leydig syndrome, obtained using fibroblasts subjected to 700 nM RSL3 to induce ferroptosis. The data indicate that compounds 1 through 4 and vatiquidon all possess protective effects (i.e., they inhibit 15-LO). In this assay, vatiquidon and compound 1 appeared to be superior to compounds 2, 3, and 4; each produced roughly equivalent protective effects in this assay. Notably, the tested compounds appeared to perform differently in this assay depending on cell type (i.e., healthy cells versus diseased cells).

[0237] E. Alastin assay

[0238] In short, ellastatin is known to induce ferroptosis, similar to RSL3. Therefore, in this study, ferroptosis was induced by adding 2 μM ellastatin to the cell culture. Aellastatin was incubated with fibroblasts for 24 hours to induce ferroptosis. Cells were co-treated with each of compounds 1 to 4 or vatiquidone as test products. Test products were applied at a range of concentrations to obtain curves. Cell viability was determined using a commercially available kit for measuring ATP production. In each graph provided, the X-axis shows the range of test product concentrations used, while the Y-axis shows cell viability (ATP production), as measured by relative luminescent units. This assay was thus used to determine whether the test products exhibited anti-ferroptosis activity (i.e., protective effect on cells). An increase in cell viability as determined by the assay was considered protective against ferroptosis.

[0239] refer to Figure 12 The study investigated whether compounds 1 through 4 and vatiquidon had a protective effect on fibroblasts from healthy individuals. In the assay, compounds 2 and 3 appeared to have superior protective effects compared to vatiquidon or compounds 1 or 4; each had approximately the same protective effect against ferroptosis in healthy cells.

[0240] refer to Figure 13 The study investigated whether compounds 1 through 4 and vatiquidon had a protective effect on fibroblasts from patients diagnosed with Riesling's syndrome. In the assays, compounds 2 and 3 appeared to have superior protective effects compared to vatiquidon or compounds 1 or 4. Vatiquidon appeared to have superior protective effects compared to compounds 1 and 4; each showed substantially similar protective effects against ferroptosis in Riesling's fibroblasts.

[0241] In summary, each of compounds 1 through 4 and vatiquidon appeared to have a protective effect against damaged cells undergoing ferroptosis, regardless of whether the cells were fibroblasts from healthy individuals or from patients diagnosed with Leydig's syndrome. In this assay, compounds 2 and 3 showed roughly equivalent protective effects and appeared to be superior in protecting healthy cells or cells from patients diagnosed with Leydig's syndrome (where cells are damaged by ellagic acid and undergo ferroptosis) compared to vatiquidon or compounds 1 or 4.

[0242] F. HETE ELISA / Alastin assay

[0243] In this assay, the inhibition of 15-LO by the test product was indirectly measured by quantifying 15(S)-HETE in the supernatant of fibroblasts undergoing ferroptosis. Ferroptosis was induced using a 2 μM concentration of ellastatin. 15(S)-HETE, a metabolite of arachidonic acid and produced by the activity of 15-LO, was measured using a commercially available ELISA kit. Data were normalized to a “no treatment” group, representing fibroblasts not damaged by ellastatin. As expected, incubation with ellastatin for 24 hours resulted in an increase in the level of 15(S)-HETE in the supernatant, as indicated by a fold increase compared to the “no treatment” control group.

[0244] refer to Figure 14 Valtiquidone and each of compounds 2 to 4 were tested in fibroblasts from healthy individuals, and they appeared to exhibit inhibitory effects on 15-LO when compared to the amount of 15(S)-HETE observed in the absence of test articles (i.e., columns labeled "Alastine + FAC + AA"). In this assay and under these conditions, valtiquidone and each of compounds 2 to 4 appeared to have substantially the same effect, with compound 2 possibly being slightly less effective.

[0245] refer to Figure 15 Valtiquidone and each of compounds 1 to 4 were tested in fibroblasts from patients diagnosed with Leishman's syndrome, and in all cases, when comparing the amount of 15(S)-HETE observed in the absence of test articles (i.e., the column labeled "Alastine + FAC + AA"), they appeared to exhibit inhibitory effects on 15-LO. In this assay and under these conditions, valtiquidone and each of compounds 1 to 4 appeared to have substantially the same effect, with compound 2 possibly being slightly less effective.

[0246] Example 2: Determination of plasma and tissue uptake in mice and rats (compounds 3 and 4)

[0247] I. Pharmacokinetics of compounds 3 and 4 after five daily subcutaneous administrations to male C57BL / 6 mice Evaluate

[0248] a) Dosing regimen: Animals: Male / C57Bl / 6 mice weighing 18-25 g were received from an approved supplier.

[0249] Fasting is not required for this study.

[0250] Record the animal's weight before administering the dose. The amount (mL / kg) of each dose delivered is based on the weight of each animal.

[0251] This was a multi-dose end-of-pipe study, with administration once daily for 5 days. Mice were sacrificed after the 5th and final doses.

[0252] Administer the dosage according to the standard operating procedures of the testing facility. Rinse after administration, if appropriate.

[0253] All dose syringes were weighed before and after administration to determine the amount of the preparation administered via gravimetric analysis.

[0254] Observe all animals during administration and each scheduled collection. Record any abnormalities.

[0255] Pre-formulated compounds 3 and 4 were provided by the research sponsor and were prepared for daily administration according to the study design described in Table 1 below.

[0256] Table 1: Research Design

[0257] b) Sample collection - post-drug administration: According to the standard operating procedures of the testing facility, after inhalation of an anesthetic, a distal blood sample was collected by cardiac puncture.

[0258] Collect blood samples into test tubes containing an appropriate anticoagulant. Store the tubes on wet ice until processed into plasma by centrifugation (3500 rpm for 10 minutes at 5°C) within 20 minutes of collection. Transfer 100 μL aliquots of the sample to individual, uniquely labeled matrix tubes and store at nominal -80°C until transfer to analytical chemistry for LC / MS / MS analysis.

[0259] Animals were euthanized immediately after each end-of-line blood collection in all groups and perfused with cold PBS. The following tissues were collected according to Table 2: heart and brain.

[0260] Plasma and tissue samples were processed for LC / MS / MS analysis to determine Figure 18-21 The amount / concentration reported in the report.

[0261] Table 2: Sample Collection Design

[0262] c) Results: The results are discussed below and in Figure 16-19 The results are presented in charts. It is also worth noting that, under the concentrations and conditions employed, the administration of compounds 3 and 4 was well tolerated.

[0263] II. Repeated (5-day) subcutaneous dose administration to male Sprague-Dawley rats Pharmacokinetic assessment of compound 3

[0264] a) Dosing regimen: Animals: Receive 225-250 g rats / Spra-Dawley / male / juveniles from an approved supplier.

[0265] Fasting is not required for this study.

[0266] Record the animal's weight before administering the dose. The amount (mL / kg) of each dose delivered is based on the weight of each animal.

[0267] This was a multi-dose end-of-pipe study, with administration once daily for 5 days. Rats were sacrificed after the 5th and final doses.

[0268] Administer the dosage according to the standard operating procedures of the testing facility. Rinse after administration, if appropriate.

[0269] All dose syringes were weighed before and after administration to determine the amount of the preparation administered via gravimetric analysis.

[0270] Observe all animals during administration and each scheduled collection. Record any abnormalities.

[0271] The pre-formulated compound 3 was provided by the research sponsor and was prepared for daily administration according to the study design described in Table 3 below.

[0272] Table 3: Research Design

[0273] b) Sample collection - post-drug administration: According to the standard operating procedures of the testing facility, after inhalation of an anesthetic, a distal blood sample was collected by cardiac puncture.

[0274] Collect blood samples into test tubes containing an appropriate anticoagulant. Store the tubes on wet ice until processed into plasma by centrifugation (3500 rpm for 10 minutes at 5°C) within 20 minutes of collection. Transfer 100 μL aliquots of the sample to individual, uniquely labeled matrix tubes and store at nominal -80°C until transfer to analytical chemistry for analysis.

[0275] Animals were euthanized immediately after each end-of-line blood collection in all groups and perfused with cold PBS. The following tissues were collected according to Table 4: heart and brain.

[0276] Plasma and tissue samples were processed for LC / MS / MS analysis to determine the amounts / concentrations reported in the figures.

[0277] Table 4: Sample Collection Design

[0278] c) Results: Discussed in Section III below, and... Figure 17 The results are presented in charts. It is also worth noting that, under the concentrations and conditions used, compound 3 was well tolerated.

[0279] III. Results of PK experiments in mice and rats

[0280] refer to Figure 16 Plasma concentrations of compound 3 (administered at both 20 mg / kg and 60 mg / kg doses) and compound 4 (administered at only 60 mg / kg dose) were plotted from 30 minutes to 24 hours post-administration. Because the route of administration was subcutaneous (SC), the drugs (i.e., each of compounds 3 and 4) needed to migrate to be absorbed into the bloodstream to provide potentially effective tissue delivery. The data showed that both compounds 3 and 4 migrated into the bloodstream of mouse plasma, and the approximate proportionate increase in plasma concentration was greater with increasing doses. Furthermore, plasma concentrations decreased over time, as expected, clearing them from the subjects.

[0281] exist Figure 17 In this study, plasma concentrations in mice obtained using compound 3 at a dose of 20 mg / kg were compared graphically with those obtained in rats at a dose of 10 mg / kg (also compound 3) during the period from 30 minutes to 24 hours after administration. Because comparisons were made between different species (i.e., mice and rats), the concentrations were calculated in mg / kg for each species. 2 The body surface area (i.e., in this case, 60 mg / m²) 2Based on this, these doses are roughly equal. (Reference) Figure 17 The data showed that the uptake of compound 3 in plasma and its clearance in both mammals were roughly equal during the test period, as SC administration was administered.

[0282] Delivering drugs directly to the heart can be challenging due to certain barriers in the body (see: Sahoo et al., Targeted Delivery of Therapeutic Agents to the Heart, *Nature Reviews Cardiology*, (2021) 18(6): 389-399). Reference Figure 18 The concentrations of compound 3 (administered at both 20 mg / kg and 60 mg / kg doses) and compound 4 (administered at 60 mg / kg dose only) in heart tissue were plotted from 30 minutes to 24 hours post-administration. The data showed that up to micrograms of drug were found per gram of heart tissue at most 2 hours post-administration (5 days after drug administration), and drug clearance from the tissue proceeded as expected over time. These data indicate that effective amounts of compounds 3 and 4 can be delivered to mouse heart tissue under these administration conditions.

[0283] Delivering drugs to the brain can be difficult because the blood-brain barrier, or BBB, protects the brain (and other central nervous system (CNS) tissues) from the penetration of certain types of molecules. (Reference) Figure 19 The concentrations of compound 3 (administered at both 20 mg / kg and 60 mg / kg doses) and compound 4 (administered at 60 mg / kg dose only) in brain tissue were plotted from 30 minutes to 24 hours post-administration. The data showed that up to micrograms of drug were found per gram of brain tissue up to 2 hours after drug administration (5 days post-administration), and drug clearance from the tissue proceeded as expected over time. These data indicate that effective amounts of compounds 3 and 4 can be delivered to mouse brain tissue under these administration conditions.

[0284] d) Summary and discussion of results: In mice, both compounds 3 and 4 were well tolerated at dose levels up to 60 mg / kg (administered via SC injection).

[0285] Through allometric growth in rodents, the dosage ratio of compound 3 was favorable (20 mg / kg in mice ≈ 10 mg / kg in rats).

[0286] Plasma exposure to compound 3 was roughly proportional to the dose (data from 20 mg / kg and 60 mg / kg in mice were compared).

[0287] Compound 4 typically has a later Tmax than compound 3, and its half-life is often longer.

[0288] Compound 3 showed higher exposures in the heart (Cmax and AUC) than in the brain; compound 4 showed no difference in exposure between the two tissues.

[0289] Based on AUC, compound 3 had slightly higher cardiac exposure than compound 4; compound 4 had slightly higher brain exposure than compound 3.

[0290] The concentrations achieved in plasma, heart, and brain tissue represent the amounts expected to be effective in treating, preventing, inhibiting, alleviating, and / or delaying the onset of Lechner's syndrome or its signs or symptoms (e.g., cardiomyopathy (i.e., hypertrophic cardiomyopathy and dilated cardiomyopathy)) and neurodevelopmental and neurodegenerative disorders (e.g., abnormalities of the brainstem, cerebellum, basal ganglia, oculomotor nerves, and cranial nerves, and their associated effects leading to dysphagia, dyspnea, dysphagia, hypotonia, developmental delay, ataxia, dysarthria, dystonia, paralysis, and seizures).

[0291] Example 3: In vivo plasma evaluation of compound 2 produced by different administration routes in rats

[0292] I. Pharmacokinetics of compound 2 after a single intravenous, subcutaneous, or oral administration in male Sprague-Dowley rats Dynamics assessment

[0293] a) Dosing regimen: Animals: Receive 225-250 g rats / Spra-Dawley / male / juveniles from an approved supplier.

[0294] Fast the animals overnight before administering the dose. Return the food 2 hours after sample collection.

[0295] Record the animal's weight before administering the dose. The amount (ml / kg) of each dose delivered is based on the weight of each animal.

[0296] This is a single-dose, continuous blood collection study in which tissue is collected for possible future analysis.

[0297] Administer the dosage according to the standard operating procedures of the testing facility. Rinse after administration, if appropriate.

[0298] All dose syringes were weighed before and after administration to determine the amount of the preparation administered via gravimetric analysis.

[0299] Observe all animals during administration and each scheduled collection. Record any abnormalities.

[0300] The pre-formulated compound 2 and vatiquidon were provided by the study sponsor and were prepared for administration according to the study design described in Table 5 below.

[0301] Table 5: Research Design

[0302] b) Sample collection - post-drug administration: Blood samples were collected via a jugular vein catheter (JVC) and transferred to tubes containing an appropriate anticoagulant. The tubes were stored on moist ice until processed into plasma by centrifugation (3500 rpm for 10 minutes at 5°C) within 20 minutes of collection. 100 μL samples were aliquoted and transferred to individual, uniquely labeled matrix tubes containing 10 μL of 5% (v / v) formic acid aqueous solution and stored at nominal -80°C until LC / MS / MS analysis.

[0303] Animals were immediately euthanized and perfused with cold PBS after the final continuous blood collection (24 hours, JVC). The following tissues were collected according to Table 6: heart and brain (stored for possible future analysis).

[0304] Plasma and tissue samples were processed for LC / MS / MS analysis to determine the amounts / concentrations reported in the figures.

[0305] Table 6: Sample Collection Design

[0306] c) Results: The results are preliminary and under continued review. However, the following observations were made regarding the results of this experiment using compound 2.

[0307] Regardless of the mode of administration (e.g., intravenous, subcutaneous, or oral), compound 2 was effectively administered (i.e., data indicate that an effective dose can be delivered to the subjects).

[0308] Regardless of the administration mode, compound 2 was well tolerated at the dosage levels examined in this study.

[0309] Example 4: Rieslings fibroblasts treated with compound 3 and those treated with RSL3 to induce glutathione stress In vitro comparison

[0310] method: Rieker fibroblasts (GM03672) were administered at a rate of 1 x 10⁻⁶ cells per 500 μL. 5 Cells were seeded at a density of 100 g / mL in intact MEM (MEM + 15% FBS + 1% penicillin / streptomycin) in 24-well tissue culture-treated plates and incubated overnight at 37°C with 5% CO2. Cells were treated with 1 μM RSL3, 1 μM RSL3 with 1 μM Compound 3, or the medium used for untreated cells at 37°C for 4 hours. After incubation, the supernatant was removed, and the cells were treated with 0.25% trypsin at 37°C for 5 minutes. The trypsin was neutralized with an aliquot of MEM. Cells were removed using a gentle pipette and clumped at 300 xg for 5 minutes. The clumps were resuspended in 500 μL Fluobrite DMEM, and the cells were transferred to 96-well V-plates. Cells were stained with 20 nM MitoTracker Green and 100 nM tetramethylrhodamine methyl ester (TMRM). The samples were incubated at 37°C for 25 minutes. Each well was mixed immediately before analysis to resuspend the precipitated cells. Fluorescence intensity was measured on a BD Accuri C6 Plus flow cytometer. At least 10,000 events were collected for each sample. Data analysis was performed using FloJo vs. 10. Gating was performed by establishing initial forward and side scatter gates. MitoTracker Green-positive and TMRM-positive cells were identified by subsequent gating. The median fluorescence intensity (MFI) of TMRM fluorescence was determined. Statistical significance between groups was determined by one-way ANOVA using a GraphPad Prism 10. A p-value less than 0.05 was considered significant. Results are in... Figure 20 The information is presented in charts.

[0311] result: refer to Figure 20The results for untreated Riesling fibroblasts and those treated with both RSL3 and compound 3 (1 μM) were comparable (i.e., essentially identical). However, those Riesling fibroblasts treated with RSL3 alone showed a significant decrease in mitochondrial membrane potential. The effect of RSL3 treatment (when used alone) on Riesling fibroblasts is consistent with the induction of ferroptosis or glutathione stress in the cell line, a well-known effect of RSL3 treatment. However, the results obtained when Riesling fibroblasts were treated with both RSL3 and compound 3 indicate that compound 3 (1 μM) has a protective effect on Riesling fibroblasts, protecting them from the loss of mitochondrial membrane potential associated with glutathione stress or ferroptosis induced by RSL3 in other ways.

[0312] refer to Figure 21 The results for untreated Riesling fibroblasts and those treated with both RSL3 and compound 4 (1 μM) were comparable (i.e., essentially identical). However, Riesling fibroblasts treated with RSL3 alone showed a significant decrease in mitochondrial membrane potential. The effect of RSL3 treatment (when used alone) on Riesling fibroblasts is consistent with the induction of ferroptosis or glutathione stress in the cell line, a well-known effect of RSL3 treatment. However, the results obtained when Riesling fibroblasts were treated with both RSL3 and compound 4 indicate that compound 4 (1 μM) has a protective effect on Riesling fibroblasts, protecting them from the loss of mitochondrial membrane potential associated with glutathione stress or ferroptosis induced by RSL3 in other ways.

[0313] Example 5: Pharmacokinetics and tissue exposure of compound 4 after five daily subcutaneous administrations to male C57BL / 6 mice. dew

[0314] This example demonstrates the pharmacokinetics and exposure-dose ratios in plasma and tissues (heart and brain) of mice administered with 60 mg / kg or 180 mg / kg of compound 4, and compares 60 mg / kg of compound 4 administered with 15% Kolliphor ELP with 5% Kolliphor ELP.

[0315] Study design details: Animals were allowed to acclimatize to the testing facility for at least two days before the study began.

[0316] The study animals were male C57BL / 6 mice, each weighing approximately 18-25 grams.

[0317] Fasting is not required for the study.

[0318] Record the animal's weight before administering the dose. The amount (mL / kg) of each dose delivered is based on the weight of each animal.

[0319] This was a multiple-dose continuous PK study, administered subcutaneously once daily for 5 days. Compound 4 was administered to two groups: Group 1 at 60 mg / kg and Group 2 at 180 mg / kg (6 mg / mL and 18 mg / mL, PBS containing 15% Kolliphor ELP, 10 mL / kg; N = 3 / group). Continuous PK time points were collected after the 5th and final administration. Mice were sacrificed after the 5th and final administration to collect blood and tissue.

[0320] All dose syringes were weighed before and after administration to determine the amount of the preparation administered via gravimetric analysis.

[0321] Observe all animals at the time of administration and each scheduled collection.

[0322] The same design was used for the third group of mice, with 60 mg / kg of 5% Kolliphor ELP administered once daily for 5 days via SC, except that 3 mice were sacrificed at each of the four PK time points (n = 12).

[0323] Table 7: Research Design

[0324] Table 8: Mixture Design

[0325] Sample collection details: Continuous blood samples are collected by cutting the tail vein.

[0326] Collect blood samples into tubes containing an anticoagulant. Store the tubes on wet ice until processed into plasma by centrifugation (3500 rpm for 10 minutes at 5°C) within 20 minutes of collection. Transfer 20 μL aliquots of the sample to individual, uniquely labeled matrix tubes and store at nominal -80°C until transfer to analytical chemistry for analysis.

[0327] Animals were euthanized immediately after each end-of-line blood collection in all groups and perfused with cold PBS. Heart and brain tissue were collected.

[0328] ○ Perfusion Method: Open the chest longitudinally with scissors to expose the thoracic organs. Make a small incision in the left ventricle with a scalpel, just large enough to insert the tip of the cannula. Attach the cannula to an appropriately sized tubing and syringe, prefill with cold PBS, and perfuse to remove any air. Insert the tip of the cannula into the left ventricle and into the ascending aorta. Use hemostatic forceps to help secure the cannula in place during perfusion. Make a second incision in the right atrium to allow fluid to drain during reflux. Manually push the syringe attached to the cannula or place it into a pump programmed to perfuse at 10 mL / min. Perfuse the animal for at least 3 minutes, or longer if necessary (until the drained fluid is clear).

[0329] Table 9: Sample Collection Design

[0330] analyze: Group 1 and Group 2 N = 3 / group; Collection relative to final drug administration: ○ Plasma: 60 mg / kg at 2 hours and 24 hours; 180 mg / kg before, at 2 hours, at 8 hours and 24 hours after administration. ○ Heart and brain 24 hours (terminal, PBS perfusion); RGA 2 bioanalysis.

[0331] Group 3 N = 3 / time point; Collection relative to final drug administration: ○ Plasma: 0.5 hours, 2 hours, 8 hours and 24 hours (terminal) ○ Heart and brain 0.5 hours, 2 hours, 8 hours and 24 hours (terminal, PBS perfusion) RGA 2 bioanalysis.

[0332] Table 10: Sample Analysis

[0333] Results - Plasma PK characteristics in mice 5 days later ( Figure 22A (Table 11-13) The 60 and 180 mg / kg SC were well tolerated over 5 days (10 mL / kg QD, PBS containing 15% Kolliphor ELP). 5% and 15% Kolliphor ELP produced similar plasma concentrations of the compound at 60 mg / kg; Tmax is approximately 0.5-2 hours; It will reach a stable state within 5 days; Plasma exposure to compound 4 is roughly proportional to the dose.

[0334] Table 11: Plasma concentration of compound 4 in mice (Group 1 and Group 2)

[0335] Table 12: Plasma concentration of compound 4 in mice (Group 3)

[0336] Table 13: Plasma PK

[0337] Results - Tissue exposure characteristics of mice 5 days later ( Figures 22B to 22C (Tables 14-16) Similar compounds were found at concentrations 4 in the heart and brain; C24 (valence) exposure is roughly proportional to dose; A QD of 180 mg / kg can maintain tissue concentrations > 1 μM.

[0338] Table 14: Concentration of Compound 4 in mouse tissues (Group 1 and Group 2)

[0339] Table 15: Concentration of Compound 4 in Mouse Tissues (Group 3)

[0340] Table 16: Tissue Exposure

[0341] equivalent

[0342] This technology is not limited to the specific embodiments described in this application, which are intended as separate illustrations of various aspects of this technology. Many modifications and variations of the technology of this invention can be made without departing from the spirit and scope of this technology, as will be apparent to those skilled in the art. Based on the above description, functionally equivalent methods and apparatuses within the scope of this technology, in addition to those methods and apparatuses listed herein, will be apparent to those skilled in the art. Such modifications and variations are intended to fall within the scope of the appended claims. This technology is limited only by the terms of the appended claims and the full scope of equivalents enjoyed by such claims. It should be understood that this technology is not limited to specific methods, reagents, compounds, compositions, or biological systems, although such specific methods, reagents, compounds, compositions, or biological systems can vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be restrictive.

[0343] Furthermore, when features or aspects of this disclosure are described in accordance with the Markush Group, those skilled in the art will recognize that this disclosure is also described in accordance with any individual member or subgroup of the Markush Group.

[0344] As those skilled in the art will understand, for all purposes, particularly for the purpose of providing a written description, all scopes disclosed herein also encompass all possible subscopes and combinations thereof. Any listed scope can be readily recognized as sufficiently describing and implementing an identical scope that can be decomposed into at least two, three, four, five, ten, etc., equal parts. As a non-limiting example, each scope discussed herein can be readily decomposed into a lower third, a middle third, and an upper third, etc. As those skilled in the art will also understand, all language such as “at most,” “at least,” “greater than,” “less than,” etc., includes the enumerated numbers and refers to a scope that can subsequently be decomposed into subscopes as discussed above. Finally, as those skilled in the art will understand, a scope includes each individual member. Thus, for example, a group having 1-3 cells means a group having 1, 2, or 3 cells. Similarly, a group having 1-5 cells means a group having 1, 2, 3, 4, or 5 cells, and so on.

[0345] All patents, patent applications, provisional applications and publications mentioned or cited herein are incorporated herein by full reference to the extent that they are not inconsistent with the express teachings in this specification, including all figures and tables.

[0346] Other embodiments are set forth in the following claims.

Claims

1. A method for treating, preventing, alleviating, suppressing, or delaying the onset of Leigh syndrome or related signs and / or symptoms in a subject in need, the method comprising administering to the subject a therapeutically effective amount of compound 1, compound 2, compound 3, compound 4, or a mixture of any two or more compounds 1 to 4, or a pharmaceutically acceptable salt, stereoisomer, mixture of stereoisomers, tautomer, hydrate, and / or solvate thereof, wherein compounds 1 to 4 have the following structure: 。 2. The method according to claim 1, wherein the signs or symptoms of Leydig syndrome include dysphagia, dyspnea, dysphagia, hypotonia, lactic acidosis, developmental delay, cardiomyopathy, seizures, ataxia, dysarthria, dystonia, paralysis, respiratory and renal impairment, color blindness and / or vision loss.

3. The method according to claim 1 or 2, wherein the subject has been diagnosed with Leydig syndrome.

4. The method according to any one of claims 1 to 3, wherein the compound is administered daily for 6 weeks or longer.

5. The method according to any one of claims 1 to 4, wherein the compound is administered orally, topically, intranasally, systemically, intravenously, subcutaneously, intraperitoneally, intradermally, intraocularly, intraocularly, intrathecally, intrathecally, via iontophoresis, through mucous membranes, intravitreal, or intramuscularly.

6. The method according to any one of claims 1 to 5, wherein the compound is administered to the subject to treat, prevent, alleviate, inhibit, or delay the onset of cardiomyopathy in the subject.

7. The method according to claim 6, wherein the cardiomyopathy is hypertrophic cardiomyopathy, left ventricular hypertrophy, dilated cardiomyopathy, pericardial effusion and / or arrhythmia / conduction abnormality.

8. The method according to claim 6, wherein the cardiomyopathy is hypertrophic cardiomyopathy.

9. The method according to any one of claims 1 to 5, wherein administering the compound to the subject reduces the lactate level in the subject's blood, urine, or cerebrospinal fluid (CSF).

10. The method according to any one of claims 1 to 5, wherein the compound is administered to the subject to treat, prevent, alleviate, inhibit, or delay the onset of multi-organ and / or central nervous system (CNS) injury in the subject, said multi-organ and / or central nervous system (CNS) injury including lesions of the brainstem, basal ganglia, and spinal cord.

11. The method according to any one of claims 1 to 5, wherein the compound is administered to the subject to treat, prevent, alleviate, inhibit, or delay the onset of color blindness and / or vision loss in the subject.

12. The method according to any one of claims 1 to 5, wherein administering the compound to the subject prolongs the lifespan of the subject.

13. The method according to any one of claims 1 to 5, wherein the compound is administered to the subject to treat, prevent, reduce, inhibit, or delay the occurrence of epileptic seizures, ataxia, dysarthria, dystonia, and / or paralysis in the subject.

14. The method according to any one of claims 1 to 13, wherein the subject is a human.

15. A composition, medicament, or formulation comprising any one or more of compounds 1 to 4, or pharmaceutically acceptable salts, stereoisomers, mixtures of stereoisomers, tautomers, hydrates, and / or solvates thereof, for the treatment, prevention, relief, inhibition, or delay of the onset of Lechner's syndrome or related signs and / or symptoms in a subject of need, wherein compounds 1 to 4 have the following structures: 。 16. The composition, drug, or formulation according to claim 15, wherein the signs or symptoms of Leydig syndrome include dysphagia, dyspnea, dysphagia, hypotonia, lactic acidosis, developmental delay, cardiomyopathy, seizures, ataxia, dysarthria, dystonia, paralysis, respiratory and renal impairment, color blindness, and vision loss.

17. The composition, drug or formulation according to claim 15 or 16, wherein the subject has been diagnosed with Leydig syndrome.

18. The composition, drug or formulation according to any one of claims 15 to 17, wherein the composition, drug or formulation is administered daily for 6 weeks or longer.

19. The composition, drug, or formulation according to any one of claims 15 to 18, wherein the composition, drug, or formulation is administered orally, topically, intranasally, systemically, intravenously, subcutaneously, intraperitoneally, intradermally, intraocularly, intraocularly, intrathecally, intrathecally, intraocularly, via iontophoresis, through mucous membranes, intravitreally, or intramuscularly.

20. The composition, drug, or formulation according to any one of claims 15 to 19, wherein administration of the composition, drug, or formulation to the subject treats, prevents, alleviates, inhibits, or delays the onset of cardiomyopathy in the subject.

21. The composition, drug or formulation according to claim 20, wherein the cardiomyopathy is hypertrophic cardiomyopathy, left ventricular hypertrophy, dilated cardiomyopathy, pericardial effusion and / or arrhythmia / conduction abnormality.

22. The composition, drug or formulation according to claim 20, wherein the cardiomyopathy is hypertrophic cardiomyopathy.

23. The composition, drug, or formulation according to any one of claims 15 to 19, wherein administration of the composition, drug, or formulation to the subject reduces lactate levels in the subject's blood, urine, or cerebrospinal fluid (CSF).

24. The composition, drug, or formulation according to any one of claims 15 to 19, wherein administration of the composition, drug, or formulation to the subject treats, prevents, alleviates, inhibits, or delays the onset of multi-organ and / or central nervous system (CNS) injury in the subject, said multi-organ and / or central nervous system (CNS) injury including lesions of the brainstem, basal ganglia, and spinal cord.

25. The composition, drug, or formulation according to any one of claims 15 to 19, wherein administration of the composition, drug, or formulation to the subject treats, prevents, alleviates, inhibits, or delays the onset of color blindness and / or vision loss in the subject.

26. The composition, drug, or formulation according to any one of claims 15 to 19, wherein administration of the composition, drug, or formulation to the subject prolongs the life of the subject.

27. The composition, drug, or formulation according to any one of claims 15 to 19, wherein administration of the composition, drug, or formulation to the subject treats, prevents, reduces, inhibits, or delays the occurrence of epileptic seizures, ataxia, dysarthria, dystonia, and / or paralysis in the subject.

28. The composition, drug or formulation according to any one of claims 15 to 27, wherein the subject is a human.

29. The use of compounds 1 to 4, or any two or more of compounds 1 to 4, or pharmaceutically acceptable salts, stereoisomers, mixtures of stereoisomers, tautomers, hydrates and / or solvates thereof, in the preparation of compositions, pharmaceuticals or formulations comprising any one of compounds 1 to 4, or any two or more of compounds 1 to 4, or pharmaceutically acceptable salts, stereoisomers, mixtures of stereoisomers, tautomers, hydrates and / or solvates thereof, for the treatment, prevention, relief, inhibition or delay of the onset of Leishman's syndrome or related signs and / or symptoms in a subject of need, wherein compounds 1 to 4 have the following structures: 。 30. The use according to claim 29, wherein the signs or symptoms of Leydig syndrome include dysphagia, dyspnea, dysphagia, hypotonia, lactic acidosis, developmental delay, cardiomyopathy, seizures, ataxia, dysarthria, dystonia, paralysis, respiratory and renal impairment, color blindness, and vision loss.

31. The use according to claim 29 or 30, wherein the subject has been diagnosed with Leydig syndrome.

32. The use according to any one of claims 29 to 31, wherein the composition, drug or formulation is administered daily for 6 weeks or longer.

33. The use according to any one of claims 29 to 32, wherein the composition, drug or formulation is administered orally, topically, intranasally, systemically, intravenously, subcutaneously, intraperitoneally, intradermally, intraocularly, intraocularly, intrathecally, intrathecally, intracranially, via iontophoresis, through mucous membranes, intravitreally, or intramuscularly.

34. The use according to any one of claims 29 to 33, wherein the administration of the composition, drug or formulation to the subject treats, prevents, alleviates, inhibits or delays the onset of cardiomyopathy in the subject.

35. The use according to claim 34, wherein the cardiomyopathy is hypertrophic cardiomyopathy, left ventricular hypertrophy, dilated cardiomyopathy, pericardial effusion and / or arrhythmia / conduction abnormality.

36. The use according to claim 34, wherein the cardiomyopathy is hypertrophic cardiomyopathy.

37. The use according to any one of claims 29 to 33, wherein administration of the composition, drug, or formulation to the subject reduces lactate levels in the subject's blood, urine, or cerebrospinal fluid (CSF).

38. The use according to any one of claims 29 to 33, wherein the administration of the composition, drug or formulation to the subject treats, prevents, alleviates, inhibits or delays the onset of multi-organ and / or central nervous system (CNS) injury in the subject, said multi-organ and / or central nervous system (CNS) injury including lesions of the brainstem, basal ganglia and spinal cord.

39. The use according to any one of claims 29 to 33, wherein the administration of the composition, drug or formulation to the subject treats, prevents, alleviates, inhibits or delays the onset of color blindness and / or vision loss in the subject.

40. The use according to any one of claims 29 to 33, wherein administration of the composition, drug, or formulation to the subject prolongs the life of the subject.

41. The use according to any one of claims 29 to 33, wherein the administration of the composition, drug or formulation to the subject treats, prevents, alleviates, inhibits or delays the occurrence of epileptic seizures, ataxia, dysarthria, dystonia and / or paralysis in the subject.

42. The use according to any one of claims 29 to 41, wherein the subject is a human.

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