JP2008534623A - Use of spirostenol for the treatment of mitochondrial disorders - Google Patents

Abstract

The present invention relates to methods and compositions involving the use of spirostenol derivatives for the treatment, prevention, or reduction of the risk of developing mitochondrial genetic diseases or diseases or conditions associated with mitochondrial genetic diseases.
[Selection] Figure 1

Description

This patent application is based on U.S. Provisional Patent Application No. 60 / 667,229 filed on April 1, 2005 and U.S. Provisional Patent Application No. 60 / 697,518 filed on July 8, 2005. And both applications are hereby incorporated by reference.

  Mitochondria are differentiated organelles present in all cells of the body except red blood cells, and are responsible for producing more than 90% of the energy the body needs to maintain life and growth through oxidative phosphorylation. When the mitochondrial respiratory chain fails, the energy level of the cell falls rapidly, leading to cell death and organ dysfunction. Depending on the organ, impaired mitochondrial physiology is associated with neurodegenerative diseases and disorders. These are diseases and disorders of the central nervous system such as Alzheimer's disease and Parkinson's disease, and diseases and disorders that affect the peripheral nervous system, such as myopathy (muscle disorders) and various kidney, liver and respiratory diseases. including.

  Depending on the severity of the mitochondrial disorder, the disease can range from mild to fatal. Whether a mitochondrial disorder is a cause or a result of the disease affected, protection of respiratory function is essential to restore impaired physiology. In addition, protection of mitochondrial respiratory function is essential for the preservation of tissues necessary for successful transplantation.

  There is no cure for mitochondrial diseases with genetic factors, but treatment can help relieve symptoms or slow or stop disease progression (M. Zeviani et al., Brain ; 127: 2153 (2004)). Although treatment is individualized depending on the constitution and severity of the disease, these palliative treatments cannot reverse the damage that has already occurred. However, a number of experimental methods are currently being evaluated, including gene therapy and drug therapy.

One method is to introduce a modified gene or gene product into the mitochondria using a protein transduction apparatus (G. Manfredi et al., Natur. Genet., 30: 394 (2002)) and sequence-specific Preventing the replication of mutated mtDNA by anti-gene peptide nucleic acids (RW
Taylor et al., Natur. Genet., 15: 212 (1997)). These efforts have not yet been used clinically.

  In cases of certain special myopathy, reduced heteroplasmic (mixed with different types of mtDNA) mutants, muscle myotoxic drugs, and controlled muscle fiber damage and mutations It is obtained by regeneration with satellite cells that do not have a body (W. Irwin et al., J. Biol. Chem., 277: 2221 (2002)). However, this treatment was ineffective in improving drooping in 5 patients with progressive extraocular palsy syndrome (PEO).

Creatine is a substrate for the synthesis of creatine phosphate and is the most abundant energy storage compound in muscle, heart and brain. An open-label study in 81 patients with various neuromuscular diseases (including 17 patients with mitochondrial disease) showed a marked improvement in ischemic isometric grip strength and non-ischemic isometric dorsiflexion torque. However, another randomized, double-blind, placebo-controlled cross-over study in 16 patients with chronic PEO or mitochondrial myopathy had no significant effect on exercise performance, eye movements, or daily activities (PF Chinnery et al. , Am. J.
Med. Genet., 106: 94 (2001)). Taken together, these data indicate that creatine is effective in some, but not all, mitochondrial diseases.

Coenzyme Q10 is ineffective in mitochondrial diseases associated with mtDNA (N. Bresolin et al., J. Neurol. Sci., 100: 70 (1990)) but leads to significant improvement in 'primary' CoQ10 deficiency . Idebenone, a short-chain analog of coenzyme Q10, appears to be effective in improving hypertrophic cardiomyopathy in Friedreich ataxia (P. Rustin et al., Lancet,
354: 477 (1999); C. Mariotti et al., Neurology, 60: 1676 (2003)).

  Thus, there remains a need for treatments for mitochondrial disorders, including mitochondrial diseases.

  The present invention provides a method of treatment that includes medication to a mammal, such as a human, who is suffering from, or has a sign of, a mitochondrial disorder that is not a neuropathology of the central nervous system. This includes administering to the mammal an effective amount of a compound of structural formula (I), or a pharmaceutically acceptable salt thereof.

R ₁ , R ₂ , R ₄ , R ₇ , R ₁₁ , R ₁₂ and R _{15 in} the formula are each independently a hydrogen atom, optionally -NR'-, -O-, -S-, -SO _{-, - SO 2 -, -} O-SO 2 -, - SO 2 -O -, - C (O) -, - C (O) -O -, - OC (O) -, - C (O) - (C ₁ -C ₈ ) alkyl group inserted with NR′- or —NR′—C (O) —, or a hydroxyl group, N (R ′) ₂ , carboxyl group, oxo group, or sulfonic acid . R ₃ is a hydroxyl group, (C ₁ -C ₈ ) alkoxy group, (C ₁ -C ₂₂ ) alkyl CO ₂ , R′O ₂ C (CH ₂ ) _2-8 CO _2- , toluene-4-sulfonyloxy group Or a benzoyloxy group, wherein (C ₁ -C ₂₂ ) alkyl group or (CH ₂ ) _2-8 is optionally 1-2 CH = CH units, 1-2 OH, and / or One to two epoxy substituents can be included. R _6, R ₈ , R ₉ , R ₁₀ , R ₁₃ , R ₁₄ , R ₁₆ and R ₁₇ are each independently a hydrogen atom, (C ₁ -C ₈ ) alkyl group, hydroxyl (C ₁ -C ₈ ) Alkyl group, (C ₁ -C ₈ ) alkoxy group, or hydroxyl group, and X is O, S, S (O), N (R ′), N (Ac) or N (toluene-4-sulfonyloxy). Group).

Here, each R ′ is individually H, (C ₁ -C ₈ ) alkyl group, phenyl group or benzyl group.
R ₁₆ and / or R ₁₇ is preferably CH ₃ .
The alkyl group is preferably a (C ₂ -C ₈ ) alkyl group.

  Accordingly, the present invention includes a therapeutic method for treating a mitochondrial disorder in a patient suffering from or showing a disease that is not a neuropathology of the central nervous system, which comprises an effective amount of structural formula (II ) Or a pharmaceutically acceptable salt thereof.

Where R ₁ , R ₂ , R ₄ , R ₇ , R ₁₁ , R ₁₂ , and R ₁₅ are each independently a hydrogen atom, a (C ₁ -C ₈ ) alkyl group, a hydroxyl group, an amino group, Group, carboxyl group, oxo group, sulfonic acid, or optionally -NH-, -N ((C ₁ -C ₈ ) alkyl)-, -O-, -S-, -SO-, -SO _2- , -O-SO _2- , -SO ₂ -O-, -C (O)-, -C (O) -O-, -OC (O)-, -C (O) -NR'-, or -NR A (C ₁ -C ₈ ) alkyl group having '-C (O)-inserted therein, wherein R ′ is H or a (C ₁ -C ₈ ) alkyl group. R ₃ is a hydroxyl group, (C ₁ -C ₆ ) alkyl CO ₂ , HO ₂ C (CH ₂ ) ₂ CO _2- , toluene-4-sulfonyloxy group or benzoyloxy group. Each of R ₆ , R ₈ , R ₉ , R ₁₀ , R ₁₃ , and R ₁₄ is independently a hydrogen atom, a (C ₁ -C ₈ ) alkyl group, a (C ₁ -C ₈ ) hydroxylalkyl group, ( C ₁ -C ₈ ) an alkoxy group or a hydroxyl group. X is an O, N (H), N (Ac) or N (toluene-4-sulfonyloxy) group.

  Spirostenol, especially 5,6-unsaturated spirostenol such as hexanoic acid = (22S, 25S)-(20S) -spirosta-5-en-3β-yl, Candaceae (Asteraceae), as detailed below Naturally occurring 22R-hydroxycholesterol derivatives not only act on Aβ, which is toxic to mitochondria, but can provide a new therapeutic method that exerts an effect by acting directly on the mitochondrial respiratory chain. Hexanoic acid = (22S, 25S)-(20S) -spirosta-5-en-3β-yl further stops uncoupling of induced oxidative phosphorylation and is a potent blocker of mitochondrial permeability transition pore Amplifies the effect of some cyclosporin A. This implies an effect on the mitochondrial permeability transition pore.

Mitochondrial disorders, including mitochondrial diseases that are not considered CNS neuropathology, and mitochondrial disorders that can be treated with this method include:
Eyeball: Retinitis pigmentosa, optic nerve atrophy Muscle: Extraocular muscle disease (droop, acquired strabismus, ocular muscle paralysis), crush muscle syndrome (compartment syndrome)
Mitochondrial dysfunction as a result of drug side effects (highly active antiretroviral therapy, HAART, etc.)
Muscle disease Heart: Heart attack, heart attack, heart disease, heart disease, cardiomyopathy, and any cardiac mitochondrial dysfunction as a result of drug side effects (such as anticancer drugs like HAART, Mega HAART, anthracyclines)
Liver: Hepatocellular dysfunction Kidney and endocrine system: tubulinitis, diabetes Respiratory disorder The present invention relates to pharmaceutical synthesis comprising an effective amount of a compound of structural formula I and / or II combined with a pharmaceutically acceptable carrier. Further provided are new compounds and compounds of structural formula I or II, such as

As described below, mitochondrial respiratory chain disorders have been described in detail in connection with Alzheimer's disease (AD). Aβ _1-42 has also been reported to uncouple the mitochondrial respiratory chain and promote the opening of the mitochondrial permeability transition pore (MPT), leading to cell death. Spirostenol, hexanoic acid = (22S, 25S)-(20S) -spirosta-5-en-3β-yl (SP-233) binds to peptides and inactivates them against Aβ _1-42 toxicity Have been reported to protect neurons. The present invention is based on the discovery that the protective effect of SP-233 is also attributed to the protection of mitochondrial function. In mitochondria isolated from rat forebrain, it has been found that picomolar concentrations of Aβ _1-42 reduce mitochondrial respiration rate due to the protective effect partially counteracted by SP-233. This protective effect of SP-233 probably comes from SP-233 directly targeting the respiratory chain as well as Aβ _1-42 .

SP-233 further stops uncoupling of oxidative phosphorylation induced by carbonyl cyanide = 3-chlorophenylhydrazone (CCCP). It also amplifies the effect of cyclosporin A (a potent blocker of the mitochondrial MPT pore), which means an effect on MPT. Respiratory chain uncoupling and mitochondrial MPT pore opening are two detrimental psychosomatic events caused by Aβ _1-42 , and these SP-233 properties may contribute to their neuroprotective effects.

In addition, using human SK-N-AS neuroblastoma, Aβ _1-42 accumulates in the mitochondrial matrix, and SP-233 completely eliminates Aβ _1-42 from the matrix. Observed. These results show that Aβ _1-42 and SP-233 have a direct effect on the mitochondrial function of human neurons, and SP-233 (when present in the culture mixture with amyloid peptide) is against Aβ-induced toxicity. Show that these cells can be protected. Taken together, these findings indicate that Aβ _1-42 has a direct toxic effect on mitochondrial function, and that SP-233 targets Aβ directly, thus protecting the respiratory chain, It shows that it provides a neuroprotective effect by providing a family of compounds that are effective in the treatment of mitochondrial diseases and disorders other than AD and other CNS neuropathologies.

  As used herein, the term mitochondrial disorder refers not only to mitochondrial diseases recognized in the art, but also to conditions where mitochondrial disorder or dysfunction plays a role in symptomatic symptoms, progression and / or prognosis. Including. The latter conditions include neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, or amyotrophic lateral sclerosis, as well as nerves caused by stroke, cerebral ischemia and other brain and spinal cord injury Includes cell death. Hexanoic acid = (22S, 25S)-(20S) -Spirostenol, including spirosta-5-en-3-yl, has been reported to protect against A-induced neurotoxicity and potential effects in AD treatment (L Lecanu et al., Steroids, 69: 1 (2004)) and also the subject of published PCT application WO / 03/077869. The mitochondrial protective effect of hexanoic acid = (22S, 25S)-(20S) -spirosta-5-en-3-yl on various stressors and in the treatment of various mitochondrial diseases is unknown to date.

  Mitochondrial disease or mitochondrial muscle disease is a group of diseases that affect mitochondria and also interferes with muscle function. Disease groups include Keynes-Sayer syndrome, Lee syndrome, mitochondrial DNA reduction syndrome (MDS), mitochondrial encephalomyopathy, lactic acidosis and stroke-like seizure symptoms (MELAS), myoclonic epilepsy with laguette red fibers (MERFF), mitochondrial neurorogastrointestinal Includes encephalomyopathy (MNGIE), neuropathy, ataxia and retinitis pigmentosa (NARP), and progressive extraocular palsy syndrome (PEO).

Symptoms of mitochondrial muscle disease include muscle weakness or exercise intolerance, cardiac disorders (cardiomyopathy) or rhythm disorders, dementia, movement disorders, stroke-like seizures, hearing loss, blindness, drooping eyelids, restricted eye movements, vomiting, and seizures including. The prognosis for these disorders ranges from progressive weakness to death. Thus, mitochondrial disorders can include ocular disorders such as optic nerve atrophy, ptosis, acquired strabismus, and ocular palsy. Mitochondrial dysfunction is due to side effects of drug therapy, hepatocellular dysfunction, endocrine tubules, and anti-cancer therapies, including muscle pressure syndrome (compartment syndrome), HAART, and drugs that damage the heart (such as anthracyclines) Can be caused by respiratory problems.

  Most mitochondrial muscle diseases develop before the age of 20, often with exercise intolerance or weakness. In physical activity, muscles are easily fatigued or weakened. Although rare, muscle spasms occur. Nausea, headache, and apnea are also associated with these disorders.

  As used herein, the term “treat” or “treatment” means any treatment of a disease or disorder associated with a mitochondrial disease or disorder in a patient and is likely to be susceptible to the disease or disorder. However, in patients who have not yet been diagnosed with the disease or disorder, prevent the onset of the disease or disorder, suppress the disease or disorder (eg, prevent the progression of the disease or disorder), Including, but not limited to, alleviating (eg, remission of symptoms of a disease or disorder) or alleviating a condition resulting from a disease or disorder (eg, suppression of symptoms of the disease or disorder). As used herein, “mitochondrial disorder” or “mitochondrial disease” is meant to include all disorders disclosed herein.

The method can also be used in vitro to prevent or reduce mitochondrial damage in transplanted or transplanted organs or tissues.
The terms “prevent” or “prevention” refer to a disease or disorder related to mitochondria in which the disease or disorder will not progress if nothing has happened in the patient, or the disorder or disease has already progressed. Means that the disorder or disease does not progress further, or that there are no symptoms that are logically observable signs of the disease.

  Preferred stereoisomers are 3S, 10R and 13S, and 20S, 22S and 25S, where the carbon skeleton is numbered consistent with the spirosten-3-ol numbering. Accordingly, a preferred compound of structural formula (I or II) is hexanoic acid = (22S, 25S)-(20S) -spirost-5-en-3β-yl (SP233). Note that the carbon atoms shown in Structural Formula (I) or (II) are saturated with hydrogen atoms unless otherwise indicated.

Unless otherwise specified, the terms “alkyl group”, prefix “alk” (as in alkoxy groups), and suffix “-alkyl” (as in hydroxyalkyl groups) are C _1-8 carbonizations, respectively. Represents a straight chain (such as a butyl group) or a branched chain (such as an isobutyl group) of hydrogen. Alkylene, alkenylene, and alkynylene represent divalent C _1-8 alkyl (such as ethylene), alkene, and alkyne radicals, respectively. The alkyl group is preferably a (C ₁ -C ₆ ) alkyl group such as a butyl group, hexyl group, methyl group, ethyl group, propyl group, or isopropyl group. The term “alkyl group” includes cycloalkyl groups, (cycloalkyl) alkyl groups, and alkyl (cycloalkyl) alkyl groups. The term “alkenyl” likewise includes an associated cycloalkenyl residue as well as an alkyl group containing 1 to 2 CH═CH units.

In particular, the (C ₁ -C ₈ ) alkyl group is a methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec-butyl group, pentyl group, 3-pentyl group, heptyl group, or octyl group It can be. The (C ₃ -C ₈ ) cycloalkyl group may be monocyclic, bicyclic, or tricyclic, and is further cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo [2.2.2] octanyl or norbornyl Groups, and various terpene and terpenoid structures. (C ₃ -C ₆ ) cycloalkyl (C ₁ -C ₂ ) alkyl group includes the above cycloalkyl group, and includes cyclopropylmethyl group, cyclobutylmethyl group, cyclopentylmethyl group, cyclohexylmethyl group, 2-cyclopropyl group An ethyl group, a 2-cyclobutylethyl group, a 2-cyclopentylethyl group, or a 2-cyclohexylethyl group can be used. A heterocycloalkyl group includes a cycloalkyl group, wherein the cycloalkyl ring system is monocyclic, bicyclic, or tricyclic, and optionally 1-2 S, non-peroxide. Or N (R ′) and 4 to 8 ring carbon atoms, for example, morpholinyl group, piperidinyl group, piperazinyl group, indanyl group, 1,3-dithian-2-yl Group, etc. All cycloalkyl or heterocycloalkyl ring systems optionally contain 1 to 3 double bonds or epoxy residues, and optionally 1 to 3 OH, (C ₁ -C ₆ It is substituted with an alkanoyloxy group, (CO), (C ₁ -C ₆ ) alkyl group, or (C ₂ -C ₆ ) alkynyl group. (C ₁ -C ₈ ) alkoxy group includes methoxy group, ethoxy group, propoxy group, isopropoxy group, butoxy group, iso-butoxy group, sec-butoxy group, pentoxy group, 3-pentoxy group, or hexyloxy group Can be included. (C ₂ -C ₆ ) alkenyl groups are vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl. A 3-pentenyl group, a 4-pentenyl group, a 1-hexenyl group, a 2-hexenyl group, a 3-hexenyl group, a 4-hexenyl group, or a 5-hexenyl group. A hydroxy (C ₁ -C ₈ ) alkyl group or a hydroxy (C ₁ -C ₈ ) alkoxy group can be an alkyl group substituted with 1 or 2 OH groups, and with these 1 or 2 OH groups, Examples of the substituted alkyl group include a hydroxymethyl group, 1-hydroxyethyl group, 2-hydroxyethyl group, 1-hydroxypropyl group, 2-hydroxypropyl group, 3-hydroxypropyl group, 1-hydroxybutyl group, 4- A hydroxybutyl group, a 3,4-dihydroxybutyl group, a 1-hydroxypentyl group, a 5-hydroxypentyl group, a 1-hydroxyhexyl group, or a 6-hydroxyhexyl group. (C ₁ -C ₂₂ ) alkylCO ₂ -can be an acetoxy group, propanoyloxy group, butanoyloxy group, isobutanoyloxy group, pentanoyloxy group, or hexanoyloxy group, or ( C ₈ -C ₂₂ ) CO _2- can also represent a residue of a natural fatty acid.

In certain embodiments of the invention, R ₁₀ and / or R ₁₂ is CH ₃ .
In another embodiment of the present invention, R ₁₆ and R ₁₇ is CH _3.
In a further embodiment of the invention, R ₁ , R ₂ and / or R ₁₂ can be H or OH.

In another embodiment, R ₁ , R ₂ , R ₄ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₁ , R ₁₂ , R ₁₄ and R ₁₅ are H.
R ₂ is, (C ₈ -C ₂₂₎ alkyl CO ₂ - or (C ₁ -C ₆₎ alkyl CO ₂ - a containing (C ₁ -C ₂₂₎ alkyl CO ₂ - can be, or OH or O It can be a (C ₁ -C ₈ ) alkyl group, or HO ₂ C (CH ₂ ) ₂ CO ₂ —.

X can be O, or NR ′ containing NH or NAc.
R ₃ may be OR ₂₃ , where R ₂₃ is a detachable hydroxy protecting group, such as a tosyl group, a mesyl group, a tri (C ₁ -C ₄ ) alkylsilyl group, THP, Eto (Et ), A benzyl group, a benzoyloxycarbonyl group, and the like. Also included in the present invention are C _3- (C ₈ -C ₂₂ ) fatty acid esters of the compounds wherein C ₃ is hydroxy substituted, with natural fatty acids being preferred.

  The various compounds of structural formulas (I) and (II) used in the practice of the present invention are shown in Table 1 below.

An effective amount of an effective compound of the invention is formulated with a pharmaceutically acceptable carrier to become a pharmaceutical composition before being approved for the treatment of a disease associated with impaired mitochondrial function. obtain. “Effective amount” or “pharmaceutically effective amount” refers to the amount of compound required to confer a therapeutic effect on a treated patient. Animal-human dose correlation (based on mg / m ² body surface area) is described in Freireich et al., Cancer Chemother. Rep., 50, 219 (1966). Body surface area can be estimated from the patient's height and weight. See, for example, Scientific Tables, Geigy Pharmaceuticals, Ardley, New York, 1970, 537. Effective doses are based on in vitro concentrations of the compound that have been found to be effective in inhibiting Aβ toxicity or otherwise protecting the mitochondria. The dosages of this compound effective for the treatment of model neuronal cell lines are disclosed below. Effective dosages will also vary by route of administration, excipient usage, and co-administration with other therapeutic agents, as will be appreciated by those skilled in the art.

The toxicity and potency of the active ingredient is determined by standard pharmaceutical procedures, for example to determine LD ₅₀ (50% lethal dose) and ED ₅₀ (50% effective dose). The dose ratio between toxic and therapeutic effects is the therapeutic index and is expressed as the ratio LD ₅₀ / ED ₅₀ . Compounds that exhibit large therapeutic indices are preferred. Although compounds that exhibit toxic side effects can be used, to design a delivery system that directs such compounds to the diseased tissue site, to minimize potential damage to uninfected cells, i.e. to reduce side effects, Caution must be taken.

  The methods, kits, compounds and pharmaceutical compositions of the present invention include crystals (such as polymorphs), enantiomers, isomers and tautomers of the described compounds and their pharmaceutically acceptable salts. Illustrative pharmaceutically acceptable salts include formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, fumaric acid, Pyruvic acid, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, mesylic acid, stearic acid, salicylic acid, p-hydroxybenzoic acid, phenylacetic acid, mandelic acid, embonic acid (pamoic acid), methanesulfonic acid, ethanesulfonic acid, benzene Prepared from sulfonic acid, pantothenic acid, toluenesulfonic acid, 2-hydroxyethanesulfonic acid, sulfanilic acid, cyclohexylaminosulfonic acid, alginic acid, b-hydroxybutyric acid, galactaric acid, and galacturonic acid.

The term “prodrug” refers to a drug or compound (active ingredient) that produces pharmacological activity as a result of conversion by metabolic processes in the body. Prodrugs are generally regarded as drug precursors and are converted to active or stronger active species through several processes, such as metabolic processes, following administration to a patient and subsequent absorption. Other products resulting from the conversion process are easily processed by the body. In general, a prodrug has a chemical functional group on it, such as an ester group or an acyl group, that reduces activity and / or renders the drug soluble or has some other property. When the chemical functional group is dissociated from the prodrug, a more active drug is born. Prodrugs are designed as reversible drug derivatives and are used as modifiers that facilitate the delivery of drugs to site-specific tissues. At present, the purpose of prodrugs is to increase the effective water solubility of therapeutic compounds to target sites where water is the primary solvent. For example, Fedorak, et al., Am. J. Physiol., 269, G210-218 (1995) describes dexamethasone-β-D-glucuronide. McLoed, et al., Gastroenterol., 106, 405-413 (1994) describes dexamethasone oxalate-dextran. Hochhaus, et al., Biomed. Chrom., 6, 283-286 (1992) describes dexamethasone sodium 21-sulfobenzoate and dexamethasone 21-isonicotinate. In addition, according to J. Larsen and H. Bundgaard, Int. J. Pharmaceutics, 37, 87 (1987), N-acylsulfonamides are evaluated as potential prodrug derivatives. According to J. Larsen et al., Int. J. Pharmaceutics, 47, 103 (1988), N-methylsulfonamide is evaluated as a prodrug derivative. Prodrugs are also described in, for example, Sinkula et al., J. Pharm. Sci., 64, 181-210 (1975). Prodrugs are also useful as synthetic intermediates in the preparation of other compounds of structural formula (I) or (II) by synthetic interconversions well known in the art. For example, a useful method for interconversion of spirostenol substituents is IT Harrison,
See Compendium of Organic Synthetic Methods, Wiley-Interscience (1971).

  The term “derivative” refers to a compound made from another compound of similar structure by exchanging or substituting one atom, molecule or group for another. For example, a hydrogen atom in a compound is substituted with an alkyl group, an acyl group, an amino group, etc. to make a derivative of the compound.

“Blood concentration” refers to the substrate concentration in plasma or serum.
“Drug absorption” or “absorption” refers to the process of movement from the site of drug administration to the systemic circulation, eg, the patient's bloodstream.

  “Bioavailability” refers to the extent to which an active ingredient (drug or metabolite) is absorbed into the systemic circulation and becomes effective at the medicinal site in the body. “Metabolism” refers to the process of chemical conversion of drugs in the body.

“Drug dynamics” refers to factors that determine the biological response observed at the drug site in response to drug concentration.
“Drug kinetics” refers to the factors that determine the achievement and maintenance of an appropriate concentration of a drug at the drug site.

“Blood half-life” refers to the time it takes for the blood concentration of a drug to decrease to 50% of its maximum concentration.
The term “about” means in the present disclosure “approximately” and encompasses parameter variables that may occur in the practice of the related art. Illustratively, the use of the word “about” indicates that dosages outside the illustrative range can be useful and safe, and such dosages are encompassed within the scope of the claims.

  The term “measurable serum concentration” refers to the serum concentration of a therapeutic agent absorbed into the bloodstream after administration (typically measured in mg, μg, or ng therapeutic amount per ml, dl, or l serum) Mean).

  The term “pharmaceutically acceptable” is used herein as an adjective, meaning that a noun modified thereby is suitable for use in a pharmaceutical product. Pharmaceutically acceptable salts include metal ions and organic ions. More preferred metal ions include, but are not limited to, suitable alkali metal (Group Ia) salts, alkaline earth metal (Group IIa) salts, and other physiologically acceptable metal ions. Examples of ions include the usual valences of aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc. Preferred organic ions include protonated tertiary amines and quaternary ammonium cations, some of which are trimethylamine, diethylamine, N, N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, There is meglumine (N-methylglucamine) and procaine. Examples of pharmaceutically acceptable acids include hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, methanesulfonic acid, acetic acid, formic acid, tartaric acid, maleic acid, malic acid, citric acid, isocitric acid, succinic acid, lactic acid , Gluconic acid, glucuronic acid, pyruvic acid, oxaloacetic acid, fumaric acid, propionic acid, aspartic acid, glutamic acid, benzoic acid, and the like, but are not limited thereto.

  The compositions of the invention are usually administered in the form of a pharmaceutical composition. Such compositions can be administered by any suitable route, eg, oral, nasogastric, rectal, transdermal, parenteral (eg, subcutaneous, intramuscular, intravenous, intrathecal, and intradermal) by injection or infusion. ), Intranasal, transmucosal, transplanted, vaginal, topical, buccal, and sublingual. The preparation typically includes buffering agents, preservatives, penetration enhancers, compatible carriers, and other therapeutic or non-therapeutic components.

The invention also includes methods of using a pharmaceutical composition comprising one or more compounds of structural formula (I) or (II) in combination with a pharmaceutically acceptable carrier or excipient. As used herein, the term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” refers to any and all solvents, dispersion media, envelopes, antifungal agents And antibacterial agents, and isotonic and absorption delaying agents. The use of such media and reagents for substrate uptake is well known in the art. The use of any medium or reagent according to the prior art is envisaged, unless it is incompatible with the composition of the present invention. Additional active ingredients can also be incorporated into the composition. In making the compositions of the present invention, the composition (s) can be mixed with pharmaceutically acceptable excipients, diluted with these excipients, or capsules, It can also be included in a carrier in the form of a package or other container. The carrier materials that can be used in preparing the compositions of the present invention are any of the excipients commonly used in pharmacy, but such carriers are compatible with the active agent and in the desired dosage form. The choice should be based on the release profile characteristics.

Illustratively, the pharmaceutical excipient is selected from the examples given below.
(A) Arabic gum, arginic acid and its salts, cellulose derivatives, methylcellulose, hydroxyethyl-cellulose, hydroxypropyl-cellulose, magnesium aluminum silicate, polyethylene glycol, rubbers, polysaccharide acids, bentonites, hydroxypropyl-methylcellulose, Gelatin, polyvinylpyrrolidone, polyvinylpyrrolidone / vinyl acetate copolymer, crospovidone, povidone, polymethacrylates, hydroxypropylmethylcellulose, hydroxypropylcellulose, starch, pregelatinized starch, ethylcellulose, tragacanth gum, dextrin, cyclodextrin, microcrystalline cellulose, sucrose Or a binder, such as glucose.

  (B) Starch, pregelatinized corn starch, pregelatinized starch, celluloses, crosslinked carboxymethylcellulose, sodium starch glycolate, crospovidone, crosslinked polyvinylpyrrolidone, croscarmellose sodium, microcrystalline cellulose, calcium, sodium alginate complex, clays Disintegrants, such as arginates, gums, or sodium starch glycolate, and any disintegrant used in tablet preparation.

  (C) Lactose, calcium carbonate, calcium phosphate, calcium hydrogen phosphate, calcium sulfate, microcrystalline cellulose, powdered cellulose, dextrose, dextran salts, dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, chloride Fillers such as sodium and polyethylene glycol.

(D) Surfactants such as sodium lauryl sulfate, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, poloxamers, bile salts, glyceryl monostearate, Pluronic ^TM series (BASF), and the like.

(E) Solubilizers such as citric acid, succinic acid, fumaric acid, malic acid, tartaric acid, maleic acid, sodium bicarbonate sodium glutarate, and sodium glutarate carbonate.
(F) A stabilizer such as any antioxidant, buffer, or acid can also be used.

  (G) Magnesium stearate, calcium hydroxide, talc, sodium stearyl fumarate, hydrogenated vegetable oil, stearic acid, glyceryl behapinate, magnesium stearate, calcium stearate, sodium stearate, stearic acid, talc, waxes, boric acid, Lubricants such as sodium benzoate, sodium acetate, sodium chloride, DL-leucine, polyethylene glycols, sodium oleate, or sodium lauryl sulfate.

(H) oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium oleate, or lauryl sulfate Wetting agents such as sodium.

  (I) Lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose, calcium hydrogen phosphate, excipient based on sucrose, powdered sugar, calcium sulfate monohydrate, calcium sulfate dihydrate, calcium lactate trihydrate Diluents such as Japanese, dextran salts, inositol, hydrolyzed grain solids, amylose, powdered cellulose, calcium carbonate, glycine, or bentonite.

  (J) Anti-caking agents or glidants such as talc, corn starch, DL-leucine, sodium lauryl sulfate, and magnesium stearate, calcium stearate, sodium stearate.

  (K) gum arabic, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerin, magnesium silicate, sodium caseinate, soybean lecithin, sodium chloride, tricalcium phosphate, dipotassium phosphate, calcium stearyl lactate, A pharmaceutically compatible carrier, such as carrageenan, monoglyceride, diglyceride, or pregelatinized starch.

  In addition, drug formulations are discussed, for example, in Remington's The Science and Practice of Pharmacy (2000). Additional considerations for drug formulation can be found in Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980. Tablets or granules containing the composition of the invention can be coated or encased in enteric coatings.

  In addition to being useful for human therapy, the present invention is also useful for other subjects including veterinary animals, reptiles, birds, exotic animals, and livestock. Includes mammals and rodents. Mammals include, for example, monkeys or lemurs, horses, dogs, pigs, or cats. Rodents include rats, mice, squirrels, or guinea pigs.

  The pharmaceutical compositions of the invention are useful when administration of an inhibitor of mitochondrial toxicity is necessary. These compositions are believed to be particularly useful in the treatment of mitochondrial diseases.

For the treatment of neurodegenerative / mitochondrial disorders, the composition of the invention is used to administer a dose of a compound of the invention in an amount sufficient to elicit a therapeutic response, such as, for example, reducing mitochondrial toxicity due to the drug. Can be provided. For example, the dose is about 5 ng to about 1000 mg, or about 100 ng to about 600 mg, or about 1 mg to about 500 mg, or about 20 mg to about 400 mg. Usually, an effective dose for body weight is in the range of about 0.0001 mg / kg to 1500 mg / kg, more preferably 1 to 1000 mg / kg, more preferably about 1 to 150 mg / kg, most preferably Is in the range of about 50 to 100 mg / kg of body weight. A single dose can be divided and administered in one to about four doses per day, and multiple doses can be administered in a single day to elicit a therapeutic effect. Illustratively, the dosage unit of the composition of the invention is, for example, about 5 ng, 50 ng, 100 ng, 500 ng, 1 mg, 10 mg, 20 mg, 40 mg, 80 mg, 100 of the compound of the invention. Containing mg, 125 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg Typical. The dosage form can be selected to suit the desired frequency of administration used to achieve a particular dose. The amount of dosage unit of the composition to be administered and the dosage regimen to treat the condition or disorder can vary depending on the various factors (subject's age, weight, gender, and symptoms, and the severity of the condition or disorder, and Depending on the route and frequency of administration) and can vary widely as is well known.

  In certain embodiments of the invention, the composition is administered to the patient in an effective amount. This means that the composition is administered such that a dose that exhibits the therapeutic effect of the compounds of the invention can be present in the patient's serum within a period of time to elicit the desired therapeutic effect. is there. Illustratively, in fasting adults (generally fasting for at least 10 hours), a therapeutically effective amount of a compound of the invention is obtained in the patient's serum from about 5 minutes after administration of the composition. The composition is administered as is. In another embodiment of the invention, a therapeutically effective amount of a compound of the invention is obtained in the patient's serum from about 10 minutes after administration of the composition to the patient. In another embodiment of the invention, a therapeutically effective amount of a compound of the invention is obtained in the patient's serum from about 20 minutes after administration of the composition to the patient.

  In yet another embodiment of the invention, a therapeutically effective amount of a compound of the invention is obtained in the patient's serum from about 30 minutes after administration of the composition to the patient. In yet another embodiment of the invention, a therapeutically effective amount of a compound of the invention is obtained in the patient's serum from about 40 minutes after administration of the composition to the patient. In certain embodiments of the invention, a therapeutically effective amount of a compound of the invention is obtained in the patient's serum between about 20 minutes and about 12 hours after administration of the composition to the patient. In another embodiment of the invention, a therapeutically effective amount of a compound of the invention is obtained in the serum of the patient within about 20 minutes to about 6 hours after administration of the composition to the patient. In yet another embodiment of the present invention, a therapeutically effective amount of a compound of the present invention will be obtained in the patient's serum within about 20 minutes to about 2 hours after administration of the composition to the patient. .

  In yet another embodiment of the present invention, a therapeutically effective amount of a compound of the present invention is obtained in the patient's serum between about 40 minutes and about 2 hours after administration of the composition to the patient. In yet another embodiment of the present invention, a therapeutically effective amount of a compound of the present invention is obtained in the patient's serum within about 40 minutes to about 1 hour after administration of the composition to the patient. become.

  In certain embodiments of the invention, the composition of the invention is administered at a dose suitable to achieve a serum concentration that is half the amount of the compound of the invention. Illustratively, after administration of the composition of the present invention, about 0.01 to about 1000 nM, or about 0.1 to about 750 nM, or about 1 to about 500 nM, or about 20 to about 1000 nM, or about 100 to about 500 A serum concentration of nM, or about 200 to about 400 nM is obtained in the patient.

  In the composition envisaged in the present invention, a therapeutic effect can be obtained by administration at an interval of about 5 minutes to about 24 hours after administration, and if desired, administration is once a day or once every two days. It is also possible. In certain embodiments of the invention, at about 10 minutes, 20 minutes, 30 minutes, or 40 minutes after administration of the composition to the patient, the patient has a half-maximum amount of the compound of the invention of about 1 μg / ml or more, Or about 5 μg / ml or more, or about 10 μg / ml or more, or about 50 μg / ml or more, or about 100 μg / ml or more, or about 500 μg / ml or more, or about 1000 μg / ml or more The composition is administered at a suitable dose so as to achieve a medium concentration.

The amount of therapeutic agent required to elicit a therapeutic effect is quantified experimentally based on, for example, the absorption rate of the reagent into the serum, the bioavailability of the reagent, and the efficacy in treating the disorder be able to. However, the specific dosage of any of the therapeutic agents of the invention to any particular patient will depend on various factors, such as the activity of the particular compound used, the age of the patient, the weight of the patient. , Overall health status, gender, diet (including whether the patient is fasting or after meals), administration time, excretion rate, drug composition, and severity of the specific disorder being treated, and It can be seen that the form of administration is included. In general, the therapeutic dose can be set to optimize safety and efficacy. Typically, dose-effect relationships obtained from initial in vitro and / or in vivo experiments are useful in deriving doses suitable for administration to patients. In general, laboratory animal studies are used as a guide to assess useful doses for the treatment of gastrointestinal disorders or diseases in accordance with the present invention. From a therapeutic protocol perspective, it should be appreciated that the dose administered will depend on a variety of factors including the particular reagent being administered, the route of administration, the particular patient condition, and the like. Generally speaking, the compound should be administered in an amount effective to achieve serum levels of the same criteria, as well as in vitro concentrations that are useful for eliciting therapeutic effects. desirable. Therefore, for the dose associated with the in vitro activity of the compound, e.g. half-maximum of 200 nM, such as high neurotoxicity due to β-amyloid and other indicators related to other indicators selected as appropriate criteria by those skilled in the art It may be desirable to administer an amount of the drug that is effective to obtain an approximate half-maximal in vivo concentration of 200 nM during a period of time that can elicit the desired therapeutic effect, such as treatment. The quantification of these parameters is well known in the art. These considerations are well known in the art and are described in standard textbooks.

To measure and quantify the effective amount of a compound of the invention delivered to a patient, standard assay methods can be used to measure the serum concentration of the compound of the invention.
The composition envisioned in the present invention exhibits a therapeutic effect at an interval of about 30 minutes to about 24 hours after administration to a patient. In some embodiments, the composition exhibits such a therapeutic effect in about 30 minutes. In another embodiment, the composition exhibits a therapeutic effect over about 24 hours, allows for once-daily administration, and improves patient compliance.

  The methods, kits, and compositions of the invention can also be used in conjunction with another therapeutic agent (eg, creatinine) for the purpose of treating or preventing mitochondrial disease (“combination therapy”). When used in conjunction with the present invention, that is, in combination therapy, additional or synergistic effects can be obtained that can reduce or eliminate many if not all of the unwanted side effects. The reduction of side effects from these drugs generally leads to, for example, the reduction of the dose required to obtain the therapeutic effect of the administered formulation.

The term “combination therapy” refers to the administration of another composition for treating or preventing a mitochondrial disorder in a patient and the composition of the present invention in conjunction with the treatment of a neurodegenerative disorder and / or a mitochondrial disorder. These adjunct activities for these therapeutic agents are included as part of specific therapies intended to obtain beneficial effects. Beneficial effects of the combination include, but are not limited to, drug kinetic or pharmacodynamic auxiliary activity resulting from the combination of therapeutic agents. The combined administration of these therapeutic agents typically takes place at a predetermined time (always substantially simultaneously, in minutes, hours, days, weeks, months, or years, depending on the selected formulation). It is done. "Combination therapy" generally refers to two or more of these therapeutic agents as part of a separate single therapy that gives concomitant and arbitrary results when used in combination with the present invention. Is not meant to encompass administering. “Combination therapy” refers to the sequential administration of these therapeutic agents, ie the administration of each therapeutic agent at a different time, and in addition, these therapeutic agents or two or more therapeutic agents are substantially substantially Means simultaneous administration. Administration that is substantially simultaneous includes, for example, administering to a patient a single tablet or capsule containing each therapeutic agent in a fixed ratio, or a single capsule or tablet of each therapeutic agent. It can be performed by administering to a patient in combination of multiple. Sequential or substantially simultaneous administration of each therapeutic agent can be performed by any suitable route. The composition of the present invention can be administered orally or nasogastrically, while other reagents used in combination can be administered by any route appropriate to the particular reagent. Such routes include, but are not limited to, the oral route, subcutaneous route, intravenous route, intramuscular route, or direct absorption from mucosal tissue. For example, the composition of the present invention can be administered orally or nasogastrically, and the therapeutic agent used in combination can be administered orally or transdermally. Continuous administration of therapeutic agents is not dangerous in the narrow sense. “Combination therapy” is further combined with other bioactive ingredients such as but not limited to analgesics, and non-medication therapy such as but not limited to surgery, It also encompasses administration of the therapeutic agents described above.

  The therapeutic compounds that make up the combination therapy can be combined into an integrated dosage form or can be in separate dosage forms intended for substantially simultaneous administration. The therapeutic agents that make up the combination therapy can also be administered sequentially with any therapeutic compound that is administered in a therapy that requires two-step administration. Thus, certain therapies require the administration of multiple therapeutic compounds sequentially, with separate active agents administered at intervals. The time interval between the multiple administration steps may further depend on the effects of feeding, depending on the properties of each therapeutic agent, such as the efficacy, solubility, bioavailability, blood half-life, and kinetic properties of each therapeutic compound, and Depending on the patient's age and condition, it can range from, for example, several minutes to several hours to several days. The optimal dosing interval can also be determined from the circadian change in the concentration of the target molecule.

  A therapeutic compound in combination therapy that is administered at the same time, substantially simultaneously, or sequentially includes, for example, one therapeutic compound by the oral route and the oral, subcutaneous, intravenous, intramuscular, or mucosal route. Includes therapies that require administration of another therapeutic agent, such as by direct absorption from the tissue. The therapeutic compound of the combination therapy is administered orally, by inhalation spray, rectal, topical, buccal, sublingual, or parenteral (eg, subcutaneous, intramuscular, intravenous, and intradermal injection) Each of the therapeutic compounds, whether administered separately or together with any of the above), is a suitable pharmaceutical formulation containing pharmaceutically acceptable excipients, diluents, or other formulation ingredients. Will be included.

  For oral administration, a pharmaceutical composition of the invention can contain a desired amount of a compound of structural formula (I) or (II), such as tablets, hard capsules or soft capsules, medicinal drops, sachets, It is administered in the form of troches, dispensing powders, granules, suspensions, elixirs, liquids, or any other form that is reasonable for oral administration. Illustratively, such pharmaceutical compositions can be prepared in the form of discrete dosage units, such as tablets or capsules, containing a predetermined amount of the active compound. Such oral dosage forms further include, for example, a buffer. Tablets, pills and the like can also be prepared by adding an enteric coat.

  Pharmaceutical compositions suitable for buccal or sublingual administration include, for example, medicinal drops containing active compounds in flavor bases such as sucrose and gum arabic, or gum tragacanth, and gelatin and glycerin, or sucrose and gum arabic Tablets containing the active compound in an inert base such as gum are included.

  Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art (eg, water). included. Such compositions can also contain, for example, wetting agents, emulsifying agents, and suspending agents, as well as sweeteners, flavors, flavorings.

Suitable liquid preparations include active compounds and β-cyclodextrin or water-soluble β-cyclodextrin derivatives (eg β-cyclodextrin = sulfobutyl ether, heptakis-2,6-di-O-methyl-β- Aqueous solutions including, but not limited to, cyclodextrin, hydroxypropyl-β-cyclodextrin, and dimethyl-β-cyclodextrin).

  The pharmaceutical composition of the present invention can also be administered by injection (intravenous, intramuscular, subcutaneous). Such injectable compositions can use, for example, saline, dextrose, or water as a suitable carrier material. If necessary, the pH value of the composition can be adjusted with an appropriate acid, base, or buffer. Suitable fillers, dispersants, wetting agents, or suspending agents (including mannitol and polyethylene glycol (such as PEG 400)) can also be included in the composition. Suitable parenteral compositions can also include lyophilized active compound in injection vials. Prior to injection, an aqueous solution may be added to dissolve the composition.

  The pharmaceutical composition can also be administered in the form of a suppository or the like. Such rectal formulations include the active compound in a total amount, for example, from about 0.075 to about 75% w / w, or from about 0.2 to about 40% w / w, or from about 0.4 to about 15% w / w. Is preferred. Carrier materials such as cocoa butter, cocoa oil, and other fats and polyethylene glycol suppository bases can be used in these compositions. Other carrier materials such as envelopes (eg, hydroxypropyl-methylcellulose coatings) and disintegrants (eg, croscarmellose sodium and cross-linked povidone) can be used if desired.

  The test compound may be in a free form or may be encapsulated in microcapsules or colloidal drug delivery systems (liposomes, microemulsions, macroemulsions, etc.).

  These pharmaceutical compositions can be prepared using any suitable pharmaceutical method, including the step of bringing into association the active compound of the invention with one or more carrier materials. In general, the compositions are a uniform and thorough mixing of the active compound with the liquid or finely divided solid carrier, or both, and thereafter the product is shaped if necessary. For example, a tablet can be prepared by optionally compressing or molding a compound powder or granule with one or more additive components. Compressed tablets are mixed with a fluid compound such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, and / or surfactant / dispersant and compressed using a suitable machine. Can be prepared. Molded tablets can be prepared by molding a compound powder moistened with an inert diluent using a suitable machine.

The tablets of the present invention can also be coated with a conventional outer coating material such as Opadry ^™ White YS-1-18027A (or another color), which accounts for about 3% of the total weight of the coated tablets. It can be the weight of the jacket. The compositions of the present invention can be formulated to be released immediately, sustained or delayed by methods well known in the art after being administered to a patient.

  If the excipient also serves as a diluent, the excipient can be a solid, semi-solid, or liquid material, which can also be a solvent, carrier, or medium for the active ingredient. Also works. Thus, the composition can be made into tablets, chewable tablets, pills, powders, medicinal drops, sachets, wafer sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solids or dissolved in liquid media) , Soft gelatin capsules, hard gelatin capsules, and sterile packaged powders.

In some embodiments of the present invention, one of the following methods or a combination thereof may be used as the manufacturing process. That is, (1) empty kneading, (2) direct compression, (3) pulverization, (4) dry granulation or non-hydrolysis granulation, (5) hydrolysis granulation, or (6) melting.
Lachman et al., The Theory and Practice of Industrial Pharmacy (1986).

  In another embodiment of the invention, the therapeutic agent of the invention is mixed with a pharmaceutical excipient to produce a solid pre-formulation composition comprising a uniform mixture of therapeutic agent and excipient. A solid composition such as a tablet can be prepared. When these pre-formulation compositions (one or more) are referred to as homogeneous, the therapeutic agent is evenly distributed throughout the composition and the composition is divided into equally useful dosage unit units ( Meaning that it can be easily subdivided into tablets, pills and capsules. Such solid pre-formulations are subdivided into the types of dosage unit as described herein.

  Compressed tablets are solid dosage forms prepared by condensing a formulation containing the active ingredient and excipients selected to aid processing and improve product properties. The term “compressed tablet” is generally a simple, uncoated tablet for oral administration that is dispensed in a single compression, or pre-compression followed by It refers to tablets that are dispensed with final compression.

  The tablets or pills of the invention can be coated or formulated into a dosage form that provides long-term active benefits. For example, a tablet or pill can contain an internal preparation and an external preparation, the latter can be configured to cover the former. In order to use enteric layers or enteric coats, a variety of polymeric acids, as well as various polymeric acids, and mixtures of polymeric acids with substances such as shellac, cetyl alcohol, and cellulose acetate are used. Substances can be used.

  For the treatment of mitochondrial disorders in patients requiring continuous administration of the composition of the invention, the use of long-term sustained release implants is suitable. As used herein, “long-term” release means that the implant is assembled and configured such that a curative amount of the active ingredient can be delivered over at least 30 days, preferably 60 days. To do. Long-term sustained release implants are well known to those skilled in the art and include some of the release systems described above.

  In another embodiment of the invention, the compound for treating a mitochondrial disorder is in the form of a kit or package comprising one or more therapeutic compounds of the invention. These therapeutic compounds of the present invention are packaged in the form of a kit or package containing doses hourly, daily, weekly, or monthly (or other cycle) to be suitable for continuous or simultaneous administration. Can be done. The present invention also includes a kit or package comprising a plurality of separately packaged dosage form units for daily administration, each dosage form unit containing at least one of the therapeutic compounds of the invention. Provide further. By using this drug delivery system, administration of all of the various embodiments of the therapeutic compound of the present invention can be facilitated. In some embodiments, the system includes multiple doses to be administered daily or weekly. The kit or package can also include other therapeutic agents that are available in combination therapy to facilitate proper administration of the dosage form. The kit or package also includes a set of instructions for the patient.

The invention is further described with reference to the detailed examples described below, using the following abbreviations:
(AChEI) acetylcholinesterase inhibitor, (Aβ) β-amyloid peptide, (ADDLs) soluble amyloid oligomer, (ANT) adenine nucleotide transferase, (APP) amyloid precursor protein, (CCCP) carbonyl cyanide 3-chlorophenylhydrazone, (FCCP) Carbonyl cyanide p-trifluoromethoxy-phenylhydrazone, (CsA) cyclosporin A, (MPT) membrane permeability transition, (MRC) mitochondrial respiration rate, (PAO) phenylarsine oxide, (SP-233) hexanoic acid = (22S, 25S)-(20S) -Spirosta-5-en-3β-yl, (SP-233) hexanoic acid = (22R, 25R)-(20α) -spirost-5-en-3β-yl.

Furthermore, the mitochondrial permeability transition (MPT) is defined as a nonspecific increase in the permeability of the inner mitochondrial membrane that occurs under adverse conditions (such as increased mitochondrial Ca ^{+ 2} content in the mitochondrial matrix or oxidative stress). Causes non-specific pore aggregates (pores) (“mega channels”) in the membrane, causing a decrease in mitochondrial membrane potential, uncoupling of mitochondrial respiration, and loss of cellular energy, all of which contribute to cell death It becomes.

[Example 1] Use of SP-233 for protection of mitochondrial function
A: Materials and methods Materials Αβ _1-42 peptide was purchased from American Peptide Company (Sunnyvale, CA, USA) and stored at -80 ° C. Anti-complex II antibody was purchased from Molecular Probes (Eugene, OR, USA) and anti-Ab _1-42 antibody was purchased from Signet Laboratories (Dedham, MA, USA). Spirostenol, hexanoic acid = (22S, 25S)-(20S) -spirosta-5-en-3β-yl (SP-233) was purchased from Interbioscreen (Moscow, Russia). Cyclosporin A (CsA) and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) were purchased from Sigma (St. Louis, MO, USA). Dulbecco's minimal basal medium (DMEM), fetal bovine serum (FBS), and SK-N-AS human neuroblastoma cells were purchased from ATCC (Manassas, VA, USA).
2. Mitochondrial isolation procedure Male Long-Evans rats weighing 230-280 g were used. Immediately after decapitation, the brain is removed and Potter- in 6 ml isolation buffer (TRIS 20 mM, sucrose 250 mM, KCl 40 mM, EGTA 2 mM, bovine serum albumin 1 mg / ml, pH 7.2, 4 ° C). Homogenization on ice was performed using an Elvejhem homogenizer (Eurostar, IKA-Verke, Staufen, Germany). The suspension was centrifuged at 2000 × g for 8 minutes to remove cell debris and nuclei. The mitochondria-containing supernatant was centrifuged at 12000 × g for 10 minutes. The resulting pellet was resuspended in 300 μl of respiration buffer (D-mannitol 300 mM, KCl 10 mM, KH2PO4 10 mM, MgCl2 5 mM, pH 7.2, 37 ° C.). This pellet is very rich in mitochondria (Zini et al. 1996). The protein concentration of the mitochondrial suspension was measured by the method of Lowry et al. 1951.
3. Preparation of another solution The Αβ _1-42 solution was re-prepared with fresh distilled water to a concentration of 500 μM. Αβ _1-42 was either freshly reconstituted or aggregates after aging by culturing the solution for 48 hours at 4 ° C. Both solutions were diluted with respiratory buffer (see above) before use and then added directly to the mitochondria at the desired concentration. In experiments using neuroblastoma cells (see below), only freshly reconstituted Αβ _1-42 solution was used. SP-233, CCCP and CsA were first dissolved in N, N-dimethylformamide (DMF) and then diluted in fresh distilled water.
4). Mitochondrial respiration monitoring Mitochondrial function was examined using Oxytherm (Hansatech, Norfolk, UK) connected to PC (Compaq, PIII 400 MHz). A 37 ° C Oxytherm thermostat was connected to the platinum-silver Clark electrode to detect O ₂ . O ₂ consumption depends on mitochondrial function and survival in the presence or absence of CCCP (uncoupler of oxidative phosphorylation) or CsA (inhibitor of MPT pore) in the presence of ADP (a precursor of ATP) Measured as a force marker. By measuring O ₂ consumption, the mitochondrial respiration rate (MRC) can be calculated and expressed as V ₃ / V ₄ . V ₄ is the base O ₂ consumption, and V ₃ is the O ₂ consumption (ATP production) after adding ADP. The effect of increasing concentrations of SP-233 (1 pM to 1 nM) on the mitochondrial respiratory chain was assessed by monitoring the gradual change of MRC. The mitochondrial concentration was adjusted to 0.4 mg protein amount / ml using a respiration buffer. Mitochondria were activated by adding malate / glutamate as a substrate for complex I of the respiratory chain. State 2 is the basal rate of mitochondrial oxygen consumption. As ATP synthesis began with the addition of ADP, the rate of oxygen consumption increased dramatically and reached State 3. When the supply of ADP is depleted, the O ₂ consumption rate returns to the baseline (State 2), which is called State 4 (Chance and Williams 1956). The control for each experiment was dependent on basal respiration in the presence of ADP.
5. Effect of SP-233 on CCCP-induced uncoupling of oxidative phosphorylation
Adding CCCP (1 μM) uncouples the respiratory chain reaction and increases O ₂ consumption. To assess the effect of SP-233 on CCCP-induced uncoupling, the mitochondrial fraction was incubated for 3 minutes at 37 ° C in the presence of SP-233 before adding malic / glutamic acid, and 1 minute later Was added.
6). Evaluation of the effect of SP-233 on CsA-induced hypoxia Hypoxia was induced by adding CsA (1 μM) to the mitochondrial culture medium (Zini et al.
al. 1996). This experiment served as a control by adding ADP in the presence of CsA and in the absence of SP-233. As described above, SP-233 or its solvent was added first and the culture was carried out for 1.5 minutes before adding CsA. The substrate malic acid / glutamic acid was then added after 1.5 minutes and ADP was added after 2.5 minutes.
7). Evaluation of protective effect of SP-233 against Αβ _1-42- induced mitochondrial dysfunction
SP-233 was cultured in the culture mixture for 3 minutes, and Αβ _1-42 was incubated at 37 ° C for 1.5 minutes before adding the substrates malic acid and glutamic acid. ADP was added 1 minute after the addition of malic acid / glutamic acid. The control of the effect of SP-233 on Αβ _1-42 was the same as in the experiment conducted in the absence of ADP.
8). Evaluation of the protective effect of SP-233 against Aβ _1-42 induced toxicity in human neuroblastoma cells Human neuroblastoma SK-N-AS cells contain 10% FBS in a 96-well plate (7x104 cells / well) The cells were seeded in DMEM medium and cultured at 5% CO ₂ and 95% humidity. Thereafter, Αβ _1-42 (0.1, 1 and 10 μM) or its solvent was added, and the culture was continued for 72 hours in the presence or absence of SP-233 (1 μM). Aβ cytotoxicity was assessed using the 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT) assay (Trevigen, Gaithersburg, MD).
9. Immunocytochemical analysis of Αβ _1-42 uptake in mitochondria of human neuroblastoma cells Human neuroblastoma SK-N-AS cells were seeded on 13-mm diameter coverslips (20,000 cells / covers) and 10% The cells were cultured overnight in DMEM medium containing FBS at 37 ° C. and 5% CO ₂ . Thereafter, Αβ _1-42 (10 μM) or its solvent was added, and the culture was continued for 3 hours in the presence or absence of SP-233 (1 μM). To assess mitochondrial Αβ _1-42 uptake, a mouse monoclonal antibody raised against mitochondrial respiratory chain complex II, anti-OxPhos complex II 70-kDa subunit (Molecular Probes, Eugene, OR, USA), and Colocalization analysis was performed using a rabbit polyclonal antibody (Signet Laboratories, Dedham, MA, USA) raised against 33-42 amino acid residues of Αβ _1-42 . Neuroblastoma cells were washed with 1X phosphate buffered saline (PBS), fixed with methanol at -20 ° C, and rinsed with 1X PBS. Subsequently, the cells were blocked with 1X PBS containing 10% donkey serum for 30 minutes, and then the primary antibody (1/200) prepared against complex II in 1X PBS containing 1.5% donkey serum and 0.01% Triton X100. And 24 hours at 4 ° C. After the specimen was washed three times with 1X PBS, a donkey anti-mouse rhodamine-labeled secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) was added at a dilution of 1/200, and the culture was continued at room temperature for 2 hours. In addition, primary antibodies raised against 33-42 amino acid residues of Αβ _1-42 were used at 1/500 dilution according to the same protocol. Specific staining was demonstrated using a donkey anti-rabbit secondary antibody labeled with the fluorescent marker Alexa Fluor ^R 488 (Molecular Probes, Eugene, OR, USA). The cover glass is then mounted on a water-soluble mounting agent containing 4 ', 6-diamidino-2-phenylindole-2-hydrochloride (DAPI) (Vect
or Laboratories. Burlingame, CA, USA). Confocal images were taken using an Olympus Fluoview BX61 Laser Scanning microscope.
10. Statistical analysis In each experiment, the results are expressed as mean ± standard deviation. Comparison between groups was performed by ANOVA according to Dunnett's test.
B: Results 1. Effects of SP-233 on the regulation of mitochondrial respiration
SP-233 was analyzed at concentrations of 10, 30, and 100 pM. Even at a low concentration of 10 pM, SP-233 significantly reduced rat brain mitochondrial MRC by 50% compared to the control group (p <0.001) (FIG. 1). Although there was no significant difference in the effects caused by different concentrations of SP-233, MRC decreased progressively over the range of concentrations from 10 to 100 pM, so the results show a correlation between concentration and effect Showed a trend.
2. Effects of SP-233 on changes in mitochondrial respiration rate caused by CCCP or CsA
1 μM uncoupler CCCP increased rat brain mitochondrial O ₂ consumption to 150% of baseline (p <0.01) (FIG. 2a). At all experimental concentrations of SP-233 (1-1000 pM), this metabolic effect of CCCP was completely inhibited (p <0.001). Inhibition of CCCP-induced uncoupling by SP-233 was related to a decrease in O ₂ consumption to 60-65% of basal levels and was concentration independent. SP-233 did not negate the decrease in MRC caused by CsA, but conversely amplified the effect of CsA in a concentration-dependent manner (FIG. 2b).
3. Effects of fresh Aβ _1-42 and old Aβ _1-42 on mitochondrial respiration rate
Aβ _1-42 significantly reduced MRC in rat brain mitochondria even at a low concentration of 0.1 pM (FIG. 3). This effect was more apparent with fresh amyloid peptides than with older ones, with 55-63% and 43-53% inhibitory effects, respectively. This difference appears to indicate that non-aggregated Aβ _1-42 penetrates much more into the mitochondria than older, aggregated peptides.
4). Effects of SP-233 on changes in mitochondrial respiration rate induced by fresh Aβ _1-42 and old Aβ _1-42 Fresh Aβ _1-42 reduced MRC by 71% compared to controls. The effect was partially inhibited by SP-233 (FIG. 4a). At the lowest experimental concentration of SP-233 (1 pM), MRC returned to approximately 40% of the control value, and MRC was significantly increased compared to that obtained with Aβ _1-42 alone (38.56).
± 0.34 vs. 28.93 ± 1.75, p <0.001). Older Aβ _1-42 reduced 51% MRC compared to control, _whereas SP-233 did not significantly inhibit this effect (FIG. 4b).
5. The effect of SP-233 on Aβ _1-42- induced toxicity in SK-N-AS human neuroblastoma cells. Figure 5 shows the effect of SP- on the decrease in Aβ _1-42- induced SK-N-AS cell viability. The influence which 233 exerts is shown. 1 μM SP-233 inhibited neurotoxicity induced by three experimental concentrations of Aβ _1-42 by 19%, 26%, and 28%, respectively (p <0.01).
6). Effect of SP-233 on Aβ _1-42 uptake into mitochondria of SK-N-AS human neuroblastoma cells
Aβ _1-42- treated neuroblastoma cells show a strong immune response that co-localizes with complex II of the mitochondrial respiratory chain, indicating that Aβ _1-42 enters and resides inside the mitochondria Prove that.
Treatment with SP-233 _{abolishes the} Aβ _1-42 immune response, indicating that SP-233 blocks Aβ _1-42 uptake into cells and mitochondria.
C: Discussion As is well known, AD is clinically characterized by progressive cognitive impairment and memory loss. Since the early days, the “memory” view of the disease has been linked to a decline in the central cholinergic pathway, reflecting a decrease in synaptic concentrations of acetylcholine (ACh). Therefore, drug discovery research focused mainly on the recovery of ACh levels in the brain, leading to the development of acetylcholinesterase inhibitors (AChEIs), tacrine, the prototype of this type of compound. However, despite the promising clinical data, the beneficial effects of tacrine are negligible, and despite the development of new production of AChEIs, represented by rivastigmine, galantamine, and donepezil, AD patients compared to tacrine. Delayed onset was not shortened. This short (one to two year) delay in further progression of AD symptoms (Tariot and Winblad, 2001; Waldemar et al. 2001; Grossberg et al. 2004) is precious to patients and their relatives, but probably cholinergic This is due to the progressive decline of sex neurons and is a limitation of the use of AChEIs. In addition, in a recent study conducted in 565 community-dwelling patients with mild to moderate AD, donepezil was not cost effective and had an effect below the minimum associated threshold (Courtney et al.
2004). Thus, although there has been no major advance in AD drug discovery, memantine, an N-methyl-D-aspartate (NMDA) receptor antagonist, has recently been approved to moderately treat severe AD (Livingston and Katona 2004), there is an urgent need to develop drugs that are more effective than AChEIs.

The etiology of AD is well documented only in the familial early-onset form of the disease and includes mutations in the APP, presenilin-1 or presenilin-2 gene. In contrast, the cause of late-onset sporadic AD, which represents about 95% of all AD cases, has not yet been established. Numerous hypotheses have emerged, which suggest the possibility that energy dysfunction resulting from mitochondrial dysfunction and oxidative damage are responsible for AD (Schulz et al.
al. 1997; Beal, 2000).

Evidence that mitochondrial dysfunction is related to the etiology of AD is the recent finding that SP-233 (natural spirostenol) protects neuronal PC12 cells from Aβ _1-42- induced neurotoxicity (Lecanu et al. 2004) Together, it triggered the study of SP-233's potential to restore isolated mitochondrial function that was damaged by exposure to Aβ _1-42 . Taken together, this study suggests that SP-233 may influence the basic mechanisms involved in Aβ and related to the pathogenesis of AD.

The effect of Aβ on mitochondrial function has been extensively studied over the past decade, and the data generated by these studies suggest various effects of peptides that can lead to mitochondrial dysfunction. Aβ _1-42 and Aβ _25-35 fragments, in neuronal mitochondria, resemble key enzymes such as respiration and succinate dehydrogenase, α-ketoglutarate dehydrogenase, pyruvate dehydrogenase, and cytochrome oxidase. It has been shown to inhibit activity (Kaneko et al. 1995; Casley et al. 2002a). Inhibition of another complex of the respiratory chain has also been reported to occur in neuronal mitochondria exposed to Aβ _25-35 (Pereira et al. 1998; Canevari et al. 1999; Casley et al. 2002b), and This same peptide fragment promotes pore opening associated with MPT (Moreira et al. 2001; Moreira et al. 2002; Bachurin et al. 2003).

The results herein above further demonstrate that Aβ _1-42 inhibits mitochondrial respiration in rat brain mitochondria, as evidenced by a decrease in MRC. This inhibition also occurs at concentrations as low as 0.1 pM Aβ _1-42 in both freshly prepared and older peptides (see FIG. 3). The inhibitory effect is more evident in the fresh form, probably because Aβ _1-42 polymers are less likely to cross the mitochondrial membrane than monomers or low polymers such as ADDLs . These latter forms are therefore more likely to have a detrimental effect on the respiratory chain and are more toxic in nature. Also, the lack of protection by SP-233 in mitochondria that received old / aggregated Aβ _1-42 (see Figure 4), SP-233 binds to the monomeric amyloid peptide aggregates It seems to be because it cannot be combined in the same way as.

Confirming past data showing the recovery effect of SP-233 on ATP synthesis in PC12 cells exposed to Aβ _1-42 (Lecanu et al. 2004), SP-233 is a freshly prepared Aβ _{1- It} can partially inhibit the decrease in MRC induced by ₄₂ and can further protect mitochondrial function. Since SP-233 is activated at very low concentrations (picomolar range), its mechanism of action appears to be relatively specific. The data in the present invention further show that SP-233 protects neurons against Aβ _1-42 neurotoxicity by binding to peptide monomers and inhibiting the formation of neurotoxic low-polymer ADDLs. Supporting recent results that show that (Lecanu et
al. 2004).

Confocal microscopic analysis performed on human neuroblastoma cells to show that Aβ _1-42 colocalizes with respiratory chain complex II suggests that Aβ _1-42 can cross both the cell and the mitochondrial membrane is doing. When both SP-233 and Aβ _1-42 are present in the culture medium, immunocytochemical staining corresponding to the amyloid peptide is completely suppressed, and this “removal” effect of SP-233 is a partial recovery of MRC. It was seen with. In addition, SP-233 partially suppressed the neurotoxic effect of Aβ _{1-42 on} the same human neuroblastoma cells. This result is particularly interesting in view of recent results (Fernandez-Vizarra et al. 2004) showing the presence of amyloid deposits in the mitochondria of post-mortem AD brain cortical pyramidal neurons. These histological features have been described as a very early phenomenon in the course of the disease, appearing before the formation of anti-spiral filaments and leading to neuronal cell death.

SP-233 binding to Aβ _1-42 and, after the “removal” effect has been demonstrated, SP-233 may directly affect mitochondria resulting in an additive protective effect on Aβ _1-42 Sex was studied. In this regard, it has been previously demonstrated that Aβ promotes brain mitochondrial MPT pore opening, causes dysfunction, and that this effect is counteracted by CsA (Moreira et al. 2001; Bachurin et al 2003; Abramov et al. 2004). This study shows that SP-233, like CsA, decreases MRC. SP-233 also amplifies the effect of CsA, suggesting that SP-233 has CsA-like properties and thereby inhibits MPT pore opening, thereby inducing Aβ _1-42 A protective effect against mitochondrial dysfunction is obtained.

SP-233 was also able to counteract the uncoupling of oxidative phosphorylation induced by CCCP (see Figure 2a). Aβ _{1-42 disrupts} mitochondrial membrane structure (Kremer et al. 2001; Rodrigues et al. 2001) and has been shown to change mitochondrial membrane fluidity in the AD brain (Mecocci et al, 1996) Both phenomena cause mitochondrial uncoupling. Thus, although the “reconjugation” effect of SP-233 has not yet been fully elucidated, this is believed to contribute to the recovery of mitochondrial function described herein. Furthermore, the combination of the removal effect of SP-233 (possibly including regulation of MPT pores) and the reconjugation effect makes SP-233 1
The reason for negating the effect of Aβ _1-42 at a low concentration of pM is considered to be clear.
[Example 2] Spirostenol, hexanoic acid = (22R, 25R) -20a-spirosta-5-en-3β-yl inhibits mitochondrial Aβ uptake in neurons and suppresses Aβ-induced mitochondrial dysfunction
A: Materials and methods material
Aβ _1-42 peptide was purchased from American Peptide Company (Sunnyvale, CA). Anti-complex II antibody was purchased from Molecular Probes (Eugene, OR), and anti-Aβ _1-42 antibody was purchased from Signet Laboratories (Dedham, MA). Spirostenol (SP-233) was purchased from Interbioscreen (Moscow, Russia). Cyclosporin A (CsA), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), carbonylcyanide p-trifluoromethoxy-phenylhydrazone (FCCP), rotenone, malonic acid, misothiazole, potassium cyanide (KCN), oligomycin, and phenyl Arsine oxide (PAO) was obtained from Sigma (St Louis, MO). Dulbecco's minimal basal medium (DMEM), fetal bovine serum (FBS), and SK-N-AS human neuroblastoma cells were purchased from ATCC (Manassas, VA).
2. Isolation of the mitochondrial fraction To isolate the mitochondrial fraction, a rat weighing 230-350 g was decapitated, the brain was immediately removed, and 6 ml of ice-cold isolation buffer (TRIS 20 mM, sucrose 250 mM, KCl 40 mM, EGTA
Homogenization on ice was performed using Potter-Elvejhem homogenizer (Eurostar, IKA-Verke, Staufen, Germany) containing 2 mM, bovine serum albumin 1 mg / ml, pH 7.2). The suspension was centrifuged at 2000 xg for 8 minutes at 4 ° C to remove cell debris and nuclei. The mitochondrial supernatant was centrifuged at 12000 xg for 10 minutes at 4 ° C. The precipitate was resuspended in 300 μl of respiration buffer (D-mannitol 300 mM, KCl 10 mM, KH ₂ PO 4 10 mM, MgCl ₂ 5 mM, pH 7.2, 37 ° C.). This precipitate is very abundant in mitochondria (Zini et al. 1996). The protein concentration of the mitochondrial suspension was measured by the Lowry method.
3. Measurement of mitochondrial respiration Mitochondrial respiration was measured using Oxytherm (Hansatech, Norfolk, UK) connected to PC (Compaq, PIII 400 MHz). To detect O ₂ , an Oxytherm 37 ° C culture vessel was connected to a platinum-silver Clark electrode. O ₂ consumption was used as a marker of mitochondrial respiration in the presence of ADP (a precursor of ATP) and in the presence or absence of the following substances: CCCP (uncoupler of oxidative phosphorylation), CsA (Inhibitors of membrane permeable transition pores), or Aβ _1-42 . By measuring O ₂ consumption, the mitochondrial respiration rate (MRC) defined by V ₃ / V ₄ can be calculated, where V ₄ is the basal O ₂ consumption and V ₃ is after adding ADP O ₂ consumption (ATP output). The final concentration of mitochondria was adjusted to 0.4 mg protein / mL using respiratory buffer. Mitochondria are activated by the addition of malate / glutamate, a substrate of the complex I of the respiratory chain. State 2 is defined as the basal rate of mitochondrial oxygen consumption. As ATP synthesis begins with the addition of ADP, oxygen consumption increases dramatically and reaches State 3. When the ADP supply is depleted, the O ₂ consumption rate returns to the baseline (State 2), which is called State 4 (Chance and Williams 1956). The effects of all experimental treatments on respiration were compared to baseline respiration or mitochondrial respiration in the presence of ADP alone.

To examine the effect of SP-233 on CCCP-induced mitochondrial dysfunction, isolated mitochondria were cultured in SP-233 for 3 minutes at 37 ° C before adding malic / glutamic acid. After 1 minute, CCCP was added. To examine the effect of SP-233 on CsA-induced mitochondrial dysfunction, isolated mitochondria were cultured in SP-233 for 1.5 minutes before adding CsA. Malic acid / glutamic acid was added after 1.5 minutes, and ADP was added after 2.5 minutes. Finally, to investigate the effect of SP-233 on mitochondrial dysfunction induced by Aβ _1-42 , the mitochondrial fraction was added for 3 minutes in SP-233 and further Aβ ₁₋ before adding malate / glutamate. Incubated at 37 ° C for 1.5 minutes in ₄₂ . One minute after adding malic / glutamic acid, ADP was added. In all experiments, SP-233, CCCP, and CsA were first dissolved in N, N-dimethylformamide (DMF) and then diluted in fresh distilled water. Aβ _1-42 used in these experiments was reconstituted with fresh distilled water to a concentration of 500 μM. Aβ _1-42 was either freshly reconstituted or agglutinated after the solution was incubated at 4 ° C. for 48 hours to become old. Both solutions were diluted with respiratory buffer before use and then added directly to mitochondria to the desired concentration.
4). Cell Culture Human neuroblastoma SK-N-AS cells were seeded in a 96-well plate (7 × 10 ⁴ cells / well) in DMEM medium containing 10% FBS and cultured at 5% CO ₂ and 95% humidity. Cultures were treated with the following substances in the presence or absence of various concentrations of SP-233. : Αβ _1-42 (0.1, 1 and 10 μM), rotenone (50 nM), malonic acid (100 mM), misothiazole (0.3 μM), KCN (12 mM), oligomycin (0.5 μg / ml), FCCP (3 μM), and PAO (0.25 μM). Cell viability was measured using the 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT) assay according to the manufacturer's instructions (Trevigen, Gaithersburg, MD ). In experiments involving Aβ _1-42 , cell viability was measured 72 hours after invasion in the presence or absence of 1 μM Aβ _1-42 . Only freshly reconstituted Aβ _1-42 solution was used. In experiments involving mitochondrial toxins (rotenone, malonic acid, misothiazole, KCN, and oligomycin), cell viability is in the presence or absence of 1, 3, 10, 30, or 100 μM Aβ _1-42 Below, measurements were taken 6 or 24 hours after invasion. SP-233 was added to the culture one hour before adding the mitochondrial toxin. In experiments involving FCCP and PAO, parallel cell cultures were performed in the presence or absence of increasing concentrations of SP-233 in the range of 1-100 μM one hour prior to the addition of FCCP or PAO. Cell viability was assessed after 24 hours. FCCP, rotenone, malonic acid, misothiazole, KCN, oligomycin, and PAO were all first dissolved in ethanol as stock solutions and then diluted in culture medium to the desired final concentration. The proportion of ethanol in the culture medium was 0.09%.
5. Immunocytochemical analysis of Aβ _1-42 Human neuroblastoma SK-N-AS cells were seeded on a 13-mm diameter cover glass (20,000 cells / cover glass) and 37 ° C in DMEM medium containing 10% FBS. And overnight with 5% CO ₂ . Thereafter, the cells were cultured with Αβ _1-42 (10 μM) or its solvent in the presence or absence of SP-233 (1 μM) for 3 hours. To evaluate mitochondrial Αβ _1-42 uptake, anti-OxPhos complex II 70-kDa subunit mouse monoclonal antibody (Molecular Probes, Eugene, OR), raised against mitochondrial respiratory chain complex II, and Αβ _{1 Colocalization} analysis was performed using a rabbit polyclonal antibody (Signet Laboratories, Dedham, Mass.) Raised against 33-42 amino acid residues of _-42 . Neuroblastoma cells were washed with phosphate buffered saline (PBS), fixed with methanol at -20 ° C, and rinsed with PBS. Subsequently, the cells were blocked with PBS containing 10% donkey serum for 30 minutes, and then cultured at 4 ° C for 24 hours with an antibody prepared against complex II (PBS containing 1.5% donkey serum and 0.01% Triton X100). (1: 200). The cells were further washed with PBS and cultured for 2 hours at room temperature with a donkey anti-mouse rhodamine-labeled secondary antibody (1: 200, Jackson ImmunoResearch, West Grove, PA). In addition, an antibody prepared against 33-42 amino acid residues of Αβ _1-42 (1: 500) and a donkey anti-rabbit secondary antibody labeled with the fluorescent marker Alexa Fluor ^R 488 (Molecular Probes, Eugene, OR) Used to continue the same continuous steps. Coverslips were mounted with a water-soluble mounting agent containing 4 ′, 6-diamidino-2-phenylindole-2-hydrochloride (DAPI; Vector Laboratories, Burlingame, Calif.). Confocal image Olympus Fluoview
Photographed using a BX61 Laser Scanning microscope.
6). Statistical analysis All results are expressed as mean ± standard deviation. Comparison between groups was performed using analysis of variance (ANOVA) according to Dunnett's test. P value <0.05 was considered significant.
B: Results 1. Effects of SP-233 on changes in mitochondrial respiration induced by CCCP or CsA First, the effect of SP-233 on mitochondrial respiration at concentrations ranging from 1 fM to 100 pM was determined by analyzing the progressive changes in MRC. Evaluation was made (FIG. 6). As the concentration of SP-233 increased, the MRC gradually decreased. Even at a low concentration of 1 nM, SP-233 significantly reduced MRC in rat brain mitochondria compared to the control group (p <0.001, FIG. 6b). At SP-233 concentrations of 10 pM or more than 10 pM, MRC decreased by 50%.

CCCP uncouples oxidative phosphorylation and increases O ₂ consumption. As shown in FIG. 7a, 1 μM CCCP increased rat brain mitochondrial O ₂ consumption to 150% of basal (p <0.01). SP-233 in the concentration range of 1-1000 pM completely counteracted this effect (p <0.001 in all), and inhibition of CCCP-induced uncoupling in the presence of these concentrations of SP-233 resulted in O ₂ consumption. It was associated with a reduction to 60-65% of the basal amount.

Hypoxia is induced by adding 1 μM CsA to the mitochondrial culture medium (Zini et al.
1996). SP-233 did not negate the reduction of MRC induced by CsA. In contrast, SP-233 amplified this effect in a concentration-dependent manner (FIG. 7b).
2. Effects of SP-233 on changes in mitochondrial respiration induced by fresh Aβ _1-42 and old Aβ _1-42
When Aβ _1-42 was added to isolated mitochondria, MRC was significantly reduced even at a concentration of 0.1 pM (FIG. 8). This effect was more apparent with fresh amyloid peptide than with the old, with the former inhibitory effect being approximately 10% greater. _{Nonaggregated} Aβ _1-42 appears to penetrate much more into the mitochondria than older, aggregated peptides. As shown in FIG. 9a, when fresh Aβ _1-42 was added to isolated mitochondria, MRC was reduced to 71% of the control value, and this decrease was partially reversed by SP-233. At the lowest experimental concentration of SP-233 (1 pM), MRC recovered to approximately 39% of the control value. MRC was significantly increased compared to that obtained with Aβ _1-42 alone (38.56 ± 0.34 vs. 28.93 ± 1.75, p <0.001). Older Aβ _1-42 reduced MRC by 51% compared to control, whereas SP-233 had no significant effect on the change in MRC induced by this older Aβ _1-42 (FIG. 9b). ).
3. SK-N-AS human neuroblastoma influence diagram 10 SP-233 to A [beta] _1-42-induced toxicity in the cells on the, SP-233 exerts against A [beta] _1-42-induced neurotoxicity in SK-N-AS cells Show the impact. The addition of 1 μM SP-233 in the presence of all three experimental concentrations of Aβ _1-42 significantly increased cell viability.
SP-233 has 19% (p <0.01 compared to Aβ _1-42 alone), 26% (p <0.01), neurotoxicity induced by 0.1, 1, and 10 μM Aβ _1-42 , respectively. And 28% (p <0.01).
4). Effect of SP-233 on Aβ _1-42 uptake into mitochondria of SK-N-AS human neuroblastoma cells FIG. 11 shows DAPI staining (a1, b1 and c1 in SK-N-AS human neuroblastoma cells). ), Representative images of immunofluorescent labels for Aβ _1-42 (a2, b2 and c2) and immunofluorescent labels for complex II 70-kDa subunit (a3, b3 and c3). The merged images are shown in Figures a4, b4 and c4. Neuroblastoma cells treated with Aβ _1-42 colocalized with the complex II label of the mitochondrial respiratory chain (FIG. 11b4) and showed a strong Aβ _1-42 immune response (FIG. 11b2). This proves that Aβ _1-42 enters the mitochondria and resides within it. The Aβ _1-42 immune response was counteracted in the presence of SP-233 (FIG. 11c2), indicating that SP-233 blocks Aβ _1-42 uptake into cells and mitochondria.
5. Neuroprotective effect of SP-233 against inhibitors of mitochondrial complex in SK-N-AS cells
SP-233 did not show any neuroprotective effect against inhibitors of complexes I, II, and III of the mitochondrial respiratory chain (FIGS. 12a, b, and c). At a concentration of 1 μM, SP-233 showed a significant protective effect against rotenone and 100 μM SP-233 showed a significant protective effect against misothiazole (both p <0.05). Although this protective effect was significant, the magnitude of the effect was small in both cases. In contrast, SP-233 exerted a significant beneficial effect on toxicity induced by the complex IV inhibitor KCN and the complex V inhibitor oligomycin (FIGS. 12d and 12e). . Low concentrations of SP-233 (1, 3, and 10 μM) are active against KCN, while high doses (30 and 100 μM) do not show beneficial effects. The protective effect of SP-233 on oligomycin was dose dependent, although 10 μM SP-233 did not show a statistically significant neuroprotective effect. These data indicate that SP-233 protects SK-N-AS cells against inhibition of complexes IV and V.
6). Neuroprotective effects of SP-233 against PAO and FCCP in SK-N-AS cells Adenine nucleotide transferase (ANT) is a channel protein located in the inner mitochondrial membrane, and in the physiological state, has a 1: 1 ratio of ATP Unload ADP. Inhibition of ANT by PAO promotes the opening of the permeable transition pore and induces apoptosis. 1 μM SP-233 reduced the toxic effect of PAO by 25% in SK-N-AS neurons (FIG. 13, p <0.01). SP-233 concentrations above 1 μM were not associated with neuroprotective effects. In addition, SP-233 exerted a slight but significant effect (p <0.01) on respiratory chain uncoupling induced by FCCP at a concentration of 100 μM (FIG. 14).
C: Consideration
AD is clinically characterized by progressive cognitive impairment and memory loss. Early on, the “memory” view of the disease has been linked to a decline in the central cholinergic pathway, reflecting a decrease in acetylcholine (ACh) synaptic levels. Therefore, drug discovery research has focused primarily on restoring ACh levels in the brain, leading to the development of acetylcholinesterase inhibitors (AChEIs). However, although these drugs are valuable to AD patients and their caregivers, AChEIs are symptomatic drugs that do not stop the progression of the disease, but only delay it for a limited time (Tariot and Winblad, 2001; Waldemar et al. 2001; Grossberg et al. 2004). Memantine, an N-methyl-D-aspartate (NMDA) receptor antagonist, has recently been approved to moderately treat severe AD (Livingston and Katona 2004), but new drugs and new treatments that target the pathogenesis of AD Laws are still urgently needed.

The etiology of AD is well documented in the familial early-onset form of the disease and includes mutations in the APP (Presenilin-1 or Presenilin-2) gene. In contrast, the cause of late-onset sporadic AD, which represents about 95% of all AD cases, has not yet been established. Numerous hypotheses have emerged, in which it has been proposed that energy dysfunction resulting from mitochondrial dysfunction and oxidative damage may be responsible for AD (Schulz et al. 1997; Beal, 2000). This mitochondrial dysfunction hypothesis, spin loss tenor derivatives SP-233 is to protect the PC12 cells of neurons from Aβ _1-42-induced neurotoxicity along with the recent discovery that (Lecanu et al. 2004), SP-233 is Aβ _1-42 It was an opportunity to study whether it was possible to restore the function of isolated mitochondria exposed to sucrose. The results herein show that SP-233 protects mitochondrial function from the toxic effects of Aβ _{1-42 in} both isolated rat brain mitochondria and human neuroblastoma cells. These findings suggest that SP-233 may influence the basic mechanisms associated with Aβ-induced neurotoxicity and related to the pathogenesis of AD.

The impact of Aβ on mitochondrial function has been extensively studied over the past decade, and the data generated by these studies suggest that various effects of peptides can cause mitochondrial dysfunction. Aβ _1-42 and Aβ _25-35 fragments, in neuronal mitochondria, resemble key enzymes such as respiration and succinate dehydrogenase, α-ketoglutarate dehydrogenase, pyruvate dehydrogenase, and cytochrome oxidase. It has been shown to inhibit activity (Kaneko et al. 1995; Casley et al. 2002a). Inhibition of the respiratory chain complex has also been reported in neuronal mitochondria exposed to Aβ _25-35 (Pereira et al.
al. 1998; Canevari et al. 1999; Casley et al. 2002b) and this same peptide fragment promotes pore opening associated with MPT (Moreira et al. 2001; Moreira et al. 2002; Bachurin et al. 2003).

In addition, the results herein show that Aβ _1-42 inhibits mitochondrial respiration in rat brain mitochondria, as evidenced by a decrease in MRC. Furthermore, this inhibition occurred even at concentrations as low as 0.1 pM Aβ _1-42 in both freshly prepared and older peptides. Since the Aβ _1-42 polymer is less likely to cross the mitochondrial membrane than monomers or low polymers such as ADDLs, this inhibitory effect appears to be more apparent in the fresh form. These latter forms are more prone to deleterious effects on the respiratory chain and are more toxic in nature. Also, the lack of protection by SP-233 in mitochondria exposed to old / aggregate Aβ _1-42 is similar to that SP-233 binds monomers to amyloid peptide aggregates. This seems to be because they cannot be combined.

Interestingly, if we look at past data showing the recovery effect of SP-233 on ATP synthesis in PC12 cells exposed to Aβ _1-42 (Lecanu et al. 2004), SP-233 is freshly prepared. It can partially inhibit the decrease in MRC induced by Aβ _1-42 . The result that SP-233 is activated at very low concentrations suggests that its mechanism of action is relatively specific. The data described herein further demonstrate that SP-233 binds to peptide monomers and inhibits the formation of neurotoxic low-polymer ADDLs, thereby preventing neuronal cells against Aβ _1-42 neurotoxicity. Supports recent results that show protection (Lecanu et al. 2004).

In demonstrating that Aβ _1-42 colocalizes with respiratory chain complex II, confocal microscopic analysis performed in human neuroblastoma cells showed that Aβ _1-42 can cross both the extracellular and mitochondrial membranes. Suggests. When both SP-233 and Aβ _1-42 were present in the culture medium, the amyloid peptide label was completely suppressed, and this “removal” effect of SP-233 was seen with partial recovery of MRC. . In addition, SP-233 partially suppressed the neurotoxic effects of Aβ _{1-42 on} the same human neuroblastoma cells. This result is particularly interesting in view of recent results (Fernandez-Vizarra et al. 2004) showing the presence of amyloid deposits in the mitochondria of post-mortem AD brain cortical pyramidal neurons. These histological features have been described as a very early phenomenon in the course of the disease, appearing before the formation of anti-spiral filaments and leading to neuronal cell death. Based on this argument, inactivation of Aβ _1-42 by SP-233 justifies the use of SP-233 in AD treatment as described herein (Lecanu et al. 2004).

SP-233 binding to Aβ _1-42 and, after the “removal” effect has been demonstrated, SP-233 may directly affect mitochondria resulting in an additive protective effect on Aβ _1-42 Sex was studied. In this regard, it has been previously demonstrated that Aβ promotes brain mitochondrial MPT pore opening, causes dysfunction, and that this effect is counteracted by CsA (Moreira et al. 2001; Bachurin et al 2003; Abramov et al. 2004). This study shows that SP-233, like CsA, decreases MRC. SP-233 also amplifies the effect of CsA, suggesting that SP-233 has CsA-like properties. That is, SP-233 inhibits MPT pore opening, thereby _providing a protective effect against Aβ _1-42- induced mitochondrial dysfunction. This hypothesis is supported by the protective effect of SP-233 against PAO (a protein that is a tyrosine phosphatase inhibitor) known to promote MPT (Korge
et al., 2001). Thus, SP-233 restored neuronal viability by 25% at a concentration of 1 μM, while higher concentrations of SP-233 were not accompanied by a protective effect. The effect of SP-233 on opening the MPT binding pore appears to occur via direct and / or indirect action (eg, by allosteric modification after insertion into the mitochondrial membrane).

SP-233 was also able to counteract the uncoupling of oxidative phosphorylation induced by CCCP in isolated mitochondrial and neuroblastoma cells. Aβ _{1-42 disrupts} mitochondrial membrane structure (Kremer et al. 2001; Rodrigues et al. 2001) and has been shown to change mitochondrial membrane fluidity in the AD brain (Mecocci et al, 1996) Both phenomena cause mitochondrial uncoupling. Thus, although the “reconjugation” effect of SP-233 has not yet been fully elucidated, this is believed to contribute to the recovery of mitochondrial function described herein. Furthermore, the ability of SP-233 to counteract the effects of Aβ _{1-42 at} a concentration as low as 1 pM is explained by a combination of the removal effect of SP-233 (possibly including regulation of MPT pores) and the reconjugation effect Seems to be able to.

In an attempt to identify another target of SP-233 in the mitochondrial respiratory chain, SP-233 may protect neuroblastoma cells against inhibitors of complex I, III, IV, and V It was shown that neuroblastoma cells cannot be protected against inhibitors of complex II (FIG. 15). The most obvious effect was observed with Complexes III and IV, but SP-233 exerts a slight but significant neuroprotective effect against rotenone and misothiazole-induced toxicity at concentrations of 1 and 100 μM, respectively. did. The importance of cell viability rescue to KCN and oligomycin compared to cell viability rescue to Aβ _1-42 is that complexes IV and V are major targets for mitochondrial dysfunction induced by amyloid peptide I suggest that. These results support previous results showing that Aβ _25-35 fragments inhibit complex IV in isolated brain mitochondria (Canevari et al., 1999). However, in contrast to this data, the same author reports that Aβ _25-35 does not affect other complexes of the respiratory chain. This apparent difference can be explained by the difference in the amyloid peptide species used and raises questions about the role of Aβ _25-35 and Aβ _1-42 in neuropathology.

Because AD is an incurable neurodegenerative disorder, most of the existing therapies based solely on AChE inhibition have serious limitations, there is an urgent need for the development of more effective treatments. SP-233, a small molecule, has previously been shown to inhibit Aβ _1-42- induced neurotoxicity by blocking the formation of ADDLs, but protects brain mitochondria from the harmful effects of Aβ _1-42 exposure This is reported here. The molecular mechanisms underlying the neuroprotective effects of SP-233 are, at least in part, the “removal” of Aβ _1-42 , the “anti-uncoupling” effect, the protective effect against mitochondrial complexes IV and V, and the This is thought to be due to the ability to inhibit opening. In conclusion, the data herein support and expand the previous results showing that SP-233 has anti-amyloid peptide properties, and further, mitochondria and respiratory chains are directly associated with Aβ _1-42 and SP-233. Specific target.
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All publications, patents and patent applications are incorporated herein by reference. In the foregoing specification, the invention has been described with reference to certain preferred embodiments and numerous details have been set forth for purposes of illustration, but those skilled in the art may make the invention take additional embodiments. And it will be apparent that some of the details described herein may be varied significantly without departing from the basic principles of the invention.

FIG. 1 shows the effect of increasing concentrations of SP-233 (10 to 100 pM) on the mitochondrial respiratory chain, measured by measuring the gradual change in the mitochondrial respiratory rate of the rat brain. This mitochondrial respiration rate (MRC) is V ₃ / V ₄ . V ₄ is the base O ₂ consumption. V ₃ is O ₂ consumption after adding ADP (ATP production). Mitochondrial concentration was adjusted to 0.4 mg protein amount / ml. It should be noted that even at a low SP-233 concentration of 10 pM, the MRC was reduced by 50% compared to the control (p <0.001). Results are expressed as mean ± standard deviation. Comparison between groups was performed by ANOVA according to Dunnett's test. FIG. 2a shows the effect of SP-233 on CCCP-induced uncoupling of oxidative phosphorylation in rat brain mitochondria. To assess the effect of SP-233 on CCCP-induced uncoupling of oxidative phosphorylation, the mitochondrial fraction was incubated with SP-233 for 3 minutes at 37 ° C before adding malic / glutamic acid, then 1 minute Later, CCCP was added. Exposure to the uncoupler CCCP (1 μM) increased O ₂ consumption to 150% of baseline (p <0.01). This change reflects an increase in oxygen consumption. At all experimental concentrations (even at 1 pM), SP-233 counteracted the metabolic effects of CCCP (p <0.001). This uncoupling inhibitory effect of SP-233 is related to a decrease in O ₂ consumption to 60 to 65% of the basal level, and is concentration-independent. FIG. 2b shows the effect of SP-233 on CsA-induced hypoxia. Hypoxia is caused by adding CsA (1 μM) to the culture medium in which mitochondria are cultured. In this experiment, ADP was added as a control in the presence of CsA and in the absence of SP-233. SP-233 or its solvent was added first and the incubation continued for 1.5 minutes before gently adding CsA. The substrates malic acid / glutamic acid were then added 1.5 minutes later and ADP 2.5 minutes later. Exposure to SP-233 did not inhibit the decrease in MRC induced by CsA. In contrast, the effect of CsA was amplified in a concentration-dependent manner. Results are expressed as mean ± standard deviation. Comparison between groups was performed by ANOVA according to Dunnett's test. FIG. 3 shows the effect of fresh Aβ _1-42 and old Aβ _{1-42 on} mitochondrial respiration rate in rat brain mitochondria. Mitochondria were incubated for 1.5 minutes at 37 ° C. in the presence of Αβ _1-42 before adding the substrate malate / glutamate. One minute after adding malic acid / glutamic acid, ADP was added. This experiment served as a control by removing ADP. Αβ _1-42 significantly reduced MRC even at concentrations as low as 0.1 pM. The effect of fresh amyloid peptide on MCR was more obvious than that of older ones, with MRC reductions of 55-63% and 43-53%, respectively. Results are expressed as mean ± standard deviation. Comparison between groups was performed by ANOVA according to Dunnett's test. FIG. 4 shows the effect of SP-233 on changes in mitochondrial respiration rate induced by fresh Aβ _1-42 and old Aβ _1-42 in rat brain mitochondria. Incubate mitochondria in the presence of SP-233 for 3 minutes and add freshly prepared Αβ _1-42 (a) or old Αβ _1-42 (b) at 37 ° C before adding malate / glutamate For 1.5 minutes. ADP was added 1 minute after the substrate malic acid / glutamic acid. Control of the effect of SP-233 on Αβ _1-42 was the same as in the experiment without ADP. In (a), fresh Αβ _1-42 reduced MRC by 71% compared to control, and this effect was partially inhibited by SP-233. The most potent effect was observed at the lowest concentration of SP-233, which returned MRC to 39% of control values (38.56 ± 0.34 vs. 28.93 ± 1.75, p <0.001). In (b), old Αβ _1-42 reduced MRC by 51% compared to control values, but SP-233 did not inhibit this effect. Results are expressed as mean ± standard deviation. Comparison between groups was performed by ANOVA according to Dunnett's test. FIG. 4 shows the effect of SP-233 on changes in mitochondrial respiration rate induced by fresh Aβ _1-42 and old Aβ _1-42 in rat brain mitochondria. Incubate mitochondria in the presence of SP-233 for 3 minutes and add freshly prepared Αβ _1-42 (a) or old Αβ _1-42 (b) at 37 ° C before adding malate / glutamate For 1.5 minutes. ADP was added 1 minute after the substrate malic acid / glutamic acid. Control of the effect of SP-233 on Αβ _1-42 was the same as in the experiment without ADP. In (a), fresh Αβ _1-42 reduced MRC by 71% compared to control, and this effect was partially inhibited by SP-233. The most potent effect was observed at the lowest concentration of SP-233, which returned MRC to 39% of control values (38.56 ± 0.34 vs. 28.93 ± 1.75, p <0.001). In (b), old Αβ _1-42 reduced MRC by 51% compared to control values, but SP-233 did not inhibit this effect. Results are expressed as mean ± standard deviation. Comparison between groups was performed by ANOVA according to Dunnett's test. FIG. 5 evaluates the protective effect of SP-233 against に 対 す る β _1-42 induced toxicity in human neuroblastoma. Cytotoxicity of Αβ was determined by placing SK-N-AS neuroblastoma in the presence or absence of SP-233 (1 μM) using the MTT assay and 2β _1-42 (0.1, 1 and 10 (μM) or after culturing in a solvent for 72 hours. SP-233 (1 μM) inhibited neurotoxicity induced by three concentrations of Αβ _1-42 . Results are expressed as mean ± standard deviation. Comparison between groups was performed by ANOVA according to Dunnett's test.

Neuroblastoma treated with Αβ _1-42 showed strong immune activity (which coexists with complex II of the mitochondrial respiratory chain, indicating that Αβ _1-42 is present inside the mitochondria).
SP-233 treatment, counteracts the immunoreactivity .alpha..beta _1-42, this indicates that the ability to prevent the .alpha..beta _1-42 falls within the mitochondria is the SP-233.
FIG. 6 shows the effect of SP-233 on mitochondrial respiration. The structural formula of the spirostenol derivative hexanoic acid = (22R, 25R) -20α-spirost-5-en-3β-yl (SP-233) is shown in (a). The effect of increasing SP-233 concentration (1 aM to 100 pM) on the mitochondrial respiratory chain was assessed by measuring the gradual change in respiratory rate in isolated rat brain mitochondria, as shown in (b) . Mitochondrial respiration rate (MRC) is defined as V ₃ / V ₄ , where V ₄ is basal O ₂ consumption and V ₃ is O ₂ consumption (ATP production) after adding ADP. Mitochondrial concentration was adjusted to 0.4 mg protein amount / ml. Of note, MRC was reduced by 40% compared to the control even at SP-233 concentrations as low as 1 fM (p <0.001). The results are expressed as mean ± standard deviation from three separate experiments performed in triplicate. FIG. 6 shows the effect of SP-233 on mitochondrial respiration. The structural formula of the spirostenol derivative hexanoic acid = (22R, 25R) -20α-spirost-5-en-3β-yl (SP-233) is shown in (a). The effect of increasing SP-233 concentration (1 aM to 100 pM) on the mitochondrial respiratory chain was evaluated by measuring the gradual change in respiratory rate in isolated rat brain mitochondria, as shown in (b) . Mitochondrial respiration rate (MRC) is defined as V ₃ / V ₄ , where V ₄ is basal O ₂ consumption and V ₃ is O ₂ consumption (ATP production) after adding ADP. Mitochondrial concentration was adjusted to 0.4 mg protein amount / ml. Of note, MRC was reduced by 40% compared to the control even at SP-233 concentrations as low as 1 fM (p <0.001). The results are expressed as mean ± standard deviation from three separate experiments performed in triplicate. FIG. 7 shows the effect of SP-233 on CCCP-induced uncoupling of oxidative phosphorylation and CsA-induced hypoxia. In (a), to assess the effect of SP-233 on CCCP-induced uncoupling of oxidative phosphorylation, the mitochondrial fraction was analyzed at 37 ° C in the presence of SP-233 before adding malate / glutamate. Incubated for 3 minutes. CCCP was added after 1 minute. Exposure to the 1 μM CCCP uncoupler increased O ₂ consumption to 150% of baseline (p <0.01). At all experimental concentrations, SP-233 counteracted the metabolic effects of CCCP (p <0.001). The uncoupling inhibitory effect of SP-233 was related to a decrease in O ₂ consumption to 60-65% of the basal level, and was concentration-independent. In (b), hypoxia was induced by adding 1 μM CsA to the culture medium for mitochondrial culture. As a control, ADP was added in the presence of CsA and in the absence of SP-233. SP-233 or its solvent was added first and incubation continued for 1.5 minutes before gently adding CsA. Substrate malic acid / glutamic acid was added after 1.5 minutes and ADP was added after 2.5 minutes. Exposure to SP-233 did not inhibit CsA-induced reduction of MRC. On the contrary, the effect of CsA was amplified in a concentration-dependent manner. The results are expressed as mean ± standard deviation from three separate experiments performed in triplicate. FIG. 7 shows the effect of SP-233 on CCCP-induced uncoupling of oxidative phosphorylation and CsA-induced hypoxia. In (a), to assess the effect of SP-233 on CCCP-induced uncoupling of oxidative phosphorylation, the mitochondrial fraction was analyzed at 37 ° C in the presence of SP-233 before adding malate / glutamate. Incubated for 3 minutes. CCCP was added after 1 minute. Exposure to the 1 μM CCCP uncoupler increased O ₂ consumption to 150% of baseline (p <0.01). At all experimental concentrations, SP-233 counteracted the metabolic effects of CCCP (p <0.001). The uncoupling inhibitory effect of SP-233 was related to a decrease in O ₂ consumption to 60-65% of the basal level, and was concentration-independent. In (b), hypoxia was induced by adding 1 μM CsA to the culture medium for mitochondrial culture. As a control, ADP was added in the presence of CsA and in the absence of SP-233. SP-233 or its solvent was added first and incubation continued for 1.5 minutes before gently adding CsA. Substrate malic acid / glutamic acid was added after 1.5 minutes and ADP was added after 2.5 minutes. Exposure to SP-233 did not inhibit CsA-induced reduction of MRC. On the contrary, the effect of CsA was amplified in a concentration-dependent manner. The results are expressed as mean ± standard deviation from three separate experiments performed in triplicate. FIG. 8 shows the effect of fresh and old Αβ _{1-42 on} mitochondrial respiration rate in rat brain mitochondria. Mitochondria were incubated for 1.5 minutes at 37 ° C. in the presence of Αβ _1-42 before adding the substrate malate / glutamate. ADP was added 1 minute after malic acid / glutamic acid. In these experiments, ADP was removed from the culture mixture. Αβ _1-42 significantly reduced MRC even at a concentration of 0.1 pM. The effect of fresh amyloid peptide on MCR was more obvious than that of older ones, with MRC reductions of 55-63% and 43-53%, respectively. The results are expressed as mean ± standard deviation from three separate experiments performed in triplicate. FIG. 9 shows the effect of SP-233 on changes in MRC induced by fresh and old Aβ _1-42 . Mitochondria were added for 3 minutes in the presence of SP-233 before addition of malate / glutamate, and 1.5 in the presence of freshly prepared Aβ _1-42 (a) or old Aβ _1-42 (b). Incubated at 37 ° C. for minutes. ADP was added 1 minute after the addition of malic / glutamic acid. Control reactions were performed in the absence of ADP. Fresh Aβ _1-42 reduced MRC by 71% compared to controls, and this effect was partially inhibited by SP-233.

The most potent effect was observed at the lowest concentration of SP-233, which returned MRC to 39% of control values (38.56 ± 0.34 vs. 28.93 ± 1.75, p <0.001). Older Aβ _1-42 reduced MRC by 51% compared to control, _whereas SP-233 did not inhibit this effect. The results are expressed as mean ± standard deviation from three separate experiments performed in triplicate.
FIG. 9 shows the effect of SP-233 on changes in MRC induced by fresh and old Aβ _1-42 . Mitochondria were added for 3 minutes in the presence of SP-233 before addition of malate / glutamate, and 1.5 in the presence of freshly prepared Aβ _1-42 (a) or old Aβ _1-42 (b). Incubated at 37 ° C. for minutes. ADP was added 1 minute after the addition of malic / glutamic acid. Control reactions were performed in the absence of ADP. Fresh Aβ _1-42 reduced MRC by 71% compared to controls, and this effect was partially inhibited by SP-233.

The most potent effect was observed at the lowest concentration of SP-233, which returned MRC to 39% of control values (38.56 ± 0.34 vs. 28.93 ± 1.75, p <0.001). Older Aβ _1-42 reduced MRC by 51% compared to control, _whereas SP-233 did not inhibit this effect. The results are expressed as mean ± standard deviation from three separate experiments performed in triplicate.
FIG. 10 evaluates the protective effect of SP-233 against Aβ _1-42 induced toxicity in human neuroblastoma. Cytotoxicity of Aβ was determined by placing SK-N-AS broth in the presence or absence of SP-233 (1 μM) using the MTT assay and Αβ _1-42 (0.1, 1 and 10 μM) Or it evaluated after culturing with a solvent for 72 hours. The results are expressed as mean ± standard deviation from three separate experiments performed in triplicate. FIG. 11 shows confocal microscopy analysis of the effect of SP-233 on mitochondrial Αβ _1-42 uptake in SK-N-AS human neuroblastoma. A representative image taken from healthy, undamaged (control) cells is shown in (a1-4). These cultures do not _accept Aβ _1-42 or SP-233. A representative image taken from SK-N-AS cells treated with 10 μM Aβ _1-42 for 3 hours is shown in (b1-4). A representative image taken from SK-N-AS cells treated with 10 μM Aβ _1-42 and 1 μM SP-233 for 3 hours is shown in (c1-4). Cells are stained with DAPI nuclear counterstain (a1, b1, and c1), Aβ _1-42 (FITC; a2, b2, and c2) and complex II 70-kDa subunit (Texas red; a3, b3, and c3). Mixed images are shown at a4, b4, and c4. Neuroblastoma treated with Aβ _1-42 showed strong antibody activity (b2) and coexisted with complex II of mitochondrial respiratory chain (b4). Furthermore, the Aβ _1-42 antibody activity was canceled by SP-233 treatment (c2). FIG. 11 shows a confocal microscopy analysis of the effect of SP-233 on mitochondrial Αβ _1-42 uptake in SK-N-AS human neuroblastoma. A representative image taken from healthy, undamaged (control) cells is shown in (a1-4). These cultures do not _accept Aβ _1-42 or SP-233. A representative image taken from SK-N-AS cells treated with 10 μM Aβ _1-42 for 3 hours is shown in (b1-4). A representative image taken from SK-N-AS cells treated with 10 μM Aβ _1-42 and 1 μM SP-233 for 3 hours is shown in (c1-4). Cells were stained with DAPI nuclear counterstain (a1, b1, and c1), Aβ _1-42 (FITC; a2, b2, and c2) and complex II 70-kDa subunit (Texas red; a3, b3, and c3). Mixed images are shown at a4, b4, and c4. Neuroblastoma treated with Aβ _1-42 showed strong antibody activity (b2) and coexisted with complex II of mitochondrial respiratory chain (b4). Moreover, the Aβ _1-42 antibody activity was canceled by SP-233 treatment (c2). FIG. 11 shows confocal microscopy analysis of the effect of SP-233 on mitochondrial Αβ _1-42 uptake in SK-N-AS human neuroblastoma. A representative image taken from healthy, undamaged (control) cells is shown in (a1-4). These cultures do not _accept Aβ _1-42 or SP-233. A representative image taken from SK-N-AS cells treated with 10 μM Aβ _1-42 for 3 hours is shown in (b1-4). A representative image taken from SK-N-AS cells treated with 10 μM Aβ _1-42 and 1 μM SP-233 for 3 hours is shown in (c1-4). Cells were stained with DAPI nuclear counterstain (a1, b1, and c1), Aβ _1-42 (FITC; a2, b2, and c2) and complex II 70-kDa subunit (Texas red; a3, b3, and c3). Mixed images are shown at a4, b4, and c4. Neuroblastoma treated with Aβ _1-42 showed strong antibody activity (b2) and coexisted with complex II of mitochondrial respiratory chain (b4). Moreover, the Aβ _1-42 antibody activity was canceled by SP-233 treatment (c2). FIG. 12 shows the neuroprotective effect of SP-233 against mitochondrial complex inhibitors in SK-N-AS cells. Five different mitochondrial toxins were added to SK-N-AS cultured cells 1 hour after addition of SP-233 or its solvent. Cell viability was measured after rotenone, malonic acid, and misothiazole were cultured for 24 hours in the absence or presence of SP-233. The incubation time for KCN and oligomycin was 6 hours. The results are expressed as mean ± standard deviation from three separate experiments performed in triplicate. Significant differences from cultured cells not treated with SP-233 are expressed as * p <0.05, ** p <0.01, *** p <0.001. FIG. 12 shows the neuroprotective effect of SP-233 against mitochondrial complex inhibitors in SK-N-AS cells. Five different mitochondrial toxins were added to SK-N-AS cultured cells 1 hour after addition of SP-233 or its solvent. Cell viability was measured after rotenone, malonic acid, and misothiazole were cultured for 24 hours in the absence or presence of SP-233. The incubation time for KCN and oligomycin was 6 hours. The results are expressed as mean ± standard deviation from three separate experiments performed in triplicate. Significant differences from cultured cells not treated with SP-233 are expressed as * p <0.05, ** p <0.01, *** p <0.001. FIG. 12 shows the neuroprotective effect of SP-233 against mitochondrial complex inhibitors in SK-N-AS cells. Five different mitochondrial toxins were added to SK-N-AS cultured cells 1 hour after addition of SP-233 or its solvent. Cell viability was measured after rotenone, malonic acid, and misothiazole were cultured for 24 hours in the absence or presence of SP-233. The incubation time for KCN and oligomycin was 6 hours. The results are expressed as mean ± standard deviation from three separate experiments performed in triplicate. Significant differences from cultured cells not treated with SP-233 are expressed as * p <0.05, ** p <0.01, *** p <0.001. FIG. 12 shows the neuroprotective effect of SP-233 against mitochondrial complex inhibitors in SK-N-AS cells. Five different mitochondrial toxins were added to SK-N-AS cultured cells 1 hour after addition of SP-233 or its solvent. Cell viability was measured after rotenone, malonic acid, and misothiazole were cultured for 24 hours in the absence or presence of SP-233. The incubation time for KCN and oligomycin was 6 hours. The results are expressed as mean ± standard deviation from three separate experiments performed in triplicate. Significant differences from cultured cells not treated with SP-233 are expressed as * p <0.05, ** p <0.01, *** p <0.001. FIG. 12 shows the neuroprotective effect of SP-233 against mitochondrial complex inhibitors in SK-N-AS cells. Five different mitochondrial toxins were added to SK-N-AS cultured cells 1 hour after addition of SP-233 or its solvent. Cell viability was measured after rotenone, malonic acid, and misothiazole were cultured for 24 hours in the absence or presence of SP-233. The incubation time for KCN and oligomycin was 6 hours. The results are expressed as mean ± standard deviation from three separate experiments performed in triplicate. Significant differences from cultured cells not treated with SP-233 are expressed as * p <0.05, ** p <0.01, *** p <0.001. FIG. 13 shows the neuroprotective effect of SP-233 on SK-N-AS cells against PAO. One hour after adding SP-233 or its solvent, PAO was added to the cultured cells. The cultured cells were cultured in PAO for 24 hours in the absence or presence of SP-233, and then cell viability was measured. The results are expressed as mean ± standard deviation from three separate experiments performed in triplicate. Significant difference from cells not treated with SP-233 is ** p <0.01. FIG. 14 shows the effect of SP-233 on FCCP-induced uncoupling of the mitochondrial respiratory chain in SK-N-AS cells. One hour after adding SP-233 or its solvent, FCCP was added to cultured SK-N-AS cells. The cultured cells were cultured in FCCP for 24 hours in the absence or presence of SP-233, and then cell viability was measured. The results are expressed as mean ± standard deviation from three separate experiments performed in triplicate. Significant difference from cells not treated with SP-233 is ** p <0.01. FIG. 15 is a schematic view of the mitochondrial SP-233 target site. The target element of SP-233 in the respiratory chain is indicated by a green arrow. A dotted arrow indicates a weak neuroprotective effect, and a green filled arrow indicates a strong protective effect. The most obvious effects were observed for KCN, an inhibitor of complex IV, oligomycin, an inhibitor of complex V, and PAO, the promoter of MPT.

Claims (31)

A method of treatment comprising administering to a mammal suffering from or having a sign of a mitochondrial disorder that is not a neuropathology of the central nervous system, wherein said mammal has an effective amount of a compound of structural formula I or pharmaceutically Administering an acceptable salt thereof, comprising:
Wherein R ₁ , R ₂ , R ₄ , R ₇ , R ₁₁ , R ₁₂ , and R ₁₅ are each independently a hydrogen atom, optionally -NR'-, -O-, -S-, -SO _{-, - SO 2 -, -} O-SO 2 -, - SO 2 -O -, - C (O) -, - C (O) -O -, - OC (O) -, - C (O) - (C ₁ -C ₈ ) alkyl group, hydroxyl group, N (R ′) ₂ , carboxyl group, oxo group, or sulfonic acid inserted with NR′- or —NR′—C (O) —,
R ₃ is a hydroxyl group, a (C ₁ -C ₈ ) alkoxy group, (C ₁ -C ₂₂ ) alkyl CO ₂ , or R′O ₂ C (CH ₂ ) _2-8 CO ₂ —,
Where (C ₁ -C ₂₂ ) alkyl group or (CH ₂ ) _2-8 is optionally 1-2 CH = CH units, 1-2 OH, and / or 1-2 epoxy substituted Groups can be included,
R ₃ is a toluene-4-sulfonyloxy group or a benzoyloxy group,
R _6, R ₈ , R ₉ , R ₁₀ , R ₁₃ , R ₁₄ , R ₁₆ and R ₁₇ are each independently a hydrogen atom, (C ₁ -C ₈ ) alkyl group, (C ₁ -C ₈ ) hydroxyl An alkyl group, a (C ₁ -C ₈ ) alkoxy group, or a hydroxyl group,
X is an O, S, S (O), N (R ′), N (Ac) or N (toluene-4-sulfonyloxy) group,
Here, each R ′ is individually H, (C ₁ -C ₈ ) alkyl group, phenyl group or benzyl group. The method of claim 1, wherein R ₁₀ and R ₁₃ are CH ₃ . The method according to claim 1 or 2, wherein one or both of R ₁₆ and R ₁₇ is CH ₃ . The method according to any one of claims 1 to 3, wherein R ₁ , R ₂ or R ₁₂ is H or OH. R ₁ , R ₂ , R ₄ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₁ , R ₁₂ , R ₁₄ and R ₁₅ are H, characterized in that they are H Any one treatment method. _{6. The process according} to claim 1, wherein R ₃ is (C ₁ -C ₂₂ ) alkylCO _2- , (C ₁ -C ₆ ) alkylCO _2- , or OH. Method of treatment.   The method according to any one of claims 1 to 6, wherein X is O.   The treatment method according to any one of claims 1 to 6, wherein X is NH. A therapeutic method for treating a mitochondrial disorder in a patient suffering from or having a sign of the disorder,
Where the disorder is not a neuropathology of the central nervous system,
Administering to said patient an effective amount of a compound of structural formula II or a pharmaceutically acceptable salt thereof,

R ₁ , R ₂ , R ₄ , R ₇ , R ₁₁ , R ₁₂ , and R ₁₅ in the formula are each independently a hydrogen atom, (C ₁ -C ₈ ) alkyl group, hydroxyl group, amino group, carboxyl group , Oxo group, sulfonic acid, or optionally -NH-, -N ((C ₁ -C ₈ ) alkyl group)-, -O-, -S-, -SO-, -SO _2- , -O- SO _2- , -SO ₂ -O-, -C (O)-, -C (O) -O-, -OC (O)-, -C (O) -NR'-, or -NR'-C A (C ₁ -C ₈ ) alkyl group having (O)-inserted therein,
Where R ′ is H or a (C ₁ -C ₈ ) alkyl group,
R ₃ is a hydroxyl group, (C ₁ -C ₆ ) alkyl CO ₂ , HO ₂ C (CH ₂ ) ₂ CO _2- , toluene-4-sulfonyloxy group or benzoyloxy group,
R ₆ , R ₈ , R ₉ , R ₁₀ , R ₁₃ , and R ₁₄ are each independently a hydrogen atom, (C ₁ -C ₈ ) alkyl group, (C ₁ -C ₈ ) hydroxylalkyl group, (C ₁ -C ₈ ) an alkoxy group or a hydroxyl group,
X is an O, N (H), N (Ac) or N (toluene-4-sulfonyloxy) group. R ₃ is OH or (C ₁ -C ₆₎ alkyl CO ₂ - characterized in that it is a process of claim 9.   11. A method according to claim 9 or claim 10, characterized in that X is O.   11. A method according to claim 9 or claim 10, characterized in that X is NH. Characterized in that R ₁₀ and R ₁₃ are CH _3, any one of the methods of claims 12 to claim 9. R _1, R ₂ or R ₁₂ is equal to or is OH, a method of any one of claims 13 claim 9. R ₁ , R ₂ , R ₄ , R ₆ , R ₇ , R ₈ , R ₉ , R ₁₁ , R ₁₂ , R ₁₄ and R ₁₅ are H, characterized in that One of the methods. A method according to any one of claims 1 or 9, comprising
The compound is (20α) -25ξ-methyl- (22R, 26) -azacyclofursta-5-en-3ξ-ol, (20ξ) -25ξ-methyl-N-acetyl- (22R, 26) -aza Cyclofurosta-5-ene-3ξ-ol, (22R, 25ξ)-(20α) -spirosta-5-ene- (2α, 3ξ) -diol, paratoluenesulfonic acid = (20α) -25ξ-methyl-N -Paratoluenesulfonyl- (22R, 26) -azacyclofursta-5-en-3ξ-yl, (22R, 25ξ)-(20α)-(14α, 20α) -spirost-5-ene- (3β, 12β ) -Diol, (22R, 25S)-(20ξ) -spirosta-5-en-3ξ-ol, benzoic acid = (22R, 25ξ)-(20α) -spirosta-5-en-3β-yl, hexanoic acid = (22S, 25S)-(20S) -Spirosta-5-en-3β-yl, (22R, 25ξ)-(20α) -Spirosta-5-ene- (1ξ, 3ξ) -diol, (22R, 25S)- (20α) -Spirosta-5-en-3β-ol, oxalic acid = (22R, 25S)-(20α) -spirost-5-en-3β-yl, and propanoic acid = (20α) -25S-methyl-N -Acetyl- (22S, 26) -azacyclofursta-5-ene-3 A method characterized in that it is selected from the group consisting of β-yl.   The process according to claim 16, characterized in that the compound is hexanoic acid = (22S, 25S)-(20S) -spirost-5-en-3β-yl.   10. The method of claim 1 or claim 9, wherein the compound is administered in a dosage form comprising a therapeutically effective amount of the compound. The method of claim 18, comprising:
The dosage form consists of tablets, soft gelatin capsules, hard gelatin capsules, suspension tablets, effervescent tablets, powders, effervescent powders, chewables, solutions, suspensions, emulsions, creams, gels, patches, and suppositories. A method characterized in that it is selected from a group.   20. The method of claim 19, wherein the dosage form further comprises a pharmaceutically acceptable excipient. 21. The method of claim 20, wherein
The pharmaceutically acceptable excipient is a binder, disintegrant, filler, surfactant, solubilizer, stabilizer, lubricant, wetting agent, diluent, anti-caking agent, glidant, or A method comprising a pharmaceutically compatible carrier.   The method according to any one of claims 1 to 21, wherein the mitochondrial disorder is a mitochondrial disease.   The method according to any one of claims 1 to 21, wherein the mitochondrial disorder is a muscle disease.   24. The method of claim 23, wherein the muscle disease is cardiomyopathy.   24. The method according to any one of claims 1 to 23, wherein the mitochondrial disorder is tubulinitis.   The method according to any one of claims 1 to 21, wherein the mitochondrial disorder is caused by compartment syndrome or muscle pressure syndrome. A method according to any one of claims 1 to 21, comprising
Neuropathology of the central nervous system includes global and local ischemia, or hemorrhagic stroke, head trauma, spinal cord injury, hypoxia-induced neuronal damage, cardiac arrest or neuronal damage resulting from neonatal difficulties, epilepsy, A method characterized in that it is selected from the group consisting of anxiety, spinal cord disorder, ALS, Alzheimer's disease, Huntington's disease, and Parkinson's disease. A pharmaceutical compound comprising at least one of a therapeutically effective amount of a compound of structural formula I or II in combination with a pharmaceutically acceptable excipient. A method of using a compound of structural formula (I) or a pharmaceutically acceptable salt thereof,

Wherein R ₁ , R ₂ , R ₄ , R ₇ , R ₁₁ , R ₁₂ , and R ₁₅ are each independently a hydrogen atom, optionally -NR'-, -O-, -S-, -SO _{-, - SO 2 -, -} O-SO 2 -, - SO 2 -O -, - C (O) -, - C (O) -O -, - OC (O) -, - C (O) - (C ₁ -C ₈ ) alkyl group, hydroxyl group, N (R ′) ₂ , carboxyl group, oxo group, or sulfonic acid inserted with NR′- or —NR′—C (O) —,
R ₃ is a hydroxyl group, a (C ₁ -C ₈ ) alkoxy group, (C ₁ -C ₂₂ ) alkyl CO ₂ , or R′O ₂ C (CH ₂ ) _2-8 CO ₂ —,
Where (C ₁ -C ₂₂ ) alkyl group or (CH ₂ ) _2-8 is optionally 1-2 CH = CH units, 1-2 OH, and / or 1-2 epoxy substituted Groups can be included,
R ₃ is a toluene-4-sulfonyloxy group or a benzoyloxy group,
R _6, R ₈ , R ₉ , R ₁₀ , R ₁₃ , R ₁₄ , R ₁₆ and R ₁₇ are each independently a hydrogen atom, (C ₁ -C ₈ ) alkyl group, (C ₁ -C ₈ ) hydroxyl An alkyl group, a (C ₁ -C ₈ ) alkoxy group, or a hydroxyl group,
X is an O, S, S (O), N (R ′), N (Ac) or N (toluene-4-sulfonyloxy) group,
Here, each R ′ is individually H, (C ₁ -C ₈ ) alkyl group, phenyl group or benzyl group,
Preparing a medicament for treating a mitochondrial disease or disorder that is not a neuropathology of the central nervous system,
Use of a compound of structural formula (I) or a pharmaceutically acceptable salt thereof. A method of using a compound of structural formula (II) or a pharmaceutically acceptable salt thereof,

R ₁ , R ₂ , R ₄ , R ₇ , R ₁₁ , R ₁₂ , and R ₁₅ in the formula are each independently a hydrogen atom, (C ₁ -C ₈ ) alkyl group, hydroxyl group, amino group, carboxyl group , Oxo group, sulfonic acid, or optionally -NH-, -N ((C ₁ -C ₈ ) alkyl group)-, -O-, -S-, -SO-, -SO _2- , -O- , SO _2- , -SO ₂ -O-, -C (O)-, -C (O) -O-, -OC (O)-, -C (O) -NR'-, or -NR'- A (C ₁ -C ₈ ) alkyl group inserted with C (O)-,
Where R ′ is H or a (C ₁ -C ₈ ) alkyl group,
R ₃ is a hydroxyl group, (C ₁ -C ₆ ) alkyl CO ₂ , HO ₂ C (CH ₂ ) ₂ CO _2- , toluene-4-sulfonyloxy group or benzoyloxy group,
R ₆ , R ₈ , R ₉ , R ₁₀ , R ₁₃ , and R ₁₄ are each independently a hydrogen atom, (C ₁ -C ₈ ) alkyl group, (C ₁ -C ₈ ) hydroxylalkyl group, (C ₁ -C ₈ ) an alkoxy group or a hydroxyl group,
X is O, N (H), N (Ac) or N (toluene-4-sulfonyloxy) group,
Preparing a medicament for treating a mitochondrial disease or disorder that is not a neuropathology of the central nervous system,
Use of a compound of structural formula (II) or a pharmaceutically acceptable salt thereof.   31. Use according to claim 29 or claim 30, characterized in that the medicament comprises a physiologically acceptable carrier.