WO2006107902A2 - Use of spirostenols to treat mitochondrial disorders - Google Patents

Use of spirostenols to treat mitochondrial disorders Download PDF

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WO2006107902A2
WO2006107902A2 PCT/US2006/012380 US2006012380W WO2006107902A2 WO 2006107902 A2 WO2006107902 A2 WO 2006107902A2 US 2006012380 W US2006012380 W US 2006012380W WO 2006107902 A2 WO2006107902 A2 WO 2006107902A2
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mitochondrial
alkyl
compound
hydroxy
effect
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PCT/US2006/012380
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WO2006107902A9 (en
WO2006107902A3 (en
WO2006107902A8 (en
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Laurent Lecanu
Laurent Tillement
Vassilios Papadopoulos
Wenguo Yao
Janet Greeson
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Samaritan Pharmaceuticals, Inc.
Georgetown University
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Priority to AU2006231451A priority Critical patent/AU2006231451A1/en
Priority to JP2008504515A priority patent/JP2008534623A/en
Priority to EP06740438A priority patent/EP1893219A2/en
Priority to CA002603127A priority patent/CA2603127A1/en
Publication of WO2006107902A2 publication Critical patent/WO2006107902A2/en
Publication of WO2006107902A3 publication Critical patent/WO2006107902A3/en
Publication of WO2006107902A9 publication Critical patent/WO2006107902A9/en
Publication of WO2006107902A8 publication Critical patent/WO2006107902A8/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • Mitochondria are specialized compartments present in every cell of the body except red blood cells, and are responsible for generating, by oxidative phosphorylation, more than 90% of the energy needed by the body to sustain life and support growth.
  • the mitochondrial respiratory chain fails, the energy level of the cell will rapidly decrease leading to the cell death and organ function impairment.
  • the impairment of the mitochondrial physiological function is involved in neurodegenerative diseases and disorders. These include those of the central nervous system, such as Alzheimer's Disease and Parkinson's Disease, and those affecting the peripheral nerves, such as muscular disorders (myopathies) and various kidney, liver and respiratory disorders.
  • the illness can range in severity from mild to lethal. Whether the mitochondrial failure is a cause or a consequence of the condition experienced, protecting respiratory function is critical to restore the impaired physiological functions. In addition, the preservation of the mitochondrial respiratory function is essential for the conservation of tissue required for a successful graft.
  • Coenzyme QlO is not effective in mtDNA-associated mitochondrial disease (N. Bresolin et al., J. Neurol. ScL, 100:70 (1990)) it leads to marked improvement in 'primary' CoQlO deficiency. Idebenone, a shorter chain analogue of Coenzyme QlO, appears to be effective in improving the hypertrophic cardiomyopathy in Friedreich's ataxia (P. Rustin et al., Lancet, 354: 477 (1999); C. Mariotti et al., Neurology, 60:1676 (2003)).
  • the present invention provides a therapeutic method comprising administering to a mammal, such as a human, afflicted with or threatened by, a mitochondrial disorder that is not a neuropathology of the central nervous system comprising administering to said mammal an effective amount of a compound of formula (T):
  • each OfR 1 , R 2 , R 4 , R 7 , R 11 , R 12 , and R 15 independently, is hydrogen, (d-C 8 )alkyl, that is optionally inserted with -NR'-, -O-, -S-, -SO-, -SO 2 -, -O- SO 2 -, -SO 2 -O-, -C(O)-, -C(O)-O-, -O-C(O)-, -C(O)-NR'-, or -NR'-C(O)-; hydroxy, N(R') 2 , carboxyl, oxo, or sulfonic acid; R 3 is hydroxy, (C 1 -C 8 )alkoxy, (C 1 -C 22 )alkylCO 2 or R'O 2 C(CH 2 ) 2-8 CO 2 -, wherein (C 1 -C 22 )alkyl or (CH 2 ) 2-8 can
  • the present invention includes therapeutic methods of treating a mitochondrial disorder in a subject afflicted with or threatened by said disorder, wherein said disorder is not a neuropathology of the central nervous system, comprising administering to the subject an effective amount of a compound of formula (II):
  • each OfR 1 , R 2 , R 4 , R 7 , R 11 , R 12 , and R 15 independently, is hydrogen, (C 1 -C 8 )allcyl, hydroxy, amino, carboxyl, oxo, sulfonic acid, or (C 1 - C 8 )alkyl that is optionally inserted with -NH-, -N((d-C 8 )alkyl)-, -O-, -S-, -SO-, -SO 2 -, -0-SO 2 -, -SO 2 -O-, -C(O)-, -C(O)-O-, -O-C(O)-, -C(O)-NR'-, or -NR'- C(O)-, wherein R' is H or (Ci-C 8 )alkyl; R 3 is hydroxy, (Ci-C 6 )alkylCO 2 -,
  • each of R 6 , R 8 , R 9 , R 10 , R 13 , and R 14 independently, is hydrogen, (d-C 8 )alkyl, hydroxyl(C 1 -C 8 )alkyl, (C 1 -C 8 )alkoxy or hydroxy; and X is O, N(H), N(Ac) or N(toluene-4- sulfonyloxy); or a pharmaceutically acceptable salt thereof.
  • spirostenols particularly 5,6 unsaturated spirostenols such as (22S,25S)-(20S)-spirost-5-en-3 ⁇ -yl hexanoate, a 22i?- hydroxycholesterol derivative naturally occurring in Gynurajaponica (asteraceae), can provide a novel therapeutic strategy that works by its direct targeting of the mitochondrial respiratory chain, as well as its targeting of A ⁇ which is toxic to mitochondria.
  • Mitochondrial disorders including mitochondrial diseases that are not considered to be CNS neuropathologies, and that can be treated with the present method include the following: eye: retinitis pigmentosa, optical nerve atrophy; muscles: disorders of the extraoccular muscles (ptosis, acquired strabismus, ophthalmoplegia), crushed muscle syndrome (compartment syndrome); mitochondrial dysfunction as a result of drug side-effect (i.e., highly active anti- retroviral therapy, HAART); myopathy; heart: heart stroke, heart attack, heart conditions, heart disease, cardiomyopathy, and any cardiac mitochondrial dysfunction as a result of drug side-effect (i.e., HAART, mega-HAART, anticancer drugs like anthracyclins); liver: hepatocellular dysfunctions; kidney and the endocrine system: tubulopathy, diabetes; and respiratory disorders.
  • eye retinitis pigmentosa, optical nerve atrophy
  • muscles disorders of the extraoccular muscles (ptosis, acquired strabismus
  • the present invention also provides novel compounds of formulas I or II and compositions, such as pharmaceutical compositions comprising an effective amount of a compound of formula I and/or II in combination with a pharmaceutically acceptable carrier.
  • Fig. 1 Effect of increasing concentrations of SP-233 (10 to 100 pM) on the mitochondrial respiratory chain, assessed by monitoring the evolution of the respiratory coefficient in rat brain mitochondria.
  • This mitochondrial respiratory coefficient (MRC) is V3/V4.
  • V4 is the basal O2 consumption
  • V3 is the O2 consumption after adding ADP (ATP production).
  • the mitochondrial concentration was adjusted to 0.4 mg protein/ml. It is noteworthy that concentrations of SP-233 as low as 10 pM induced a 50% decrease in the MRC, compared to control (p ⁇ 0.001). Results are expressed as mean ⁇ SD; comparisons between groups were made by an ANOVA followed by a Dunnett's test.
  • the mitochondrial fraction was incubated for 3 min at 37°C in presence of SP-233 before adding malate/glutamate, followed 1 min later by the addition of CCCP.
  • Exposure to the uncoupling agent CCCP (1 ⁇ M) increased the O2 consumption to 150% of the basal value (p ⁇ 0.01), a change that reflected an increase in oxygen consumption.
  • SP-233 abolished the metabolic effect of CCCP (pO.OOl).
  • Fig. 3 Effects of fresh and aged A ⁇ i-42 on the mitochondrial respiratory coefficient of rat brain mitochondria. Mitochondria were incubated in the presence of A ⁇ i-42 for 1.5 min at 37°C before adding the substrates malate/glutamate. ADP was added 1 min after malate/glutamate. This experiment was controlled by omitting ADP. A/5i-42 significantly decreased the MRC even at the low concentration of 0.1 pM. The effect of the fresh amyloid peptide on the MCR was more pronounced than that of the aged form, the respective decreases in MRC being 55-63% and 43-53%. Results are expressed as mean ⁇ SD; comparisons between groups were made by an ANOVA followed by a Dunnett's test.
  • Fig. 4 Effects of SP-233 on modifications of the mitochondrial respiratory coefficient induced by fresh or aged AjSi -42 in rat brain mitochondria. Mitochondria were incubated in the presence of SP-233 for 3 min and in the presence of freshly prepared (a) or aged (b) A ⁇ i-42 for 1.5 min at 37 0 C before addition of malate/glutamate. ADP was added 1 min after the substrates malate/glutamate. The control for the effect of SP-233 versus AjSi -42 was the same as for the experiment without ADP. (a): Fresh A ⁇ i-42 reduced the MRC by 71% compared to the control, and this effect was partially inhibited by SP-233.
  • Fig. 5 Assessment of the protective effect of SP-233 against AjSi-42- induced toxicity in human neuroblastoma cells.
  • the cellular toxicity of AjS was assessed after 72 h incubation of SK-N-AS neuroblastoma cells with A ⁇ i-42 (0.1 , 1 and 10 ⁇ M) or vehicle and in the presence or absence of SP-233 (1 ⁇ M) using the MTT assay.
  • SP-233 (1 ⁇ M) prevented the neurotoxicity induced by the three concentrations of AjSi -42. Results are expressed as mean ⁇ SD; comparisons between groups were made by an ANOVA followed by Dunnett's test.
  • Neuroblastoma cells treated with AjSi-42 displayed strong immunoreactivity (which co-localized with complex II of the mitochondrial respiratory chain, indicating that A j Si -42 was present inside the mitochondria).
  • Treatment with SP-233 abolished AjSi -42 immunoreactivity, indicating its ability to block the entry of AjSi -42 into the mitochondria.
  • Fig. 6 Effect of SP-233 on mitochondrial respiration.
  • the Chemical formula of the spirostenol derivative (22R,25R)-20 ⁇ -spirost-5-en-3 ⁇ -yl hexanoate (SP-233) is shown in (a).
  • Fig. 7 Effect of SP-233 on CCCP-induced uncoupling of oxidative phosphorylation and on CsA-induced hypoxia
  • the mitochondrial fraction was incubated for 3 min at 37°C in presence of SP-233 before adding malate/glutamate.
  • CCCP was added 1 min later. Exposure to the uncoupling agent 1 ⁇ M CCCP increased the O 2 consumption to 150% of the basal value (p ⁇ 0.01). At all concentrations tested, SP-233 abolished the metabolic effect of CCCP (pO.001).
  • SP-233 's inhibitory effect on "uncoupling” was associated with a decrease in O 2 consumption to 60-65% of the basal level and was concentration-independent
  • ADP was added in presence of CsA and in the absence of SP-233.
  • SP-233, or its vehicle, was added first, and incubation was continued for 1.5 min before CsA was gently added.
  • the substrate malate/glutamate was added 1.5 min later, and ADP was added 2.5 min later.
  • Exposure to SP-233 did not inhibit the decrease in the MRC induced by CsA.
  • CsA' s effect was amplified in a concentration-dependent manner. Results are expressed as means ⁇ SD from three independent experiments performed in triplicate.
  • Fig. 8 Effects of fresh and aged A ⁇ l-42 on the mitochondrial respiratory coefficient of rat brain mitochondria. Mitochondria were incubated in the presence OfAB 1 ⁇ 42 for 1.5 min at 37°C before adding the substrates malate/glutamate. ADP was added 1 min after malate/glutamate. In these experiments ADP was omitted from the incubation mixture. AB 1-42 significantly decreased the MRC, even at a concentration of 0.1 pM. The effect of the fresh amyloid peptide on the MCR was more pronounced than that of the aged form, the respective decreases in MRC being 55-63% and 43-53%. Results are expressed as means ⁇ SD from three independent experiments performed in triplicates.
  • Fig. 9 Effects of SP-233 on changes in the MRC induced by fresh or aged AB 1-42 . Mitochondria were incubated in the presence of SP-233 for 3 min and in the presence of freshly prepared (a) or aged (b) AB 1-42 for 1.5 min at 37 0 C before addition of malate/glutamate. ADP was added 1 min after the addition of malate/glutamate. Controls reactions were performed in the absence of ADP. Fresh AB 1-42 reduced the MRC by 71% compared to the control, and this effect was partially inhibited by SP-233.
  • FIG. 11 Confocal microscopy analysis of the effect of SP-233 on mitochondrial uptake of AB 1-42 in SK-N-AS human neuroblastoma cells. Representative images taken from healthy, uninjured (control) cells are shown in (al-4). These cultures did not receive any AB 1-42 or SP-233. Representative images taken from SK-N-AS cells treated with ABj -42 10 ⁇ M for 3 hours are shown in (bl-4). Representative images taken from SK-N-AS cells treated with AB 1-42 10 ⁇ M and SP-233 1 ⁇ M for 3 hours are shown in (cl-4).
  • Fig. 14 Effect of SP-233 on FCCP-induced uncoupling of the mitochondrial respiratory chain in SK-N-AS cells.
  • FCCP was added to SK-N- AS cultures 1 hour after the addition of SP-233 or its solvent. Cultures were incubated in FCCP for 24 hours in the absence or presence of SP-233, before the cell viability measurements were performed. Results are expressed as means ⁇ SD from three independent experiments performed in triplicate. ** p ⁇ 0.01 compared to cells not treated with SP-233.
  • Fig. 15 Schematic representation of the mitochondrial sites targeted by SP-233.
  • the elements of the respiratory chain targeted by SP-233 are indicated with a green arrow. Dotted arrows denote a poor neuroprotective effect, and full green arrows denote a strong protective effect. The most pronounced effect was observed against KCN, an inhibitor of complex IV, oligomycin, an inhibitor of the complex V, and PAO, a promoter of the MPT.
  • mitochondrial disorder encompasses art recognized mitochondrial diseases as well as conditions in which mitochondrial disorders or dysfunction plays a role in the onset symptomology, progression and/or outcome.
  • the latter conditions include neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, or myotrophic lateral sclerosis as well as neuronal death induced by stroke, cerebral ischemia and other brain and spinal cord injuries.
  • Mitochondrial diseases or mitochondrial myopathies are a group of diseases affecting the mitochondria, that also interfere with the function of muscles.
  • the group includes Kearns-Sayre syndrome, Leigh's syndrome, mitochondrial DNA depletion syndrome (MDS), mitochondrial encephalomyopathy, lactic acidosis and strokelike episodes (MELAS), myoclonus epilepsy with ragged red fibers (MERFF), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), neuropathy, ataxia and retinitis pigmentosa (NARP), and progressive external ophthalmoplegia (PEO).
  • mitochondrial myopathies include muscle weakness or exercise intolerance, heart failure (cardiomyopathy) or rhythm disturbances, dementia, movement disorders, stroke-like episodes, deafness, blindness, droopy eyelids, limited mobility of the eyes, vomiting, and seizures.
  • the prognosis for these disorders ranges in severity from progressive weakness to death.
  • mitochondrial disorders can include disorders of the eye, such as optic nerve atrophy, ptosis, acquired stabimus, and ophthalomplegia.
  • Mitochondrial dysfunction can be caused by crushed muscle syndrome (or compartment syndrome), the side effects of drug therapies such as HAART and anti-cancer therapies involving drugs that damage the heart (e.g., anthracycline), hepatocellular dysfunction, endocrine tubulopathy and respiratory disorders.
  • drug therapies such as HAART and anti-cancer therapies involving drugs that damage the heart (e.g., anthracycline), hepatocellular dysfunction, endocrine tubulopathy and respiratory disorders.
  • Most mitochondrial myopathies occur before the age of 20, and often begin with exercise intolerance or muscle weakness. During physical activity, muscles may become easily fatigued or weak. Muscle cramping is rare, but may occur. Nausea, headache, and breathlessness are also associated with these disorders.
  • treat refers to any treatment of a disorder or disease associated with a disease or disorder related to mitochondria, in a subject, and includes, but is not limited to, preventing the disorder or disease from occurring in a subject who may be predisposed to the disorder or disease, but has not yet been diagnosed as having the disorder or disease; inhibiting the disorder or disease, for example, arresting the development of the disorder or disease; relieving the disorder or disease, for example, causing regression of the disorder or disease; or relieving the condition caused by the disease or disorder, for example, stopping the symptoms of the disease or disorder.
  • mitochondrial disorder or “mitochondrial disease” is intended to encompass all disorders disclosed herein.
  • the present method can also be used to prevent or reduce mitochondrial disorders in vitro, as in organs or tissues awaiting or undergoing transplantation.
  • prevent in relation to a disease or disorder related to mitochondria, in a subject, means no disease or disorder development if none had occurred, or no further disorder or disease development if there had already been development of the disorder or disease, or no symptoms to logically observable signs of the disease.
  • Preferred stereoisomers are 3S, as well as 1Oi? and 13 S, and are also 2OS, 22S and 25S wherein the carbon skeleton is numbered in accord with spirosten- 3-ol numbering.
  • a preferred compound of formula (I or II) is (225,255)- (20iS)-spirost-5-en-3 ⁇ -yl hexanoate (SP233). Note that the carbon atoms shown in formula (I) or (II) are saturated with hydrogen unless otherwise indicated.
  • alkyl refers to a Ci - 8 hydrocarbon chain, linear (e.g., butyl) or branched (e.g., isobutyl).
  • Alkylene, alkenylene, and alkynylene refer to divalent Ci -8 alkyl (e.g., ethylene), alkene, and alkyne radicals, respectively.
  • alkyl is (Ci-C 6 )alkyl, such as butyl, hexyl, methyl, ethyl, propyl or isopropyl.
  • alkyl includes cycloakyl, (cycloalkyl)alkyl and alkyl(cycloalkyl)alkyl.
  • (C 1 -C 8 )alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, heptyl or octyl;
  • (C 3 -C 8 )cycloalkyl can be monocyclic, bicyclic or tricyclic and includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.2] octanyl or norbornyl, as well as various terpene and terpenoid structures.
  • (C 3 -C 6 )cycloalkyl(C!-C 2 )alkyl includes the foregoing cycloalkyl and can be cyclopropymiethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl, 2- cyclopentylethyl, or 2-cyclohexylethyl.
  • Heterocycloalkyl includes cycloalkyl wherein the cycloalkyl ring system is monocyclic, bicyclic or tricyclic and optionally comprises 1-2 S, non-peroxide O or N(R') as well as 4-8 ring carbon atoms; such as morpholinyl, piperidinyl, piperazinyl, indanyl, l,3-dithian-2-yl, and the like.
  • Any cycloalkyl or heterocycloalkyl ring system optionally includes 1-3 double bonds or epoxy moieties and optionally is substituted with 1-3 OH, (d-C ⁇ alkanoyloxy, (CO), (Q-C ⁇ alkyl or (C 2 -C 6 )alkynyl.
  • (C 1 -C 8 )AIkOXy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy;
  • (C 2 -C 6 )alkenyl can be vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl;
  • hydroxy(C 1 -C 8 )alkyl or hydroxy (C 1 -C 8 )alkoxyl can be alkyl substituted with 1 or 2 OH groups, such as alkyl substituted with 1 or 2 OH groups such as hydroxymethyl, 1-hydroxyethyl, 2-hydroxy
  • R 10 and/or R 12 are CH 3 .
  • R 16 and R 17 are CH 3 .
  • R 1 , R 2 and/or R 12 can be 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.
  • R 2 can be (C r C 22 )alkylCO 2 -, including (C 8 -C 22 )alkylCO 2 - or (C 1 - C 6 )alkylCO 2 -, or can be OH or CXQ-CsOalkyl, or HO 2 C(CH 2 ) 2 CO 2 -.
  • X can be O or NR' including NH or NAc.
  • R 3 may also be OR 23 wherein R 23 is a removable hydroxy-protecting group such as tosyl, mesyl, M(C 1 -GOaIkVlSiIyI, THP, Eto(Et), benzyl, benzoyloxycarbony and the like.
  • R 23 is a removable hydroxy-protecting group such as tosyl, mesyl, M(C 1 -GOaIkVlSiIyI, THP, Eto(Et), benzyl, benzoyloxycarbony and the like.
  • C 3 -(C 8 -C 22 ) fatty acid esters of compounds in which C 3 is hydroxysubstituted are also within the invention, wherein the fatty acid is preferably naturally occurring.
  • Table 1 Shown below in Table 1 are several compounds of formula (I) and (II) described above that can be used to practice this invention: Table 1.
  • An effective amount of an efficacious compound of the invention can be formulated with a pharmaceutically acceptable carrier to form a pharmaceutical composition before being administered for treatment of a disease related to impaired mitochondrial function.
  • “An effective amount” or “pharmacologically effective amount” refers to the amount of the compound which is required to confer therapeutic effect on the treated subject. The interrelationship of dosages for animals and humans (based on milligrams per square meter of body surface) is described by Freireich et al., Cancer Chemother. Rep., 50, 219 (1966). Body surface area may be approximately determined from height and weight of the patient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, New York, 1970, 537.
  • Effective doses can be based on in vitro concentrations of the present compounds found to be effective to inhibit the toxicity of A ⁇ or to otherwise protect mitochondria. Doses of the present compounds useful to treat a model neuronal cell line are disclosed below. Effective doses will also vary, as recognized by those skilled in the art, depending on the route of administration, the excipient usage, and the optional co-administration with other therapeutic agents. Toxicity and therapeutic efficacy of the active ingredients can be determined by standard pharmaceutical procedures, e.g., for detem ⁇ ning LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 5 Q/ED 50 .
  • Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. Included in the methods, kits, combinations and pharmaceutical compositions of the present invention are the crystalline forms (e.g., polymorphs), enantiomeric forms, isomeric forms and tautomers of the described compounds and the pharmaceutically-acceptable salts thereof.
  • crystalline forms e.g., polymorphs
  • enantiomeric forms e.g., isomeric forms and tautomers of the described compounds and the pharmaceutically-acceptable salts thereof.
  • Illustrative pharmaceutically acceptable salts are prepared from formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, stearic, salicylic, p-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, toluenesulfonic, 2-hydroxyethanesulfonic, sulfanilic, cyclohexylaminosulfonic, algenic, b- hydroxybutyric, galactaric and galacturonic acids.
  • prodrug refers to a drug or compound (active moiety) that elicits the pharmacological action results from conversion by metabolic processes within the body.
  • Prodrugs are generally considered drug precursors that, following administration to a subject and subsequent absorption, are converted to an active or a more active species via some process, such as a metabolic process. Other products from the conversion process are easily disposed of by the body.
  • Prodrugs generally have a chemical group present on the prodrug which renders it less active and/or confers solubility or some other property to the drug, such as an ester or acyl group. Once the chemical group has been cleaved from the prodrug the more active drug is generated.
  • Prodrugs may be designed as reversible drug derivatives and utilized as modifiers to enhance drug transport to site-specific tissues.
  • the design of prodrugs to date has been to increase the effective water solubility of the therapeutic compound for targeting to regions where water is the principal solvent.
  • Fedorak, et al., Am. J. Physiol, 269, G210-218 (1995) describe dexamethasone- beta-D-glucuronide.
  • McLoed, et al., Gastroenterol., 106, 405-413 (1994) describe dexamethasone-succinate-dextrans. Hochhaus, et al., Biomed.
  • Prodrugs are also useful as synthetic intermediates in the preparation of other compounds of formulas (I) or (II), by synthetic interconversions known to the art. For example, see, LT. Harrison, Compendium of Organic Synthetic Methods, Wiley-Interscience (1971), for methods useful to interconvert spirostenol substituents.
  • derivative refers to a compound that is produced from another compound of similar structure by the replacement or substitution of one atom, molecule or group by another.
  • a hydrogen atom of a compound may be substituted by alkyl, acyl, amino, etc., to produce a derivative of that compound.
  • “Plasma concentration” refers to the concentration of a substance in blood plasma or blood serum.
  • “Drug absorption” or “absorption” refers to the process of movement from the site of administration of a drug toward the systemic circulation, for example, into the bloodstream of a subject.
  • Bioavailability refers to the extent to which an active moiety (drug or metabolite) is absorbed into the general circulation and becomes available at the site of drug action in the body.
  • Methodabolism refers to the process of chemical transformations of drugs in the body.
  • “Pharmacodynamics” refers to the factors which determine the biologic response observed relative to the concentration of drug at a site of action.
  • “Pharmacokinetics” refers to the factors which determine the attainment and maintenance of the appropriate concentration of drug at a site of action.
  • Plasma half-life refers to the time required for the plasma drug concentration to decrease by 50% from its maximum concentration.
  • measurable serum concentration means the serum concentration (typically measured in mg, ⁇ g, or ng of therapeutic agent per ml, dl, or 1 of blood serum) of a therapeutic agent absorbed into the bloodstream after administration.
  • compositions include metallic ions and organic ions. More preferred metallic ions include, but are not limited to appropriate alkali metal (Group Ia) salts, alkaline earth metal (Group Ha) salts and other physiological acceptable metal ions. Exemplary ions include aluminum, calcium, lithium, magnesium, potassium, sodium and zinc in their usual valences.
  • Preferred organic ions include protonated tertiary amines and quaternary ammonium cations, including in part, trimethylamine, diethylamine, N 5 N 1 - dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine.
  • Exemplary pharmaceutically acceptable acids include without limitation 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 oxalacetic acid, fumaric acid, propionic acid, aspartic acid, glutamic acid, benzoic acid, and the like.
  • the compositions of the present invention are usually administered in the form of pharmaceutical compositions.
  • compositions can be administered by any appropriate route including, but not limited to, oral, nasogastric, rectal, transdermal, parenteral (for example, subcutaneous, intramuscular, intravenous, intramedullary and intradermal injections, or infusion techniques administration), intranasal, transmucosal, implantation, vaginal, topical, buccal, and sublingual.
  • parenteral for example, subcutaneous, intramuscular, intravenous, intramedullary and intradermal injections, or infusion techniques administration
  • intranasal transmucosal
  • implantation vaginal
  • vaginal topical
  • buccal and sublingual
  • preparations may routinely contain buffering agents, preservatives, penetration enhancers, compatible carriers and other therapeutic or non-therapeutic ingredients.
  • the present invention also includes methods employing a pharmaceutical composition that contains one or more compound of formula I or II associated with pharmaceutically acceptable carriers or excipients.
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable excipients” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for ingestible substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the compositions, its use is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • compositions(s) can be mixed with a pharmaceutically acceptable excipient, diluted by the excipient or enclosed within such a carrier, which can be in the form of a capsule, sachet, or other container.
  • a pharmaceutically acceptable excipient diluted by the excipient or enclosed within such a carrier, which can be in the form of a capsule, sachet, or other container.
  • the carrier materials that can be employed in making the composition of the present invention are any of those commonly used excipients in pharmaceutics and should be selected on the basis of compatibility with the active drug and the release profile properties of the desired dosage form.
  • compositions are chosen below as examples:
  • Binders such as acacia, alginic acid and salts thereof, cellulose derivatives, methylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, magnesium aluminum silicate, polyethylene glycol, gums, polysaccharide acids, bentonites, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, polyvinylpyrrolidone/vinyl acetate copolymer, crospovidone, povidone, polymethacrylates, hydroxypropylmethylcellulose, hydroxypropylcellulose, starch, pregelatinized starch, ethylcellulose, tragacanth, dextrin, cyclodextrins, microcrystalline cellulose, sucrose, or glucose, and the like.
  • Binders such as acacia, alginic acid and salts thereof, cellulose derivatives, methylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, magnesium aluminum silicate, polyethylene glycol,
  • Disintegration agents such as starches, pregelatinized corn starch, pregelatinized starch, celluloses, cross-linked carboxymethylcellulose, sodium starch glycolate, crospovidone, cross-linked polyvinylpyrrolidone, croscarmellose sodium, microcrystalline cellulose, a calcium, a sodium alginate complex, clays, alginates, gums, or sodium starch glycolate, and any disintegration agents used in tablet preparations.
  • (c) Filling agents such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.
  • Surfactants such as sodium lauryl sulfate, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, PluronicTM line (BASF), and the like.
  • Solubilizer such as citric acid, succinic acid, fumaric acid, malic acid, tartaric acid, maleic acid, glutaric acid sodium bicarbonate and sodium carbonate and the like.
  • Stabilizers such as any antioxidation agents, buffers, or acids, and the like, can also be utilized.
  • Lubricants such as magnesium stearate, calcium hydroxide, talc, sodium stearyl fumarate, hydrogenated vegetable oil, stearic acid, glyceryl behapate, magnesium, calcium and sodium stearates, stearic acid, talc, waxes, boric acid, sodium benzoate, sodium acetate, sodium chloride, DL-leucine, polyethylene glycols, sodium oleate, or sodium lauryl sulfate, and the like.
  • Lubricants such as magnesium stearate, calcium hydroxide, talc, sodium stearyl fumarate, hydrogenated vegetable oil, stearic acid, glyceryl behapate, magnesium, calcium and sodium stearates, stearic acid, talc, waxes, boric acid, sodium benzoate, sodium acetate, sodium chloride, DL-leucine, polyethylene glycols, sodium oleate, or sodium lauryl sulfate, and the like.
  • wetting agents such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium oleate, or sodium lauryl sulfate, and the like.
  • Diluents such lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose, dibasic calcium phosphate, sucrose-based diluents, confectioner's sugar, monobasic calcium sulfate monohydrate, calcium sulfate dihydrate, calcium lactate trihydrate, dextrates, inositol, hydrolyzed cereal solids, amylose, powdered cellulose, calcium carbonate, glycine, or bentonite, and the like.
  • Anti-adherents or glidants such as talc, corn starch, DL-leucine, sodium lauryl sulfate, and magnesium, calcium, or sodium stearates, and the like.
  • Pharmaceutically compatible carrier comprises acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerine, magnesium silicate, sodium caseinate, soy lecithin, sodium chloride, tricalcium phosphate, dipotassium phosphate, sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, or pregelatinized starch, and the like.
  • compositions are discussed in, for example, Remington's The Science and Practice of Pharmacy (2000). Another discussion of drug formulations can be found in Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N. Y., 1980.
  • the tablets or granules comprising the inventive compositions may be film coated or enteric-coated.
  • Mammal includes a primate, for example, a monkey, or a lemur, a horse, a dog, a pig, or a cat.
  • a rodent includes a rat, a mouse, a squirrel, or a guinea pig.
  • compositions of the present invention are useful where administration of an inhibitor of mitochondrial toxicity is indicated. It is believed that these compositions will be particularly effective in the treatment of mitochondrial diseases.
  • compositions of the invention can be used to provide a dose of a compound of the present invention in an amount sufficient to elicit a therapeutic response, e.g., reduction of drug-induced mitochondrial toxicity, for example a dose of about 5 ng to about 1000 nig, or about 100 ng to about 600 mg, or about 1 mg to about 500 mg, or about 20 mg to about 400 mg.
  • a dosage effective amount will range from about 0.0001 mg/kg to 1500 mg/kg, more preferably 1 to 1000 mg/kg, more preferably from about 1 to 150 mg/kg of body weight, and most preferably about 50 to 100 mg/kg of body weight.
  • a dose can be administered in one to about four doses per day, or in as many doses per day to elicit a therapeutic effect.
  • a dosage unit of a composition of the present invention can typically contain, for example, about 5 ng, 50 ng 100 ng, 500 ng, 1 mg, 10 mg, 20 mg, 40 mg, 80 mg, 100 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 of a compound of the present invention.
  • the dosage form can be selected to accommodate the desired frequency of administration used to achieve the specified dosage.
  • the amount of the unit dosage form of the composition that is administered and the dosage regimen for treating the condition or disorder depends on a variety of factors, including, the age, weight, sex and medical condition, of the subject, the severity of the condition or disorder, the route and frequency of administration, and this can vary widely, as is well known.
  • the composition is administered to a subject in an effective amount, that is, the composition is administered in an amount that achieves a therapeutically effective dose of a compound of the present invention in the blood serum of a subject for a period of time to elicit a desired therapeutic effect.
  • the composition in a fasting adult human (fasting for generally at least 10 hours) is administered to achieve a therapeutically effective dose of a compound of the present invention in the blood serum of a subject from about 5 minutes after administration of the composition.
  • a therapeutically effective dose of the compound of the present invention is achieved in the blood serum of a subject at about 10 minutes from the time of administration of the composition to the subject
  • a therapeutically effective dose of the compound of the present invention is achieved in the blood serum of a subject at about 20 minutes from the time of administration of the composition to the subject.
  • a therapeutically effective dose of the compound of the present invention is achieved in the blood serum of a subject at about 30 minutes from the time of administration of the composition to the subject.
  • a therapeutically effective dose of the compound of the present invention is achieved in the blood serum of a subject at about 40 minutes from the time of administration of the composition to the subject.
  • a therapeutically effective dose of the compound of the present invention is achieved in the blood serum of a subject at about 20 minutes to about 12 hours from the time of administration of the composition to the subject.
  • a therapeutically effective dose of the compound of the present invention is achieved in the blood serum of a subject at about 20 minutes to about 6 hours from the time of administration of the composition to the subject. In yet another embodiment of the present invention, a therapeutically effective dose of the compound of the present invention is achieved in the blood serum of a subject at about 20 minutes to about 2 hours from the time of administration of the composition to the subject.
  • a therapeutically effective dose of the compound of the present invention is achieved in the blood serum of a subject at about 40 minutes to about 2 hours from the time of administration of the composition to the subject.
  • a therapeutically effective dose of the compound of the present invention is achieved in the blood serum of a subject at about 40 minutes to about 1 hour from the time of administration of the composition to the subject.
  • a composition of the present invention is administered at a dose suitable to provide a blood serum concentration with a half maximum dose of a compound of the present invention.
  • a blood serum concentration of about 0.01 to about 1000 iiM, 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 nM, or about 200 to about 400 nM is achieved in a subject after administration of a composition of the present invention.
  • compositions of the present invention provide a therapeutic effect over an interval of about 5 minutes to about 24 hours after administration, enabling once-a-day or twice-a-day administration if desired, hi one embodiment of the present invention, the composition is administered at a dose suitable to provide an average blood serum concentration with a half maximum dose of a compound of the present invention of at least about 1 ⁇ ,g/ml, or at least about 5 ⁇ g/ml, or at least about 10 ⁇ .g/ml, or at least about 50 ⁇ g/ml, or at least about 100 ⁇ g/ml, or at least about 500 ⁇ g/ml, or at least about 1000 jUg/ml in a subject about 10, 20, 30, or 40 minutes after administration of the composition to the subject.
  • the amount of therapeutic agent necessary to elicit a therapeutic effect can be experimentally determined based on, for example, the absorption rate of the agent into the blood serum, the bioavailability of the agent, and the potency for treating the disorder. It is understood, however, that specific dose levels of the therapeutic agents of the present invention for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, and diet of the subject (including, for example, whether the subject is in a fasting or fed state), the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration. Treatment dosages generally may be titrated to optimize safety and efficacy.
  • dosage-effect relationships from in vitro and/or in vivo tests initially can provide useful guidance on the proper doses for subject administration.
  • Studies in animal models generally may be used for guidance regarding effective dosages for treatment of gastrointestinal disorders or diseases in accordance with the present invention.
  • the dosage to be administered will depend on several factors, including the particular agent that is administered, the route administered, the condition of the particular subject, etc.
  • a compound is found to demonstrate in vitro activity at, for example, a half-maximum effective dose of 200 nM
  • serum compound of the present invention concentrations can be measured using standard assay techniques.
  • Contemplated compositions of the present invention provide a therapeutic effect over an interval of about 30 minutes to about 24 hours after administration to a subject, hi one embodiment compositions provide such therapeutic effect in about 30 minutes, hi another embodiment compositions provide therapeutic effect over about 24 hours, enabling once-a-day administration to improve patient compliance.
  • present methods, kits, and compositions can also be used in combination ("combination therapy") with another pharmaceutical agent that is indicated for treating or preventing a mitochondrial disease, such as, for example, creatinine.
  • a mitochondrial disease such as, for example, creatinine.
  • an additive or synergistic effect may be achieved such that many if not all of unwanted side effects can be reduced or eliminated.
  • the reduced side effect profile of these drugs is generally attributed to, for example, the reduced dosage necessary to achieve a therapeutic effect with the administered combination.
  • composition therapy embraces the administration of a composition of the present invention in conjunction with another pharmaceutical agent that is indicated for treating or preventing a mitochondrial disorder in a subject, as part of a specific treatment regimen intended to provide a beneficial effect from the co-action of these therapeutic agents for the treatment of a neurodegenerative and/or a mitochondrial disorder.
  • the beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents.
  • Administration of these therapeutic agents in combination typically is carried out over a defined time period (usually substantially simultaneously, minutes, hours, days, weeks, months or years depending upon the combination selected).
  • “Combination therapy” generally is not intended to encompass the administration of two or more of these therapeutic agents as part of separate monotherapy regimens that incidentally and arbitrarily result in the combinations of the present invention.
  • “Combination therapy” is intended to embrace administration of these therapeutic agents in a sequential manner, that is, where each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the therapeutic agents, in a substantially simultaneous manner.
  • Substantially simultaneous administration can be accomplished, for example, by administering to the subject a single tablet or capsule having a fixed ratio of each therapeutic agent or in multiple, single capsules, or tablets for each of the therapeutic agents.
  • Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route.
  • composition of the present invention can be administered orally or nasogastric, while the other therapeutic agent of the combination can be administered by any appropriate route for that particular agent, including, but not limited to, an oral route, a percutaneous route, an intravenous route, an intramuscular route, or by direct absorption through mucous membrane tissues.
  • the composition of the present invention is administered orally or nasogastric and the therapeutic agent of the combination may be administered orally, or percutaneously.
  • the sequence in which the therapeutic agents are administered is not narrowly critical.
  • “Combination therapy” also can embrace the administration of the therapeutic agents as described above in further combination with other biologically active ingredients, such as, but not limited to, an analgesic, for example, and with non- drug therapies, such as, but not limited to, surgery.
  • the therapeutic compounds which make up the combination therapy may be a combined dosage form or in separate dosage forms intended for substantially simultaneous administration.
  • the therapeutic compounds that make up the combination therapy may also be administered sequentially, with either therapeutic compound being administered by a regimen calling for two step administration.
  • a regimen may call for sequential administration of the therapeutic compounds with spaced-apart administration of the separate, active agents.
  • the time period between the multiple administration steps may range from, for example, a few minutes to several hours to days, depending upon the properties of each therapeutic compound such as potency, solubility, bioavailability, plasma half-life and kinetic profile of the therapeutic compound, as well as depending upon the effect of food ingestion and the age and condition of the subject. Circadian variation of the target molecule concentration may also determine the optimal dose interval.
  • the therapeutic compounds of the combined therapy may involve a regimen calling for administration of one therapeutic compound by oral route and another therapeutic compound by an oral route, a percutaneous route, an intravenous route, an intramuscular route, or by direct absorption through mucous membrane tissues, for example.
  • a regimen calling for administration of one therapeutic compound by oral route and another therapeutic compound by an oral route, a percutaneous route, an intravenous route, an intramuscular route, or by direct absorption through mucous membrane tissues for example.
  • each such therapeutic compound will be contained in a suitable pharmaceutical formulation of pharmaceutically- acceptable excipients, diluents or other formulations components.
  • the pharmaceutical composition can contain a desired amount of a compound of formula (I) or (II) and be in the form of, for example, a tablet, a hard or soft capsule, a lozenge, a cachet, a troche, a dispensable powder, granules, a suspension, an elixir, a liquid, or any other form reasonably adapted for oral administration.
  • a pharmaceutical composition can be made in the form of a discrete dosage unit containing a predetermined amount of the active compound such as a tablet or a capsule.
  • Such oral dosage forms can further comprise, for example, buffering agents. Tablets, pills and the like additionally can be prepared with enteric coatings.
  • compositions suitable for buccal or sublingual administration include, for example, lozenges comprising the active compound in a flavored base, such as sucrose, and acacia or tragacanth, and pastilles comprising the active compound in an inert base such as gelatin and glycerin or sucrose and acacia.
  • Liquid dosage forms for oral administration can include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water.
  • Such compositions can also comprise, for example, wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.
  • suitable liquid dosage forms include, but are not limited to, aqueous solutions comprising the active compound and beta-cyclodextrin or a water soluble derivative of beta-cyclodextrin such as sulfobutyl ether beta- cyclodextrin; heptakis-2,6-di-O-methyl-beta-cyclodextrin; hydroxypropyl-beta- cyclodextrin; and dimethyl-beta-cyclodextrin.
  • beta-cyclodextrin such as sulfobutyl ether beta- cyclodextrin; heptakis-2,6-di-O-methyl-beta-cyclodextrin; hydroxypropyl-beta- cyclodextrin; and dimethyl-beta-cyclodextrin.
  • compositions of the present invention can also be administered by injection (intravenous, intramuscular, subcutaneous).
  • injectable compositions can employ, for example, saline, dextrose, or water as a suitable carrier material.
  • the pH value of the composition can be adjusted, if necessary, with suitable acid, base, or buffer.
  • suitable bulking, dispersing, wetting or suspending agents including mannitol and polyethylene glycol (such as PEG 400), can also be included in the composition.
  • a suitable parenteral composition can also include an active compound lyophilized in injection vials.
  • Aqueous solutions can be added to dissolve the composition prior to injection.
  • the pharmaceutical compositions can be administered in the form of a suppository or the like.
  • Such rectal formulations preferably contain the active compound in a total amount of, for example, about 0.075 to about 75% w/w, or about 0.2 to about 40% w/w, or about 0.4 to about 15% w/w.
  • Carrier materials such as cocoa butter, theobroma oil, and other oil and polyethylene glycol suppository bases can be used in such compositions.
  • Other carrier materials such as coatings (for example, hydroxypropyl methylcellulose film coating) and disintegrants (for example, croscarmellose sodium and cross-linked povidone) can also be employed if desired.
  • the subject compounds may be free or entrapped in microcapsules, in colloidal drug delivery systems such as liposomes, microemulsions, and macroemulsions.
  • compositions can be prepared by any suitable method of pharmaceutics, which includes the step of bringing into association active compound of the present invention and a carrier material or carriers materials.
  • the compositions are uniformly and intimately admixing the active compound with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • a tablet can be prepared by compressing or molding a powder or granules of the compound, optionally with one or more accessory ingredients.
  • Compressed tablets can be prepared by compressing, in a suitable machine, the compound in a free-flowing form, such as a powder or granules optionally mixed with a binding agent, lubricant, inert diluent and/or surface active/dispersing agent(s). Molded tablets can be made by molding, in a suitable machine, the powdered compound moistened with an inert liquid diluent.
  • Tablets of the present invention can also be coated with a conventional coating material such as OpadryTM White YS-1-18027A (or another color) and the weight fraction of the coating can be about 3% of the total weight of the coated tablet.
  • a conventional coating material such as OpadryTM White YS-1-18027A (or another color) and the weight fraction of the coating can be about 3% of the total weight of the coated tablet.
  • the compositions of the present invention can be formulated so as to provide quick, sustained or delayed release of the compositions after administration to the patient by employing procedures known in the art.
  • the excipient when it serves as a diluent, it can be a solid, semi-solid or liquid material, which acts as a vehicle, carrier or medium for the active ingredient.
  • the compositions can be in the form of tablets, chewable tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), soft and hard gelatin capsules and sterile packaged powders.
  • the manufacturing processes may employ one or a combination of methods including: (1) dry mixing, (2) direct compression, (3) milling, (4) dry or non-aqueous granulation, (5) wet granulation, or (6) fusion.
  • solid compositions such as tablets, are prepared by mixing a therapeutic agent of the present invention with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of the therapeutic agent and the excipient.
  • compositions(s) when referring to these preformulation compositions(s) as homogeneous, it is meant that the therapeutic agent is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms, such as tablets, pills and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described herein.
  • Compressed tablets are solid dosage forms prepared by compacting a formulation containing an active ingredient and excipients selected to aid the processing and improve the properties of the product.
  • the term "compressed tablet” generally refers to a plain, uncoated tablet for oral ingestion, prepared by a single compression or by pre-compaction tapping followed by a final compression.
  • the tablets or pills of the present invention may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
  • the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.
  • enteric layers or coatings including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
  • long-term sustained release implant may be suitable for treatment of mitochondrial disorders in patients who need continuous administration of the compositions of the present invention.
  • Long-term release as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredients for at least 30 days, and preferably 60 days.
  • Long-term sustained release implants are well known to those of ordinary skill in the art and include some of the release systems described above.
  • the compound for treating a mitochondrial disorder comes in the form of a kit or package containing one or more of the therapeutic compounds of the present invention.
  • These therapeutic compounds of the present invention can be packaged in the form of a kit or package in which hourly, daily, weekly, or monthly (or other periodic) dosages are arranged for proper sequential or simultaneous administration.
  • the present invention further provides a kit or package containing a plurality of separately-packaged dosage units, adapted for successive daily administration, each dosage unit comprising at least one of the therapeutic compounds of the present invention.
  • This drug delivery system can be used to facilitate administering any of the various embodiments of the therapeutic compounds of the present invention.
  • the system contains a plurality of dosages to be administered daily or weekly.
  • the kit or package can also contain the agents utilized in combination therapy to facilitate proper administration of the dosage forms.
  • the kits or packages also contain a set of instructions for the subject.
  • AChEI acetylcholinesterase inhibitor
  • a ⁇ /?-amyloid peptide
  • ADDLs amyloid-derived diffusible ligands
  • ANT adenine nucleotide translocase
  • APP amyloid precursor protein
  • CCCP carbonyl cyanide 3-chlorophenylhydrazone
  • FCCP carbonyl cyanide p-trifluoromethoxy-phenylhydrazone
  • CsA cyclosporin A
  • MPT membrane permeability transition
  • MRC mitochondrial respiratory coefficient
  • PAO phenylarsine oxide
  • Mitochondrial permeability transition may be further defined as a nonspecific increase in the permeability of the inner mitochondrial membrane that occurs under adverse conditions, such as an increase in mitochondrial Ca +2 content of the mitochondrial matrix or oxidative stress, and which causes the assembly (opening) of non-specific pores ("megachannels") in the mitochondrial inner membrane, leading to a loss of mitochondrial membrane potential, an uncoupling of mitochondrial respiration, and cellular energy failure, all of which can contribute to cell death.
  • MPT Mitochondrial permeability transition
  • Ap 1 - ⁇ 2 peptide was purchased from American Peptide Company (Sunnyvale, CA, USA) and stored at -80°C.
  • the antibody anti-complex II was purchased from Molecular Probes (Eugene, OR, USA) and the antibody anti- Ab 1-42 was from Signet Laboratories (Dedham, MA, USA).
  • the spirostenol, (22S,25S)-(20S)-s ⁇ irost-5-en-3/3-yl hexanoate (SP-233) was purchased from Interbioscreen (Moscow, Russia).
  • Cyclosporin A (CsA) and carbonyl cyanide 3- chlorophenylhydrazone (CCCP) were obtained from Sigma (St. Louis, MO, USA).
  • Dulbecco's Minimum Essential Medium (DMEM), fetal bovine serum (FBS) and SK-N-AS human neuroblastoma cells were purchased form ATCC (Manassas, VA, USA).
  • the pellet was resuspended in 300 ⁇ l of respiratory buffer (D-mannitol 300 mM, KCl 10 mM, KH2PO4 10 mM, MgC12 5 mM; pH 7.2 at 37°C). This pellet is highly enriched in mitochondria (Zini et al. 1996). The protein concentration of the mitochondrial suspension was determined by the method of Lowry et al. 1951.
  • Ap 1- ⁇ 2 solution was reconstituted with fresh distilled water to provide a concentration of 500 ⁇ M.
  • Ap 1 ⁇ 2 was used as freshly reconstituted or in an aggregated form after having been "aged” by incubating the solution for 48 h at 4 0 C. Both solutions were diluted with the respiratory buffer (see above) before use and then added directly to the mitochondria at the desired concentration. In experiments conducted with neuroblastoma cells (see below), only the freshly reconstituted A ⁇ i_ 42 solution was used. SP-233, CCCP and CsA were first dissolved in N,N-dimethylformamide (DMF) and then diluted in fresh distilled water.
  • DMF N,N-dimethylformamide
  • Mitochondrial functions were studied using an Oxytherm (Hansatech, Norfolk, UK) linked to a PC (Compaq, PIII 400 MHz).
  • the 37°C thermostated incubation chamber of the Oxytherm was connected to a platinum-silver Clarck electrode for O 2 detection.
  • O 2 consumption was monitored as a marker of mitochondrial function and viability in the presence of ADP (precursor for ATP) in the presence or absence of CCCP (an uncoupling agent for oxidative phosphorylation), or CsA (an inhibitor of the MPT pore).
  • Measurement of O 2 consumption permitted calculation of the mitochondrial respiratory coefficient (MRC) which is V 3 /V 4 .
  • MRC mitochondrial respiratory coefficient
  • V 4 is the basal O 2 consumption
  • V 3 is the O 2 consumption after adding ADP (ATP production).
  • ADP ATP production
  • the effect of increasing concentrations of SP-233 (1 pM to 1 nM) on the mitochondrial respiratory chain was assessed by monitoring the evolution of MRC.
  • the concentration of mitochondria was adjusted to 0.4 mg protein/ml using respiratory buffer.
  • Mitochondria were activated by addition of malate/glutamate as substrates of complex I of the respiratory chain.
  • State 2 is the baseline rate of mitochondrial oxygen consumption.
  • ATP synthesis was initiated by addition of ADP, the rate of oxygen consumption increased dramatically to reach state 3.
  • the rate of O 2 consumption returned to baseline (state 2) which was called state 4 (Chance and Williams 1956). Controls for each experiment corresponded to basal respiration in the presence of ADP.
  • SP-233 was present in the incubation mixture for 3 min and Ap 1- ⁇ 2 for 1.5 min at 37°C before addition of the substrates malate/glutamate. ADP was added 1 min after addition of malate/glutamate. Control for the effect of SP-233 versus Ap 1 ⁇ 2 was the same as for the experiment conducted in the absence of ADP.
  • SP-233 was studied at concentrations of 10, 30 and 100 pM. At a concentration as low as 10 pM, SP-233 induced a significant 50% decrease in the MRC of rat brain mitochondria, as compared to the control group (pO.OOl) (Fig. 1). Although the differences in the effects induced by various concentrations of SP-233 are not significant, the results do tend to display a concentration-effect relationship since the MRC was progressively decreased over the concentration range of 10-100 pM.
  • the uncoupling agent CCCP at 1 ⁇ M increased the O 2 consumption of rat brain mitochondria to 150% of the basal value (p ⁇ 0.01) (Fig. 2a). All concentrations of SP-233 that were tested (1-1000 pM) completely inhibited this metabolic effect of CCCP (p ⁇ 0.001). The inhibition of CCCP-induced uncoupling by SP-233 was associated with decreases in O 2 consumption values to 60-65% of the basal level, and was concentration-independent. SP-233 did not counteract the decrease in the MRC induced by CsA, but on the contrary, it amplified this effect of CsA in a concentration-dependent manner (Fig. 2b).
  • AjS 1-42 significantly decreased the MRC of rat brain mitochondria even when it was present at a concentration as low as 0.1 pM (Fig. 3). This effect was more pronounced with the fresh amyloid peptide than with the aged form, the respective inhibitory effects being 55-63% and 43-53%. This difference may be revealing that the non-aggregated form of AjS 1-42 penetrates into the mitochondria to a greater extent than the aged, aggregated form of the peptide.
  • Fresh A / 3 1-42 reduced the MRC by 71% compared to the control, and this effect was partially inhibited by SP-233 (Fig. 4a).
  • the MRC was restored to about 40% of the control value, and the MRC was significantly increased in comparison with the value obtained with AjS 1-42 alone (38.56 ⁇ 0.34 versus 28.93 ⁇ 1.75, pO.OOl).
  • Aged AjS 1-42 reduced the MRC by 51% compared to the control but SP-233 did not significantly prevent this effect (Fig. 4b). 5. Effect of SP-233 on A/3i. 42 -induced toxicity on SK-N-AS human neuroblastoma cells
  • Fig. 5 shows the effect of SP-233 on the AjS 1 -42 induced decrease in SK- N-AS cell viability.
  • SP-233 1 ⁇ M prevented the neurotoxicity induced by the three concentrations of A/3 1-42 used by 19%, 26% and 28%, respectively (p ⁇ .01).
  • AD Alzheimer's disease
  • AD The etiology of AD is well documented only for the familial early-onset form of the disease, which involves a mutation of the APP, presenilin-1 or presenilin-2 gene, hi contrast, the cause of the late-onset sporadic form of AD, which represents about 95% of all AD cases, remains to be established. Many theories have emerged, and among these it has been proposed that an energetic failure due to mitochondrial impairment and oxidative damage may be at the origin of AD (Schulz et al. 1997; Beal, 2000).
  • AjSi -42 and the fragment A/3 25-35 have been shown to inhibit respiration and the activities of key enzymes, such as succinate dehydrogenase, ⁇ -ketoglutarate dehydrogenase, pyruvate dehydrogenase and cytochrome oxidase, in neuronal mitochondria (Kaneko et al. 1995; Casley et al. 2002a). Inhibition of the different complexes of the respiratory chain has also been reported to occur in neuronal mitochondria exposed to AjS 25-35 (Pereira et al.
  • results presented herein above show further that AjS 1-42 inhibits mitochondrial respiration of rat brain mitochondria, as revealed by a decrease in the MRC, and that this inhibition occurs at AjS 1-42 concentrations as low as 0.1 pM with both freshly prepared and aged forms of the peptide (see Fig. 3).
  • the inhibitory effect is more pronounced with the fresh form probably because polymeric forms of A/3 1-42 would traverse mitochondrial membranes to a lesser extend than the monomeric form or oligomeric forms such as ADDLs, these latter forms therefore being more likely to exert their detrimental effects on the respiratory chain and being de facto more toxic.
  • the lack of protection by SP-233 of mitochondria submitted to the aged/aggregated form of AjS 1-42 might be due to an inability of SP-233 to bind the aggregated form of the amyloid peptide in the same way that it binds the monomeric form.
  • SP-233 was able to partially prevent the decrease in MRC induced by freshly prepared A 1 S 1-42 and protect mitochondrial function, confirming previous data which showed a restoring effect of SP-233 on the ATP synthesis of PC 12 cells exposed to A 1 S 1-42 (Lecanu et al. 2004).
  • SP-233 was active in very low concentrations (picomolar range), its mechanism of action might be relatively specific.
  • the present data also confirms recent results which showed that SP-233 protected neuronal cells against A 1 S 1-42 neurotoxicity by binding the monomeric form of the peptide and inhibiting the formation of the neurotoxic oligomeric ADDLs (Lecanu et al. 2004).
  • SP-233 was also able to abolish the uncoupling of oxidative phosphorylation induced by CCCP (see Fig. 2a). It has been shown that AJS J-42 disrupts mitochondrial membrane structure (Kremer et al. 2001; Rodrigues et al. 2001) and that mitochondrial membrane fluidity may be altered in AD brain (Mecocci et al, 1996), both phenomena leading to mitochondrial uncoupling. Therefore, even though the "recoupling" effect of SP-233 cannot yet be fully clarified, it might contribute to the restoration of mitochondrial function described herein.
  • Example 2 The Spirostenol (22R,25RV20a-spirost-5-en-3fi-yI Hexanoate Blocks Mitochondrial Uptake of AB in Neuronal Cells and Prevents AB- Induced Impairment of Mitochondrial Function A. Materials and methods 1. Materials
  • ABi -42 peptide was purchased from American Peptide Company (Sunnyvale, CA).
  • the anti-complex II antibody was purchased from Molecular Probes (Eugene, OR), and the antibody anti-AJ31-42 was from Signet Laboratories (Dedham, MA).
  • Spirostenol (SP-233) was purchased from Interbioscreen (Moscow, Russia).
  • Cyclosporin A carbonyl cyanide 3- chlorophenylhydrazone (CCCP), carbonyl cyanide p-trifluoromethoxy- phenylhydrazone (FCCP), rotenone, malonate, myxothiazol, potassium cyanide (KCN), oligomycin, and phenylarsine oxide (PAO) were obtained from Sigma (St Louis, MO).
  • Dulbecco's Minimum Essential Medium (DMEM), fetal bovine serum (FBS), and SK-N-AS human neuroblastoma cells were purchased form ATCC (Manassas, VA).
  • the pellet was resuspended in 300 ⁇ l of respiratory buffer (D-mannitol 300 mM, KCl 10 mM, KH 2 PO4 10 mM, MgCl 2 5 mM; pH 7.2 at 37°C). This pellet is highly enriched in mitochondria (Zini et al. 1996).
  • the protein concentration of the mitochondrial suspension was determined by the method of Lowry.
  • Mitochondrial respiration was measured using an Oxytherm (Hansatech, Norfolk, UK) linked to a PC (Compaq, PIII 400 MHz).
  • the 37°C incubation chamber of the Oxytherm was connected to a platinum-silver Clarck electrode for O 2 detection.
  • O 2 consumption was employed as a marker of mitochondrial respiration in the presence of ADP (precursor for ATP) and in the presence or absence of the following: CCCP (an uncoupling agent for oxidative phosphorylation), CsA (an inhibitor of the membrane permeability transition pore), or AB 1-42 .
  • ADP was added 1 min after addition of malate/glutamate.
  • SP-233, CCCP, and CsA were first dissolved in N,N-dimethylformamide (DMF) and then diluted in fresh distilled water.
  • AB 1 ⁇ 2 employed in these experiments was reconstituted with fresh distilled water to a concentration of 500 ⁇ M.
  • AB 1-42 was used as freshly reconstituted or in an aggregated form after having been "aged” by incubating the solution for 48 h at 4°C. Both solutions were diluted with the respiratory buffer before use and then added directly to the mitochondria to achieve the desired concentration.
  • Human neuroblastoma SK-N-AS cells were seeded in 96-well plates (7xlO 4 cells/well) in DMEM containing 10% FBS and were cultured in 5% CO .2 and 95% humidity. Cultures were treated with the following in the presence or absence of varying concentrations of SP-233: AB 1-42 (0.1, 1, and 10 ⁇ M), rotenone (50 nM), malonate (100 mM), myxothiazol (0.3 ⁇ M), KCN (12 mM), oligomycin (0.5 ⁇ g/ml), FCCP (3 ⁇ M), and PAO (0.25 ⁇ M).
  • SP-233 was added to cultures 1 hour prior to the addition of the mitochondrial toxins.
  • parallel cell cultures were incubated with or without increasing concentrations of SP-233, ranging from 1 to 100 ⁇ M, one hour prior to the addition of FCCP or PAO. Cell viability was assessed 24 hours later.
  • FCCP, rotenone, malonate, myxothiazol, KCN, oligomycin, and PAO were all first dissolved as a stock solution in ethanol before being diluted in culture medium to reach the indicated final concentrations. The percentage of ethanol in the culture medium was 0.09%.
  • Human neuroblastoma SK-N-AS cells were seeded on 13 -mm diameter coverslips (20,000 cells/coverslip) and incubated overnight at 37 0 C, at 5% CO 2 , in DMEM containing 10% FBS. Cultures were then incubated with AB 1-42 (10 ⁇ M) or its vehicle for 3 h in the presence or absence of SP-233 (1 ⁇ M).
  • Coverslips were mounted with an aqueous mounting medium containing 4',6-diamidino-2-phenylindole-2-hydrochloride (DAPI; Vector Laboratories, Burlingame, CA). Confocal images were acquired using an Olympus Fluoview BX61 Laser Scanning microscope.
  • DAPI 4',6-diamidino-2-phenylindole-2-hydrochloride
  • CCCP uncouples oxidative phosphorylation and increases O 2 consumption.
  • 1 ⁇ M CCCP increases O 2 consumption in rat brain mitochondria to 150% of the basal value (pO.Ol).
  • Concentrations of SP-233 ranging from 1-1000 pM completely abolished this effect (pO.OOl for all), and the inhibition of CCCP-induced uncoupling in the presence of all of these SP-233 concentrations was associated with a decrease in O 2 consumption values to 60-65% of the basal level.
  • the MRC was significantly increased in comparison to the value obtained with AB 1- 42 alone (38.56 ⁇ 0.34 vs. 28.93 ⁇ 1.75, ⁇ 0.001).
  • Aged AB 1-42 reduced the MRC by 51% compared to the control, but SP-233 did not significantly affect this aged ABi -42 -induced change in the MRC (Fig. 9b).
  • SP-233 decreased neurotoxicity induced by 0.1, 1, and lO ⁇ M AB 1-42 , by 19% (pO.Ol, compared to AB 1-42 alone), 26% (pO.Ol) and 28% (pO.Ol), respectively.
  • Figure 11 shows representative images of the DAPI staining (al, bl and cl), the immunofluorescent labeling OfAB 1-42 (a2, b2 and c2), and the immunofluorescent labeling of the complex II 70-kDa subunit (a3, b3 and c3) in SK-N-AS human neuroblastoma cells. Merged images are shown in Figure a4, b4, and c4.
  • Neuroblastoma cells treated with AB 1-42 displayed strong AB 1-42 immunoreactivity (Fig. 1 Ib2) that co-localized with labeling of complex II of the mitochondrial respiratory chain (Fig. Ilb4), providing evidence that AB 1-42 entered, and was present inside, the mitochondria.
  • a BI-42 immunoreactivity was abrogated in the presence of SP-233 (Fig. 1 Ic2), indicating that SP-233 blocked the entry OfAB 1-42 into the cell and the mitochondria.
  • SP-233 did not display any neuroprotective effect against the inhibitors of complexes I, II, and III of the mitochondria respiratory chain (Fig. 12 a, b, and c). At concentrations of 1 ⁇ M, SP-233 significantly protected against rotenone and 100 ⁇ M SP-233 significantly protected against myxoyhiazol (p ⁇ 0.05 for both). Although this protective effect was significant, in both cases the magnitude of the effect was small. In contrast, SP-233 exerted a pronounced beneficial effect against toxicity induced by KCN, a complex IV inhibitor, and oligomycin, a complex V inhibitor (Fig. 12d and 12e).
  • Adenine nucleotide translocase is a channel protein located in the inner membrane of the mitochondria that, under physiological conditions, exports ATP and imports ADP with a 1 : 1 ratio.
  • the inhibition of ANT by PAO promotes the opening of the permeability transition pore, which leads to apoptosis.
  • One micro molar SP-233 reduced the toxic effect of PAO on SK-N- AS neuronal cells by 25% (Fig. 13, p ⁇ .01). Concentrations of SP-233 above l ⁇ M were not associated with any neuroprotective effects.
  • SP-233 exerted a small, but significant (p ⁇ 0.01), effect on FCCP-induced uncoupling of the respiratory chain when administered at the concentration of 100 ⁇ M (Fig. 14).
  • AD Alzheimer's disease
  • AchEIs acetylcholinesterase inhibitors
  • N-methyl-D- aspartate (NMDA) receptor antagonist has recently been approved for treating moderately to severe AD (Livingston and Katona 2004), there is an urgent need for new drugs and new therapeutic strategies that target the etiological causes of the AD.
  • AD The etiology of AD is well documented for the familial, early-onset form of the disease, which involves a mutation of the APP (presenilin-1 or presenilin- 2) gene, hi contrast, the cause of the late-onset sporadic form of AD, which represents about 95% of all AD cases, remains to be established.
  • APP preilin-1 or presenilin- 2 gene
  • Many theories have emerged, and among these is the proposal that an energetic failure due to mitochondrial impairment and oxidative damage maybe at the origin of AD (Schulz et al. 1997; Beal, 2000). This mitochondrial dysfunction theory, together with the recent finding that the spirostenol derivative, SP-233, can protect neuronal PC 12 cells against AB 1-42 -induced neurotoxicity (Lecanu et al.
  • AB 1-42 and the fragment AB 25-35 have been shown to inhibit respiration and the activities of key enzymes such as succinate dehydrogenase, ⁇ -ketoglutarate dehydrogenase, pyruvate dehydrogenase, and cytochrome oxidase, in neuronal mitochondria (Kaneko et al. 1995; Casley et al. 2002a). Inhibition of the respiratory chain complexes has also been reported in neuronal mitochondria exposed to AB 25-35 (Pereira et al. 1999; Canevari et al.
  • SP-233 was able to partially prevent the decrease in MRC induced by freshly prepared AB 1-42 , confirming previous results showing a restorative effect of SP-233 on ATP synthesis in PC12 cells exposed to AB 1-42 (Lecanu et al. 2004).
  • the finding that SP-233 is active at very low concentrations suggests that its mechanism of action might be relatively specific.
  • the data described herein also confirmed recent results showing that SP-233 protects neuronal cells against AB 1-42 neurotoxicity by binding to the monomeric form of the peptide and inhibiting the formation of the neurotoxic oligomeric ADDLs (Lecanu et al. 2004).
  • SP-233 a protein tyrosine phosphatase inhibitor known to promote the MPT (Korge et al., 2001).
  • SP-233 at concentrations of 1 ⁇ M, restored neuronal cell viability by 25%; whereas, higher concentrations of SP-233 were not associated with a protective effect.
  • SP-233 's effect on MPT-linked pore opening may occur via direct and/or indirect action (e.g., by allosteric modification after its insertion into mitochondrial membranes).
  • SP-233 was also able to abolish CCCP-induced uncoupling of oxidative phosphorylation in isolated mitochondria and in neuroblastoma cells. It has been shown that ABi -42 disrupts mitochondrial membrane structure (Kremer et al. 2001; Rodrigues et al. 2001) and that mitochondrial membrane fluidity may be altered in AD brain (Mecocci et al, 1996); both phenomena lead to mitochondrial uncoupling. Therefore, even though the "re-coupling" effect of SP-233 has not been fully clarified, it might contribute to the restoration of mitochondrial function described herein.
  • SP-233 is able to protect neuroblastoma cells against inhibitors of complexes I, III, IV, and V, but not against inhibitors of complex II (Fig. 15).
  • Beta-amyloid peptides induce mitochondrial dysfunction and oxidative stress in astrocytes and death of neurons through activation of NADPH oxidase. J. Neurosci. 24, 565-575. Bachurin S. O., Shevtsova E. P., Kireeva E. G., Oxenkrug G. F. and Sablin S. O. (2003) Mitochondria as a target for neurotoxins and neuroprotective agents. Ann. NY. Acad. ScL 993, 334-344. Beal M. F. (1992) Does impairment of energy metabolism result in excitotoxic neuronal death in neurodegenerative disease? Ann. Neurol. 31, 119-130. Beal M. F. (2000) Energetics in the pathogenesis of neurodegenerative diseases.
  • Beta-amyloid fragment 25-35 causes mitochondrial dysfunction in primary cortical neurons. Neurobiol. Dis. 10, 258-267. Courtney C, Farrell D., Gray R. et al ⁇ AD2000 Collaborative Group. (2004) Long-term donepezil treatment in 565 patients with Alzheimer's disease (AD2000): randomised double-blind trial. Lancet 363, 2105-2115. Eckert G. P., Wood W. G. and Muller W. E. (2001) Effects of aging and ⁇ - amyloid on the properties of brain synaptic and mitochondrial membranes. J. Neural. Transm. 108, 1051-1064.
  • Kaneko L Yamada N., Sakuraba Y., Kamenosono M. and Tutumi S. (1995) Suppression of mitochondrial succinate dehydrogenase, a primary target of beta-amyloid, and its derivative recemized at Ser residue. J. Neurochem. 65, 2585-2593. Kim S. H., Vlkolinsky R., Cairns N. and Lubec G. (2000) Decreased levels of complex HI core protein 1 and complex V beta chain in brains from patients with Alzheimer's disease and Down syndrome. Cell. MoI. Life ScL 57, 1810- 1816.
  • Alzheimer type a hypothesis. Neurobiol. Aging 11, 567-571. Waldemar G., Winblad B., Engedal K., Soininen H., Verhey F., Wimo A., Wetterholm A. L., Zhang R., Haglund A. and Subbiah P. (2001) Benefits of donepezil on cognition, function and neuropsychiatric symptoms in patients with mild and moderate Alzheimer's disease over one year, in Alzheimer's

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Abstract

The present invention relates to methods and compositions involving the use of spirostenol derivatives for treating, preventing or reducing the risk of developing a mitochondrial disorder or disease or the symptoms associated with a mitochondrial disorder or disease.

Description

USE OF SPIROSTENOLS TO TREAT MITOCHONDRIAL DISORDERS
Related Application This application claims priority from U.S. Provisional Application Serial
No. 60/667,229 filed April 1, 2005 and to U.S. Provisional Application Serial No. 60/697,518 filed July 8, 2005, which applications are herein incorporated by reference.
Background of the Invention Mitochondria are specialized compartments present in every cell of the body except red blood cells, and are responsible for generating, by oxidative phosphorylation, more than 90% of the energy needed by the body to sustain life and support growth. When the mitochondrial respiratory chain fails, the energy level of the cell will rapidly decrease leading to the cell death and organ function impairment. Depending on the organ considered, the impairment of the mitochondrial physiological function is involved in neurodegenerative diseases and disorders. These include those of the central nervous system, such as Alzheimer's Disease and Parkinson's Disease, and those affecting the peripheral nerves, such as muscular disorders (myopathies) and various kidney, liver and respiratory disorders.
Depending on how severe the mitochondrial disorder is, the illness can range in severity from mild to lethal. Whether the mitochondrial failure is a cause or a consequence of the condition experienced, protecting respiratory function is critical to restore the impaired physiological functions. In addition, the preservation of the mitochondrial respiratory function is essential for the conservation of tissue required for a successful graft.
There are no cures for mitochondrial diseases which have a genetic basis, but treatment can help reduce symptoms or delay or prevent the progression of the disease (M. Zeviani et al, Brain; 127: 2153 (2004)). Treatment is individualized for each patient, depending on the nature and the severity of the disorder, although these palliative treatments will not reverse the damage already sustained. However, a number of experimental strategies are currently being assessed, including gene therapy and pharmacological intervention.
One strategy consists of the introduction of modified genes or gene products into mitochondria via the protein import machinery (G. Manfredi et al., Natur. Genet, 30:394 (2002)) and inhibition of replication of mutant mtDNA by sequence-specific antigenomic peptide-nucleic acids (R.W. Taylor et al., Natur. Genet, 15:212 (1997)). These approaches are not yet employed clinically.
In selected, isolated myopathy cases, reduction of heteroplasmic mutant load was obtained by controlled muscle fiber damage and regeneration by mutation-free satellite cells, using myotoxic drugs (W. Irwin et al., J Biol. Chem., 277:2221 (2002)). However, this treatment was not effective in improving ptosis in five patients with progressive external ophthalmoplegia (PEO). Creatine is the substrate for the synthesis of phosphocreatine and is the most abundant energy storage compound in muscle, heart and brain. An open trial of 81 patients with various neuromuscular disorders (including 17 with mitochondrial diseases) showed significant improvement of ischaemic isometric handgrip strength and non-ischemic isometric dorsiflexion torque. Another placebo-controlled, double-blind, randomized crossover trial in 16 patients with chronic PEO or mitochondrial myopathy, however, did not find significant effects on exercise performance, eye movements, or activities of daily life (P. F. Chinnery et al., Am. J. Med. Genet, 106:94 (2001)). Taken together, these data suggest that creatine may be effective in some, but not all mitochondrial diseases.
While Coenzyme QlO is not effective in mtDNA-associated mitochondrial disease (N. Bresolin et al., J. Neurol. ScL, 100:70 (1990)) it leads to marked improvement in 'primary' CoQlO deficiency. Idebenone, a shorter chain analogue of Coenzyme QlO, appears to be effective in improving the hypertrophic cardiomyopathy in Friedreich's ataxia (P. Rustin et al., Lancet, 354: 477 (1999); C. Mariotti et al., Neurology, 60:1676 (2003)).
Thus, there is a continuing need for methods to treat mitrochondrial disorders, including mitochondrial diseases.
Summary of the Invention
The present invention provides a therapeutic method comprising administering to a mammal, such as a human, afflicted with or threatened by, a mitochondrial disorder that is not a neuropathology of the central nervous system comprising administering to said mammal an effective amount of a compound of formula (T):
Figure imgf000004_0001
wherein each OfR1, R2, R4, R7, R11, R12, and R15, independently, is hydrogen, (d-C8)alkyl, that is optionally inserted with -NR'-, -O-, -S-, -SO-, -SO2-, -O- SO2-, -SO2-O-, -C(O)-, -C(O)-O-, -O-C(O)-, -C(O)-NR'-, or -NR'-C(O)-; hydroxy, N(R')2, carboxyl, oxo, or sulfonic acid; R3 is hydroxy, (C1-C8)alkoxy, (C1-C22)alkylCO2 or R'O2C(CH2)2-8CO2-, wherein (C1-C22)alkyl or (CH2)2-8 can optionally comprise 1-2 CH=CH units, 1-2 OH, and/or 1-2 epoxy substituents, toluene-4-sulfonyloxy or benzoyloxy; each of R6, R8, R9, R10, R13, Ri4, Ri6 and Ri7, independently, is hydrogen, (Ci-C8)alkyl, hydroxyl(Ci-C8)alkyl, (Ci- C8)alkoxy, or hydroxy; and X is O, S, S(O), N(R'), N(Ac) or N(toluene-4- sulfonyloxy), wherein each R' is individually H, (Ci-C8)alkyl, phenyl or benzyl; or a pharmaceutically acceptable salt thereof. Ri6 and/or Ri7 are preferably CH3. Alkyl is preferably (C2-C8)alkyl.
Therefore, the present invention includes therapeutic methods of treating a mitochondrial disorder in a subject afflicted with or threatened by said disorder, wherein said disorder is not a neuropathology of the central nervous system, comprising administering to the subject an effective amount of a compound of formula (II):
Figure imgf000004_0002
(II) wherein each OfR1, R2, R4, R7, R11, R12, and R15, independently, is hydrogen, (C1-C8)allcyl, hydroxy, amino, carboxyl, oxo, sulfonic acid, or (C1- C8)alkyl that is optionally inserted with -NH-, -N((d-C8)alkyl)-, -O-, -S-, -SO-, -SO2-, -0-SO2-, -SO2-O-, -C(O)-, -C(O)-O-, -O-C(O)-, -C(O)-NR'-, or -NR'- C(O)-, wherein R' is H or (Ci-C8)alkyl; R3 is hydroxy, (Ci-C6)alkylCO2-,
HO2C(CH2)2CO2-, toluene-4-sulfonyloxy or benzoyloxy; each of R6, R8, R9, R10, R13, and R14, independently, is hydrogen, (d-C8)alkyl, hydroxyl(C1-C8)alkyl, (C1-C8)alkoxy or hydroxy; and X is O, N(H), N(Ac) or N(toluene-4- sulfonyloxy); or a pharmaceutically acceptable salt thereof. As discussed in detail below, spirostenols, particularly 5,6 unsaturated spirostenols such as (22S,25S)-(20S)-spirost-5-en-3β-yl hexanoate, a 22i?- hydroxycholesterol derivative naturally occurring in Gynurajaponica (asteraceae), can provide a novel therapeutic strategy that works by its direct targeting of the mitochondrial respiratory chain, as well as its targeting of Aβ which is toxic to mitochondria. (22S,25S)-(20S)-Spirost-5-en-3β-yl hexanoate also abolished the induced uncoupling of oxidative phosphorylation and amplified the effect of cyclosporin A, a potent blocker of mitochondrial membrane permeability transition pore, which suggest an effect on membrane permeability transition pore. Mitochondrial disorders, including mitochondrial diseases that are not considered to be CNS neuropathologies, and that can be treated with the present method include the following: eye: retinitis pigmentosa, optical nerve atrophy; muscles: disorders of the extraoccular muscles (ptosis, acquired strabismus, ophthalmoplegia), crushed muscle syndrome (compartment syndrome); mitochondrial dysfunction as a result of drug side-effect (i.e., highly active anti- retroviral therapy, HAART); myopathy; heart: heart stroke, heart attack, heart conditions, heart disease, cardiomyopathy, and any cardiac mitochondrial dysfunction as a result of drug side-effect (i.e., HAART, mega-HAART, anticancer drugs like anthracyclins); liver: hepatocellular dysfunctions; kidney and the endocrine system: tubulopathy, diabetes; and respiratory disorders.
The present invention also provides novel compounds of formulas I or II and compositions, such as pharmaceutical compositions comprising an effective amount of a compound of formula I and/or II in combination with a pharmaceutically acceptable carrier. Brief Description of the Figures
Fig. 1 Effect of increasing concentrations of SP-233 (10 to 100 pM) on the mitochondrial respiratory chain, assessed by monitoring the evolution of the respiratory coefficient in rat brain mitochondria. This mitochondrial respiratory coefficient (MRC) is V3/V4. V4 is the basal O2 consumption, V3 is the O2 consumption after adding ADP (ATP production). The mitochondrial concentration was adjusted to 0.4 mg protein/ml. It is noteworthy that concentrations of SP-233 as low as 10 pM induced a 50% decrease in the MRC, compared to control (p<0.001). Results are expressed as mean ± SD; comparisons between groups were made by an ANOVA followed by a Dunnett's test.
Fig. 2 (a): 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 for 3 min at 37°C in presence of SP-233 before adding malate/glutamate, followed 1 min later by the addition of CCCP. Exposure to the uncoupling agent CCCP (1 μM) increased the O2 consumption to 150% of the basal value (p<0.01), a change that reflected an increase in oxygen consumption. At all concentrations tested (even at 1 pM), SP-233 abolished the metabolic effect of CCCP (pO.OOl). This inhibitory effect of SP-233 on "uncoupling" was associated with decreases in the O2 consumption to 60-65% of the basal level and was concentration-independent, (b): Effect of SP-233 on CsA-induced hypoxia. Hypoxia was induced by adding CsA (1 μM) to the medium bathing the mitochondria. This test was controlled by adding ADP in presence of CsA and absence of SP-233. SP-233, or its vehicle, was added first, and incubation was continued for 1.5 min before CsA was gently added. Then, the substrate malate/glutamate was added 1.5 min later and ADP 2.5 min later. Exposure to SP-233 did not inhibit the decrease in the MRC induced by CsA, but on the contrary, the effect of CsA was amplified in a concentration- dependent manner. Results are expressed as mean ± SD; comparisons between groups were made by an ANOVA followed by a Dunnett's test.
Fig. 3 Effects of fresh and aged Aβi-42 on the mitochondrial respiratory coefficient of rat brain mitochondria. Mitochondria were incubated in the presence of Aβi-42 for 1.5 min at 37°C before adding the substrates malate/glutamate. ADP was added 1 min after malate/glutamate. This experiment was controlled by omitting ADP. A/5i-42 significantly decreased the MRC even at the low concentration of 0.1 pM. The effect of the fresh amyloid peptide on the MCR was more pronounced than that of the aged form, the respective decreases in MRC being 55-63% and 43-53%. Results are expressed as mean ± SD; comparisons between groups were made by an ANOVA followed by a Dunnett's test.
Fig. 4 Effects of SP-233 on modifications of the mitochondrial respiratory coefficient induced by fresh or aged AjSi -42 in rat brain mitochondria. Mitochondria were incubated in the presence of SP-233 for 3 min and in the presence of freshly prepared (a) or aged (b) Aβi-42 for 1.5 min at 370C before addition of malate/glutamate. ADP was added 1 min after the substrates malate/glutamate. The control for the effect of SP-233 versus AjSi -42 was the same as for the experiment without ADP. (a): Fresh Aβi-42 reduced the MRC by 71% compared to the control, and this effect was partially inhibited by SP-233. The most potent effect was observed with the lowest concentration of SP-233 which restored the MRC to 39% of the control value (38.56 ± 0.34 versus 28.93 ± 1.75, pO.001). (b): Aged AjSi -42 reduced the MRC by 51 % compared to the control value, but SP-233 did not prevent this effect. Results are expressed as mean ± SD; comparisons between groups were made by an ANOVA followed by a Dunnett's test.
Fig. 5 Assessment of the protective effect of SP-233 against AjSi-42- induced toxicity in human neuroblastoma cells. The cellular toxicity of AjS was assessed after 72 h incubation of SK-N-AS neuroblastoma cells with Aβi-42 (0.1 , 1 and 10 μM) or vehicle and in the presence or absence of SP-233 (1 μM) using the MTT assay. SP-233 (1 μM) prevented the neurotoxicity induced by the three concentrations of AjSi -42. Results are expressed as mean ± SD; comparisons between groups were made by an ANOVA followed by Dunnett's test. Neuroblastoma cells treated with AjSi-42 displayed strong immunoreactivity (which co-localized with complex II of the mitochondrial respiratory chain, indicating that AjSi -42 was present inside the mitochondria). Treatment with SP-233 abolished AjSi -42 immunoreactivity, indicating its ability to block the entry of AjSi -42 into the mitochondria. Fig. 6 Effect of SP-233 on mitochondrial respiration. The Chemical formula of the spirostenol derivative (22R,25R)-20α-spirost-5-en-3β-yl hexanoate (SP-233) is shown in (a). The effect of increasing concentrations of SP-233 (1 aM to 100 pM) on the mitochondrial respiratory chain, assessed by monitoring the evolution of the respiratory coefficient in isolated rat brain mitochondria, is shown in (b). This mitochondrial respiratory coefficient (MRC) is defined as V3/V4 where V4 is the basal O2 consumption and V3 is the O2 consumption after adding ADP (ATP production). The mitochondrial concentration was adjusted to 0.4 mg protein/ml. Note that concentrations of SP-233 as low as 1 fJVI resulted in a 40% decrease in the MRC, compared to the control (p<0.001). Results are expressed as means ± SD from three independent experiments performed in triplicate.
Fig. 7 Effect of SP-233 on CCCP-induced uncoupling of oxidative phosphorylation and on CsA-induced hypoxia, (a) To assess the effect of SP-233 on CCCP-induced uncoupling of oxidative phosphorylation, the mitochondrial fraction was incubated for 3 min at 37°C in presence of SP-233 before adding malate/glutamate. CCCP was added 1 min later. Exposure to the uncoupling agent 1 μM CCCP increased the O2 consumption to 150% of the basal value (p<0.01). At all concentrations tested, SP-233 abolished the metabolic effect of CCCP (pO.001). SP-233 's inhibitory effect on "uncoupling" was associated with a decrease in O2 consumption to 60-65% of the basal level and was concentration-independent, (b) Hypoxia was induced by adding 1 μM CsA to the medium bathing the mitochondria. As a control, ADP was added in presence of CsA and in the absence of SP-233. SP-233, or its vehicle, was added first, and incubation was continued for 1.5 min before CsA was gently added. The substrate malate/glutamate was added 1.5 min later, and ADP was added 2.5 min later. Exposure to SP-233 did not inhibit the decrease in the MRC induced by CsA. On the contrary, CsA' s effect was amplified in a concentration-dependent manner. Results are expressed as means ± SD from three independent experiments performed in triplicate.
Fig. 8 Effects of fresh and aged Aβl-42 on the mitochondrial respiratory coefficient of rat brain mitochondria. Mitochondria were incubated in the presence OfAB1^42 for 1.5 min at 37°C before adding the substrates malate/glutamate. ADP was added 1 min after malate/glutamate. In these experiments ADP was omitted from the incubation mixture. AB1-42 significantly decreased the MRC, even at a concentration of 0.1 pM. The effect of the fresh amyloid peptide on the MCR was more pronounced than that of the aged form, the respective decreases in MRC being 55-63% and 43-53%. Results are expressed as means ± SD from three independent experiments performed in triplicates.
Fig. 9 Effects of SP-233 on changes in the MRC induced by fresh or aged AB1-42. Mitochondria were incubated in the presence of SP-233 for 3 min and in the presence of freshly prepared (a) or aged (b) AB 1-42 for 1.5 min at 370C before addition of malate/glutamate. ADP was added 1 min after the addition of malate/glutamate. Controls reactions were performed in the absence of ADP. Fresh AB1-42 reduced the MRC by 71% compared to the control, and this effect was partially inhibited by SP-233.
The most potent effect was observed with the lowest concentration of SP- 233 examined, which restored the MRC to 39% of the control value (38.56 ± 0.34 vs. 28.93 ± 1.75, p<0.001). Aged AB1-42 reduced the MRC by 51% compared to the control value, but SP-233 did not prevent this effect. Results are expressed as means ± SD from three independent experiments performed in triplicate. Fig. 10 Assessment of the protective effect of SP-233 against AB1-42- induced toxicity in human neuroblastoma cells. The cellular toxicity of AB was assessed 72 h after incubation of SK-N-AS cultures with AB 1-42 (0.1, 1 and 10 μM) or vehicle and in presence or absence of SP-233 (1 μM) using the MTT assay. Results are expressed as means ± SD from three independent experiments performed in triplicate.
Fig. 11 Confocal microscopy analysis of the effect of SP-233 on mitochondrial uptake of AB 1-42 in SK-N-AS human neuroblastoma cells. Representative images taken from healthy, uninjured (control) cells are shown in (al-4). These cultures did not receive any AB1-42 or SP-233. Representative images taken from SK-N-AS cells treated with ABj-42 10 μM for 3 hours are shown in (bl-4). Representative images taken from SK-N-AS cells treated with AB1-42 10 μM and SP-233 1 μM for 3 hours are shown in (cl-4). Cells were stained with DAPI nuclear counterstain (al, bl, and cl) and labeled for AB1-42 (FITC; a2, b2, and c2) and complex II 70-kDa subunit (Texas red; a3, b3, and c3). Merged images are shown in a4, b4, and c4. Neuroblastoma cells treated with AB1-42 displayed strong immunoreactivity (b2) that co-localized with complex II of the mitochondrial respiratory chain (b4), and treatment with SP- 233 abolished AB1-42 immunoreactivity (c2). Fig. 12 Neuroprotective effects of SP-233 against mitochondrial complex inhibitors in SK-N-AS cells. Five different mitochondrial toxins were added to SK-N-AS cultures 1 hour after the addition of SP-233 or its solvent. Rotenone, malonate, and myxothiazol were incubated for 24 hours in absence or presence of SP-233 before the cell viability measures were carried out. The duration of incubation for KCN and oligomycin was 6 hours. Results are expressed as means ± SD from three independent experiments performed in triplicate. * ρ<0.05, ** ρ<0.01, *** pO.OOl compared to cultures not treated with SP-233. Fig. 13 Neuroprotective effects of SP-233 on SK-N-AS neuronal cells against PAO. PAO was added to cultures 1 hour after the addition of SP-233 or its solvent. Cultures were incubated in PAO for 24 hours in the absence or presence of SP-233, prior to cell viability measurements. Results are expressed as means ± SD from three independent experiments performed in triplicate. ** pO.Ol compared to cells not treated with SP-233.
Fig. 14 Effect of SP-233 on FCCP-induced uncoupling of the mitochondrial respiratory chain in SK-N-AS cells. FCCP was added to SK-N- AS cultures 1 hour after the addition of SP-233 or its solvent. Cultures were incubated in FCCP for 24 hours in the absence or presence of SP-233, before the cell viability measurements were performed. Results are expressed as means ± SD from three independent experiments performed in triplicate. ** p<0.01 compared to cells not treated with SP-233.
Fig. 15 Schematic representation of the mitochondrial sites targeted by SP-233. The elements of the respiratory chain targeted by SP-233 are indicated with a green arrow. Dotted arrows denote a poor neuroprotective effect, and full green arrows denote a strong protective effect. The most pronounced effect was observed against KCN, an inhibitor of complex IV, oligomycin, an inhibitor of the complex V, and PAO, a promoter of the MPT. Detailed Description of the Invention
As discussed hereinbelow, impairment of the mitochondrial respiratory chain has been extensively described in relation to Alzheimer's disease (AD), and it has been reported that Aβi-42 uncouples the mitochondrial respiratory chain and promotes the opening of the membrane permeability transition (MPT) pores of mitochondria, leading to cell death. The spirostenol (22S,25S)-(20S)- spirost-5-en-3/3-yl hexanoate (SP-233) has been reported to protect neuronal cells against AjS1-42 toxicity by binding and inactivating the peptide. The present invention is based on the discovery that the protective effect of SP-233 also results in protection of mitochondrial function. It was found that picomolar concentrations OfAjS1-42 decreased the mitochondrial respiratory coefficient in mitochondria isolated from rat forebrain by a protective effect that was partially reversed by SP-233. This protective effect of SP-233 probably results from its direct targeting of the respiratory chain, as well as its targeting of AjS1-42. SP-233 also abolished the uncoupling of oxidative phosphorylation induced by carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and amplified the effect of cyclosporin A (a potent blocker of mitochondrial MPT pores), suggesting an effect on MPT. These properties of SP-233 might contribute to its neuroprotective effect, since the uncoupling of the respiratory chain and the opening of mitochondrial MPT pores are two deleterious events that could be triggered by A/31-42.
Using human SK-N-AS neuroblastoma cells, it was further observed, using confocal microscopy, that AjS1-42 accumulated in the mitochondrial matrix and that SP-233 completely scavenged AjS1-42 from the matrix. These results indicate that AjS1-42 and SP-233 exert direct effects on the mitochondrial function of human neuronal cells, and that SP-233 (when present in the incubation mixture together with the amyloid peptide) can protect these cells against A/3- induced toxicity. Collectively, these findings indicate that A1S1-42 may exert a direct toxic effect on mitochondrial function, and that SP-233 exerts its neuroprotective effects by targeting A/3 directly, thereby protecting the respiratory chain, and providing a family of compounds useful to treat mitochondrial diseases and disorders other than AD and other CNS neuropathologies. As used herein, the term mitochondrial disorder encompasses art recognized mitochondrial diseases as well as conditions in which mitochondrial disorders or dysfunction plays a role in the onset symptomology, progression and/or outcome. The latter conditions include neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, or myotrophic lateral sclerosis as well as neuronal death induced by stroke, cerebral ischemia and other brain and spinal cord injuries. The use of spirostenols including (22S,25S)-(20S)-spirost-5-en-3-yl hexanoate to protect against A-induced neurotoxicity and their potential use in AD therapy has been reported (L. Lecanu et al., Steroids, 69: 1 (2004)) and is the subject of published PCT application WO/03/077869. The use of (22S,25S)-(20S)-spirost-5-en-3-yl hexanoate to protect mitochondria against a variety of stressors and its use in the therapy of various mitochondrial diseases has not been previously known.
Mitochondrial diseases or mitochondrial myopathies are a group of diseases affecting the mitochondria, that also interfere with the function of muscles. The group includes Kearns-Sayre syndrome, Leigh's syndrome, mitochondrial DNA depletion syndrome (MDS), mitochondrial encephalomyopathy, lactic acidosis and strokelike episodes (MELAS), myoclonus epilepsy with ragged red fibers (MERFF), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), neuropathy, ataxia and retinitis pigmentosa (NARP), and progressive external ophthalmoplegia (PEO). The symptoms of mitochondrial myopathies include muscle weakness or exercise intolerance, heart failure (cardiomyopathy) or rhythm disturbances, dementia, movement disorders, stroke-like episodes, deafness, blindness, droopy eyelids, limited mobility of the eyes, vomiting, and seizures. The prognosis for these disorders ranges in severity from progressive weakness to death. Thus, mitochondrial disorders can include disorders of the eye, such as optic nerve atrophy, ptosis, acquired stabimus, and ophthalomplegia. Mitochondrial dysfunction can be caused by crushed muscle syndrome (or compartment syndrome), the side effects of drug therapies such as HAART and anti-cancer therapies involving drugs that damage the heart (e.g., anthracycline), hepatocellular dysfunction, endocrine tubulopathy and respiratory disorders. Most mitochondrial myopathies occur before the age of 20, and often begin with exercise intolerance or muscle weakness. During physical activity, muscles may become easily fatigued or weak. Muscle cramping is rare, but may occur. Nausea, headache, and breathlessness are also associated with these disorders.
The term "treat" or "treatment" as used herein refers to any treatment of a disorder or disease associated with a disease or disorder related to mitochondria, in a subject, and includes, but is not limited to, preventing the disorder or disease from occurring in a subject who may be predisposed to the disorder or disease, but has not yet been diagnosed as having the disorder or disease; inhibiting the disorder or disease, for example, arresting the development of the disorder or disease; relieving the disorder or disease, for example, causing regression of the disorder or disease; or relieving the condition caused by the disease or disorder, for example, stopping the symptoms of the disease or disorder. As used herein, "mitochondrial disorder" or "mitochondrial disease" is intended to encompass all disorders disclosed herein. The present method can also be used to prevent or reduce mitochondrial disorders in vitro, as in organs or tissues awaiting or undergoing transplantation.
The term "prevent" or "prevention," in relation to a disease or disorder related to mitochondria, in a subject, means no disease or disorder development if none had occurred, or no further disorder or disease development if there had already been development of the disorder or disease, or no symptoms to logically observable signs of the disease.
Preferred stereoisomers are 3S, as well as 1Oi? and 13 S, and are also 2OS, 22S and 25S wherein the carbon skeleton is numbered in accord with spirosten- 3-ol numbering. Thus, a preferred compound of formula (I or II) is (225,255)- (20iS)-spirost-5-en-3β-yl hexanoate (SP233). Note that the carbon atoms shown in formula (I) or (II) are saturated with hydrogen unless otherwise indicated.
Unless defined otherwise, each of the term "alkyl," the prefix "alk" (as in alkoxy), and the suffix "-alkyl" (as in hydroxyalkyl) refers to a Ci -8 hydrocarbon chain, linear (e.g., butyl) or branched (e.g., isobutyl). Alkylene, alkenylene, and alkynylene refer to divalent Ci-8 alkyl (e.g., ethylene), alkene, and alkyne radicals, respectively. Preferably, alkyl is (Ci-C6)alkyl, such as butyl, hexyl, methyl, ethyl, propyl or isopropyl. The term "alkyl" includes cycloakyl, (cycloalkyl)alkyl and alkyl(cycloalkyl)alkyl. The term "alkenyl" likewise includes alkyl comprising 1-2 CH=CH units, as well as the corresponding cycloalkenyl moieties.
Specifically, (C1-C8)alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, heptyl or octyl; (C3-C8)cycloalkyl can be monocyclic, bicyclic or tricyclic and includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.2] octanyl or norbornyl, as well as various terpene and terpenoid structures. (C3-C6)cycloalkyl(C!-C2)alkyl includes the foregoing cycloalkyl and can be cyclopropymiethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl, 2- cyclopentylethyl, or 2-cyclohexylethyl. Heterocycloalkyl includes cycloalkyl wherein the cycloalkyl ring system is monocyclic, bicyclic or tricyclic and optionally comprises 1-2 S, non-peroxide O or N(R') as well as 4-8 ring carbon atoms; such as morpholinyl, piperidinyl, piperazinyl, indanyl, l,3-dithian-2-yl, and the like. Any cycloalkyl or heterocycloalkyl ring system optionally includes 1-3 double bonds or epoxy moieties and optionally is substituted with 1-3 OH, (d-C^alkanoyloxy, (CO), (Q-C^alkyl or (C2-C6)alkynyl. (C1-C8)AIkOXy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy; (C2-C6)alkenyl can be vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl; hydroxy(C1-C8)alkyl or hydroxy (C1-C8)alkoxyl can be alkyl substituted with 1 or 2 OH groups, such as alkyl substituted with 1 or 2 OH groups such as hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2- hydroxypropyl, 3-hydroxypropyl, 1-hydroxybutyl, 4-hydroxybutyl, 3,4- dihydroxybutyl, 1-hydroxypentyl, 5-hydroxypentyl, 1-hydroxyhexyl, or 6- hydroxyhexyl; (d-C22)alkylCO2- can be acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy or hexanoyloxy, or (C8-C22)CO2- can represent the residue of a naturally occurring fatty acid.
In one embodiment of the invention, R10 and/or R12 are CH3. In another embodiment of the invention, R16 and R17 are CH3.
In a further embodiment of the invention, R1, R2 and/or R12 can be H or OH.
In another embodiment, R1, R2, R4, R6, R7, R8, R9, R11, R12, R14 and R15 are H. R2 can be (CrC22)alkylCO2-, including (C8-C22)alkylCO2- or (C1- C6)alkylCO2-, or can be OH or CXQ-CsOalkyl, or HO2C(CH2)2CO2-.
X can be O or NR' including NH or NAc.
R3 may also be OR23 wherein R23 is a removable hydroxy-protecting group such as tosyl, mesyl, M(C1-GOaIkVlSiIyI, THP, Eto(Et), benzyl, benzoyloxycarbony and the like. C3-(C8-C22) fatty acid esters of compounds in which C3 is hydroxysubstituted are also within the invention, wherein the fatty acid is preferably naturally occurring.
Shown below in Table 1 are several compounds of formula (I) and (II) described above that can be used to practice this invention: Table 1.
Figure imgf000015_0001
An effective amount of an efficacious compound of the invention can be formulated with a pharmaceutically acceptable carrier to form a pharmaceutical composition before being administered for treatment of a disease related to impaired mitochondrial function. "An effective amount" or "pharmacologically effective amount" refers to the amount of the compound which is required to confer therapeutic effect on the treated subject. The interrelationship of dosages for animals and humans (based on milligrams per square meter of body surface) is described by Freireich et al., Cancer Chemother. Rep., 50, 219 (1966). Body surface area may be approximately determined from height and weight of the patient. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, New York, 1970, 537. Effective doses can be based on in vitro concentrations of the present compounds found to be effective to inhibit the toxicity of Aβ or to otherwise protect mitochondria. Doses of the present compounds useful to treat a model neuronal cell line are disclosed below. Effective doses will also vary, as recognized by those skilled in the art, depending on the route of administration, the excipient usage, and the optional co-administration with other therapeutic agents. Toxicity and therapeutic efficacy of the active ingredients can be determined by standard pharmaceutical procedures, e.g., for detemπning LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD5Q/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. Included in the methods, kits, combinations and pharmaceutical compositions of the present invention are the crystalline forms (e.g., polymorphs), enantiomeric forms, isomeric forms and tautomers of the described compounds and the pharmaceutically-acceptable salts thereof. Illustrative pharmaceutically acceptable salts are prepared from formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, stearic, salicylic, p-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, toluenesulfonic, 2-hydroxyethanesulfonic, sulfanilic, cyclohexylaminosulfonic, algenic, b- hydroxybutyric, galactaric and galacturonic acids.
The term "prodrug" refers to a drug or compound (active moiety) that elicits the pharmacological action results from conversion by metabolic processes within the body. Prodrugs are generally considered drug precursors that, following administration to a subject and subsequent absorption, are converted to an active or a more active species via some process, such as a metabolic process. Other products from the conversion process are easily disposed of by the body. Prodrugs generally have a chemical group present on the prodrug which renders it less active and/or confers solubility or some other property to the drug, such as an ester or acyl group. Once the chemical group has been cleaved from the prodrug the more active drug is generated. Prodrugs may be designed as reversible drug derivatives and utilized as modifiers to enhance drug transport to site-specific tissues. The design of prodrugs to date has been to increase the effective water solubility of the therapeutic compound for targeting to regions where water is the principal solvent. For example, Fedorak, et al., Am. J. Physiol, 269, G210-218 (1995), describe dexamethasone- beta-D-glucuronide. McLoed, et al., Gastroenterol., 106, 405-413 (1994), describe dexamethasone-succinate-dextrans. Hochhaus, et al., Biomed. Chrom., 6, 283-286 (1992), describe dexamethasone-21-sulphobenzoate sodium and dexamethasone-21-isonicotinate. Additionally, J. Larsen and H. Bundgaard, Int. J. Pharmaceutics, 37, 87 (1987) describe the evaluation of N-acylsulfonamides as potential prodrug derivatives. J. Larsen et al., Int. J. Pharmaceutics, 47, 103 (1988) describe the evaluation of N-methylsulfonamides as potential prodrug derivatives. 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 formulas (I) or (II), by synthetic interconversions known to the art. For example, see, LT. Harrison, Compendium of Organic Synthetic Methods, Wiley-Interscience (1971), for methods useful to interconvert spirostenol substituents.
The term "derivative" refers to a compound that is produced from another compound of similar structure by the replacement or substitution of one atom, molecule or group by another. For example, a hydrogen atom of a compound may be substituted by alkyl, acyl, amino, etc., to produce a derivative of that compound.
"Plasma concentration" refers to the concentration of a substance in blood plasma or blood serum. "Drug absorption" or "absorption" refers to the process of movement from the site of administration of a drug toward the systemic circulation, for example, into the bloodstream of a subject.
"Bioavailability" refers to the extent to which an active moiety (drug or metabolite) is absorbed into the general circulation and becomes available at the site of drug action in the body. "Metabolism" refers to the process of chemical transformations of drugs in the body.
"Pharmacodynamics" refers to the factors which determine the biologic response observed relative to the concentration of drug at a site of action.
"Pharmacokinetics" refers to the factors which determine the attainment and maintenance of the appropriate concentration of drug at a site of action.
"Plasma half-life" refers to the time required for the plasma drug concentration to decrease by 50% from its maximum concentration.
The use of the term "about" in the present disclosure means "approximately," and encompasses variations in parameters that would arise during practice of the relevant art. Illustratively, the use of the term "about" indicates that dosages outside the cited ranges may also be effective and safe, and such dosages are also encompassed by the scope of the present claims.
The term "measurable serum concentration" means the serum concentration (typically measured in mg, μg, or ng of therapeutic agent per ml, dl, or 1 of blood serum) of a therapeutic agent absorbed into the bloodstream after administration.
The term "pharmaceutically acceptable" is used adjectivally herein to mean that the modified noun is appropriate for use in a pharmaceutical product. Pharmaceutically acceptable salts include metallic ions and organic ions. More preferred metallic ions include, but are not limited to appropriate alkali metal (Group Ia) salts, alkaline earth metal (Group Ha) salts and other physiological acceptable metal ions. Exemplary ions include aluminum, calcium, lithium, magnesium, potassium, sodium and zinc in their usual valences. Preferred organic ions include protonated tertiary amines and quaternary ammonium cations, including in part, trimethylamine, diethylamine, N5N1- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. Exemplary pharmaceutically acceptable acids include without limitation 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 oxalacetic acid, fumaric acid, propionic acid, aspartic acid, glutamic acid, benzoic acid, and the like. The compositions of the present invention are usually administered in the form of pharmaceutical compositions. These compositions can be administered by any appropriate route including, but not limited to, oral, nasogastric, rectal, transdermal, parenteral (for example, subcutaneous, intramuscular, intravenous, intramedullary and intradermal injections, or infusion techniques administration), intranasal, transmucosal, implantation, vaginal, topical, buccal, and sublingual. Such preparations may routinely contain buffering agents, preservatives, penetration enhancers, compatible carriers and other therapeutic or non-therapeutic ingredients.
The present invention also includes methods employing a pharmaceutical composition that contains one or more compound of formula I or II associated with pharmaceutically acceptable carriers or excipients. As used herein, the terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipients" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for ingestible substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the compositions, its use is contemplated. Supplementary active ingredients can also be incorporated into the compositions. In making the compositions of the present invention, the compositions(s) can be mixed with a pharmaceutically acceptable excipient, diluted by the excipient or enclosed within such a carrier, which can be in the form of a capsule, sachet, or other container. The carrier materials that can be employed in making the composition of the present invention are any of those commonly used excipients in pharmaceutics and should be selected on the basis of compatibility with the active drug and the release profile properties of the desired dosage form.
Illustratively, pharmaceutical excipients are chosen below as examples:
(a) Binders such as acacia, alginic acid and salts thereof, cellulose derivatives, methylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, magnesium aluminum silicate, polyethylene glycol, gums, polysaccharide acids, bentonites, hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, polyvinylpyrrolidone/vinyl acetate copolymer, crospovidone, povidone, polymethacrylates, hydroxypropylmethylcellulose, hydroxypropylcellulose, starch, pregelatinized starch, ethylcellulose, tragacanth, dextrin, cyclodextrins, microcrystalline cellulose, sucrose, or glucose, and the like.
(b) Disintegration agents such as starches, pregelatinized corn starch, pregelatinized starch, celluloses, cross-linked carboxymethylcellulose, sodium starch glycolate, crospovidone, cross-linked polyvinylpyrrolidone, croscarmellose sodium, microcrystalline cellulose, a calcium, a sodium alginate complex, clays, alginates, gums, or sodium starch glycolate, and any disintegration agents used in tablet preparations.
(c) Filling agents such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.
(d) Surfactants such as sodium lauryl sulfate, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, Pluronic™ line (BASF), and the like.
(e) Solubilizer such as citric acid, succinic acid, fumaric acid, malic acid, tartaric acid, maleic acid, glutaric acid sodium bicarbonate and sodium carbonate and the like.
(f) Stabilizers such as any antioxidation agents, buffers, or acids, and the like, can also be utilized.
(g) Lubricants such as magnesium stearate, calcium hydroxide, talc, sodium stearyl fumarate, hydrogenated vegetable oil, stearic acid, glyceryl behapate, magnesium, calcium and sodium stearates, stearic acid, talc, waxes, boric acid, sodium benzoate, sodium acetate, sodium chloride, DL-leucine, polyethylene glycols, sodium oleate, or sodium lauryl sulfate, and the like.
(h) Wetting agents such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium oleate, or sodium lauryl sulfate, and the like.
(i) Diluents such lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose, dibasic calcium phosphate, sucrose-based diluents, confectioner's sugar, monobasic calcium sulfate monohydrate, calcium sulfate dihydrate, calcium lactate trihydrate, dextrates, inositol, hydrolyzed cereal solids, amylose, powdered cellulose, calcium carbonate, glycine, or bentonite, and the like.
(j) Anti-adherents or glidants such as talc, corn starch, DL-leucine, sodium lauryl sulfate, and magnesium, calcium, or sodium stearates, and the like.
(k) Pharmaceutically compatible carrier comprises acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerine, magnesium silicate, sodium caseinate, soy lecithin, sodium chloride, tricalcium phosphate, dipotassium phosphate, sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, or pregelatinized starch, and the like.
Additionally, drug formulations are discussed in, for example, Remington's The Science and Practice of Pharmacy (2000). Another discussion of drug formulations can be found in Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N. Y., 1980. The tablets or granules comprising the inventive compositions may be film coated or enteric-coated.
Besides being useful for human treatment, the present invention is also useful for other subjects including veterinary animals, reptiles, birds, exotic animals and farm animals, including mammals, rodents, and the like. Mammal includes a primate, for example, a monkey, or a lemur, a horse, a dog, a pig, or a cat. A rodent includes a rat, a mouse, a squirrel, or a guinea pig.
The pharmaceutical compositions of the present invention are useful where administration of an inhibitor of mitochondrial toxicity is indicated. It is believed that these compositions will be particularly effective in the treatment of mitochondrial diseases.
For treatment of a neurodegenerative disorder/mitochondrial disorder, compositions of the invention can be used to provide a dose of a compound of the present invention in an amount sufficient to elicit a therapeutic response, e.g., reduction of drug-induced mitochondrial toxicity, for example a dose of about 5 ng to about 1000 nig, or about 100 ng to about 600 mg, or about 1 mg to about 500 mg, or about 20 mg to about 400 mg. Typically a dosage effective amount will range from about 0.0001 mg/kg to 1500 mg/kg, more preferably 1 to 1000 mg/kg, more preferably from about 1 to 150 mg/kg of body weight, and most preferably about 50 to 100 mg/kg of body weight. A dose can be administered in one to about four doses per day, or in as many doses per day to elicit a therapeutic effect. Illustratively, a dosage unit of a composition of the present invention can typically contain, for example, about 5 ng, 50 ng 100 ng, 500 ng, 1 mg, 10 mg, 20 mg, 40 mg, 80 mg, 100 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 of a compound of the present invention. The dosage form can be selected to accommodate the desired frequency of administration used to achieve the specified dosage. The amount of the unit dosage form of the composition that is administered and the dosage regimen for treating the condition or disorder depends on a variety of factors, including, the age, weight, sex and medical condition, of the subject, the severity of the condition or disorder, the route and frequency of administration, and this can vary widely, as is well known. In one embodiment of the present invention, the composition is administered to a subject in an effective amount, that is, the composition is administered in an amount that achieves a therapeutically effective dose of a compound of the present invention in the blood serum of a subject for a period of time to elicit a desired therapeutic effect. Illustratively, in a fasting adult human (fasting for generally at least 10 hours) the composition is administered to achieve a therapeutically effective dose of a compound of the present invention in the blood serum of a subject from about 5 minutes after administration of the composition. In another embodiment of the present invention, a therapeutically effective dose of the compound of the present invention is achieved in the blood serum of a subject at about 10 minutes from the time of administration of the composition to the subject, m another embodiment of the present invention, a therapeutically effective dose of the compound of the present invention is achieved in the blood serum of a subject at about 20 minutes from the time of administration of the composition to the subject. hi yet another embodiment of the present invention, a therapeutically effective dose of the compound of the present invention is achieved in the blood serum of a subject at about 30 minutes from the time of administration of the composition to the subject. Li still another embodiment of the present invention, a therapeutically effective dose of the compound of the present invention is achieved in the blood serum of a subject at about 40 minutes from the time of administration of the composition to the subject. In one embodiment of the present invention, a therapeutically effective dose of the compound of the present invention is achieved in the blood serum of a subject at about 20 minutes to about 12 hours from the time of administration of the composition to the subject. In another embodiment of the present invention, a therapeutically effective dose of the compound of the present invention is achieved in the blood serum of a subject at about 20 minutes to about 6 hours from the time of administration of the composition to the subject. In yet another embodiment of the present invention, a therapeutically effective dose of the compound of the present invention is achieved in the blood serum of a subject at about 20 minutes to about 2 hours from the time of administration of the composition to the subject.
In still another embodiment of the present invention, a therapeutically effective dose of the compound of the present invention is achieved in the blood serum of a subject at about 40 minutes to about 2 hours from the time of administration of the composition to the subject. And in yet another embodiment of the present invention, a therapeutically effective dose of the compound of the present invention is achieved in the blood serum of a subject at about 40 minutes to about 1 hour from the time of administration of the composition to the subject.
In one embodiment of the present invention, a composition of the present invention is administered at a dose suitable to provide a blood serum concentration with a half maximum dose of a compound of the present invention. Illustratively, a blood serum concentration of about 0.01 to about 1000 iiM, 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 nM, or about 200 to about 400 nM is achieved in a subject after administration of a composition of the present invention.
Contemplated compositions of the present invention provide a therapeutic effect over an interval of about 5 minutes to about 24 hours after administration, enabling once-a-day or twice-a-day administration if desired, hi one embodiment of the present invention, the composition is administered at a dose suitable to provide an average blood serum concentration with a half maximum dose of a compound of the present invention of at least about 1 μ,g/ml, or at least about 5 μg/ml, or at least about 10 μ.g/ml, or at least about 50 μg/ml, or at least about 100 μg/ml, or at least about 500 μg/ml, or at least about 1000 jUg/ml in a subject about 10, 20, 30, or 40 minutes after administration of the composition to the subject.
The amount of therapeutic agent necessary to elicit a therapeutic effect can be experimentally determined based on, for example, the absorption rate of the agent into the blood serum, the bioavailability of the agent, and the potency for treating the disorder. It is understood, however, that specific dose levels of the therapeutic agents of the present invention for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, and diet of the subject (including, for example, whether the subject is in a fasting or fed state), the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration. Treatment dosages generally may be titrated to optimize safety and efficacy. Typically, dosage-effect relationships from in vitro and/or in vivo tests initially can provide useful guidance on the proper doses for subject administration. Studies in animal models generally may be used for guidance regarding effective dosages for treatment of gastrointestinal disorders or diseases in accordance with the present invention. In terms of treatment protocols, it should be appreciated that the dosage to be administered will depend on several factors, including the particular agent that is administered, the route administered, the condition of the particular subject, etc. Generally speaking, one will desire to administer an amount of the compound that is effective to achieve a serum level commensurate with the concentrations found to be effective in vitro for a period of time effective to elicit a therapeutic effect. Thus, where a compound is found to demonstrate in vitro activity at, for example, a half-maximum effective dose of 200 nM, one will desire to administer an amount of the drug that is effective to provide about a half-maximum effective dose of 200 nM concentration in vivo for a period of time that elicits a desired therapeutic effect, for example, treating a disorder related to high beta-amyloid-induced neurotoxicity and other indicators as are selected as appropriate measures by those skilled in the art. Determination of these parameters is well within the skill of the art. These considerations are well known in the art and are described in standard textbooks. hi order to measure and determine the effective amount of a compound of the present invention to be delivered to a subject, serum compound of the present invention concentrations can be measured using standard assay techniques.
Contemplated compositions of the present invention provide a therapeutic effect over an interval of about 30 minutes to about 24 hours after administration to a subject, hi one embodiment compositions provide such therapeutic effect in about 30 minutes, hi another embodiment compositions provide therapeutic effect over about 24 hours, enabling once-a-day administration to improve patient compliance.
The present methods, kits, and compositions can also be used in combination ("combination therapy") with another pharmaceutical agent that is indicated for treating or preventing a mitochondrial disease, such as, for example, creatinine. When used in conjunction with the present invention, that is, in combination therapy, an additive or synergistic effect may be achieved such that many if not all of unwanted side effects can be reduced or eliminated. The reduced side effect profile of these drugs is generally attributed to, for example, the reduced dosage necessary to achieve a therapeutic effect with the administered combination.
The phrase "combination therapy" embraces the administration of a composition of the present invention in conjunction with another pharmaceutical agent that is indicated for treating or preventing a mitochondrial disorder in a subject, as part of a specific treatment regimen intended to provide a beneficial effect from the co-action of these therapeutic agents for the treatment of a neurodegenerative and/or a mitochondrial disorder. The beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents. Administration of these therapeutic agents in combination typically is carried out over a defined time period (usually substantially simultaneously, minutes, hours, days, weeks, months or years depending upon the combination selected). "Combination therapy" generally is not intended to encompass the administration of two or more of these therapeutic agents as part of separate monotherapy regimens that incidentally and arbitrarily result in the combinations of the present invention. "Combination therapy" is intended to embrace administration of these therapeutic agents in a sequential manner, that is, where each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the therapeutic agents, in a substantially simultaneous manner. Substantially simultaneous administration can be accomplished, for example, by administering to the subject a single tablet or capsule having a fixed ratio of each therapeutic agent or in multiple, single capsules, or tablets for each of the therapeutic agents. Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route. The composition of the present invention can be administered orally or nasogastric, while the other therapeutic agent of the combination can be administered by any appropriate route for that particular agent, including, but not limited to, an oral route, a percutaneous route, an intravenous route, an intramuscular route, or by direct absorption through mucous membrane tissues. For example, the composition of the present invention is administered orally or nasogastric and the therapeutic agent of the combination may be administered orally, or percutaneously. The sequence in which the therapeutic agents are administered is not narrowly critical. "Combination therapy" also can embrace the administration of the therapeutic agents as described above in further combination with other biologically active ingredients, such as, but not limited to, an analgesic, for example, and with non- drug therapies, such as, but not limited to, surgery.
The therapeutic compounds which make up the combination therapy may be a combined dosage form or in separate dosage forms intended for substantially simultaneous administration. The therapeutic compounds that make up the combination therapy may also be administered sequentially, with either therapeutic compound being administered by a regimen calling for two step administration. Thus, a regimen may call for sequential administration of the therapeutic compounds with spaced-apart administration of the separate, active agents. The time period between the multiple administration steps may range from, for example, a few minutes to several hours to days, depending upon the properties of each therapeutic compound such as potency, solubility, bioavailability, plasma half-life and kinetic profile of the therapeutic compound, as well as depending upon the effect of food ingestion and the age and condition of the subject. Circadian variation of the target molecule concentration may also determine the optimal dose interval.
The therapeutic compounds of the combined therapy whether administered simultaneously, substantially simultaneously, or sequentially, may involve a regimen calling for administration of one therapeutic compound by oral route and another therapeutic compound by an oral route, a percutaneous route, an intravenous route, an intramuscular route, or by direct absorption through mucous membrane tissues, for example. Whether the therapeutic compounds of the combined therapy are administered orally, by inhalation spray, rectally, topically, buccally, sublingually, or parenterally (for example, subcutaneous, intramuscular, intravenous and intradermal injections), separately or together, each such therapeutic compound will be contained in a suitable pharmaceutical formulation of pharmaceutically- acceptable excipients, diluents or other formulations components. For oral administration, the pharmaceutical composition can contain a desired amount of a compound of formula (I) or (II) and be in the form of, for example, a tablet, a hard or soft capsule, a lozenge, a cachet, a troche, a dispensable powder, granules, a suspension, an elixir, a liquid, or any other form reasonably adapted for oral administration. Illustratively, such a pharmaceutical composition can be made in the form of a discrete dosage unit containing a predetermined amount of the active compound such as a tablet or a capsule. Such oral dosage forms can further comprise, for example, buffering agents. Tablets, pills and the like additionally can be prepared with enteric coatings. Pharmaceutical compositions suitable for buccal or sublingual administration include, for example, lozenges comprising the active compound in a flavored base, such as sucrose, and acacia or tragacanth, and pastilles comprising the active compound in an inert base such as gelatin and glycerin or sucrose and acacia.
Liquid dosage forms for oral administration can include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions can also comprise, for example, wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.
Examples of suitable liquid dosage forms include, but are not limited to, aqueous solutions comprising the active compound and beta-cyclodextrin or a water soluble derivative of beta-cyclodextrin such as sulfobutyl ether beta- cyclodextrin; heptakis-2,6-di-O-methyl-beta-cyclodextrin; hydroxypropyl-beta- cyclodextrin; and dimethyl-beta-cyclodextrin.
The pharmaceutical compositions of the present invention can also be administered by injection (intravenous, intramuscular, subcutaneous). Such injectable compositions can employ, for example, saline, dextrose, or water as a suitable carrier material. The pH value of the composition can be adjusted, if necessary, with suitable acid, base, or buffer. Suitable bulking, dispersing, wetting or suspending agents, including mannitol and polyethylene glycol (such as PEG 400), can also be included in the composition. A suitable parenteral composition can also include an active compound lyophilized in injection vials. Aqueous solutions can be added to dissolve the composition prior to injection. The pharmaceutical compositions can be administered in the form of a suppository or the like. Such rectal formulations preferably contain the active compound in a total amount of, for example, about 0.075 to about 75% w/w, or about 0.2 to about 40% w/w, or about 0.4 to about 15% w/w. Carrier materials such as cocoa butter, theobroma oil, and other oil and polyethylene glycol suppository bases can be used in such compositions. Other carrier materials such as coatings (for example, hydroxypropyl methylcellulose film coating) and disintegrants (for example, croscarmellose sodium and cross-linked povidone) can also be employed if desired. The subject compounds may be free or entrapped in microcapsules, in colloidal drug delivery systems such as liposomes, microemulsions, and macroemulsions.
These pharmaceutical compositions can be prepared by any suitable method of pharmaceutics, which includes the step of bringing into association active compound of the present invention and a carrier material or carriers materials. In general, the compositions are uniformly and intimately admixing the active compound with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the product. For example, a tablet can be prepared by compressing or molding a powder or granules of the compound, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing, in a suitable machine, the compound in a free-flowing form, such as a powder or granules optionally mixed with a binding agent, lubricant, inert diluent and/or surface active/dispersing agent(s). Molded tablets can be made by molding, in a suitable machine, the powdered compound moistened with an inert liquid diluent.
Tablets of the present invention can also be coated with a conventional coating material such as Opadry™ White YS-1-18027A (or another color) and the weight fraction of the coating can be about 3% of the total weight of the coated tablet. The compositions of the present invention can be formulated so as to provide quick, sustained or delayed release of the compositions after administration to the patient by employing procedures known in the art.
When the excipient serves as a diluent, it can be a solid, semi-solid or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, chewable tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), soft and hard gelatin capsules and sterile packaged powders.
In one embodiment of the present invention, the manufacturing processes may employ one or a combination of methods including: (1) dry mixing, (2) direct compression, (3) milling, (4) dry or non-aqueous granulation, (5) wet granulation, or (6) fusion. Lachman et al., The Theory and Practice of Industrial Pharmacy (1986). In another embodiment of the present invention, solid compositions, such as tablets, are prepared by mixing a therapeutic agent of the present invention with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of the therapeutic agent and the excipient. When referring to these preformulation compositions(s) as homogeneous, it is meant that the therapeutic agent is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms, such as tablets, pills and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described herein. Compressed tablets are solid dosage forms prepared by compacting a formulation containing an active ingredient and excipients selected to aid the processing and improve the properties of the product. The term "compressed tablet" generally refers to a plain, uncoated tablet for oral ingestion, prepared by a single compression or by pre-compaction tapping followed by a final compression.
The tablets or pills of the present invention may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. A variety of materials can be used for such enteric layers or coatings, including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
Use of a long-term sustained release implant may be suitable for treatment of mitochondrial disorders in patients who need continuous administration of the compositions of the present invention. "Long-term" release, as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredients for at least 30 days, and preferably 60 days. Long-term sustained release implants are well known to those of ordinary skill in the art and include some of the release systems described above.
In another embodiment of the present invention, the compound for treating a mitochondrial disorder comes in the form of a kit or package containing one or more of the therapeutic compounds of the present invention. These therapeutic compounds of the present invention can be packaged in the form of a kit or package in which hourly, daily, weekly, or monthly (or other periodic) dosages are arranged for proper sequential or simultaneous administration. The present invention further provides a kit or package containing a plurality of separately-packaged dosage units, adapted for successive daily administration, each dosage unit comprising at least one of the therapeutic compounds of the present invention. This drug delivery system can be used to facilitate administering any of the various embodiments of the therapeutic compounds of the present invention. In one embodiment, the system contains a plurality of dosages to be administered daily or weekly. The kit or package can also contain the agents utilized in combination therapy to facilitate proper administration of the dosage forms. The kits or packages also contain a set of instructions for the subject.
The invention will be further described by reference to the following detailed examples wherein the following abbreviations are used: AChEI, acetylcholinesterase inhibitor; Aβ, /?-amyloid peptide; ADDLs, amyloid-derived diffusible ligands; ANT, adenine nucleotide translocase; APP, amyloid precursor protein; CCCP, carbonyl cyanide 3-chlorophenylhydrazone; FCCP, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone; CsA, cyclosporin A; MPT, membrane permeability transition; MRC, mitochondrial respiratory coefficient; PAO, phenylarsine oxide; SP-233, (22S,25S)-(20S)-spiiost-5-en-3j8- yl hexanoate; SP-233, (22R,25R)-(20θ!)-spirost-5-en-3/3-yl hexanoate.
Mitochondrial permeability transition (MPT) may be further defined as a nonspecific increase in the permeability of the inner mitochondrial membrane that occurs under adverse conditions, such as an increase in mitochondrial Ca+2 content of the mitochondrial matrix or oxidative stress, and which causes the assembly (opening) of non-specific pores ("megachannels") in the mitochondrial inner membrane, leading to a loss of mitochondrial membrane potential, an uncoupling of mitochondrial respiration, and cellular energy failure, all of which can contribute to cell death. Example 1. Use of SP-233 to Protect Mitochondrial Function A. Materials and methods 1. Materials
Ap1-^2 peptide was purchased from American Peptide Company (Sunnyvale, CA, USA) and stored at -80°C. The antibody anti-complex II was purchased from Molecular Probes (Eugene, OR, USA) and the antibody anti- Ab1-42 was from Signet Laboratories (Dedham, MA, USA). The spirostenol, (22S,25S)-(20S)-sρirost-5-en-3/3-yl hexanoate (SP-233) was purchased from Interbioscreen (Moscow, Russia). Cyclosporin A (CsA) and carbonyl cyanide 3- chlorophenylhydrazone (CCCP) were obtained from Sigma (St. Louis, MO, USA). Dulbecco's Minimum Essential Medium (DMEM), fetal bovine serum (FBS) and SK-N-AS human neuroblastoma cells were purchased form ATCC (Manassas, VA, USA).
2. Mitochondrial isolation procedure
Male Long-Evans rats weighing 230-280 g were used. After decapitation, the brain was rapidly removed and homogenized on ice in a Potter-Elvejhem homogenizer (Eurostar, IKA-Verke, Staufen, Germany) containing 6 ml of isolation buffer (TRIS 20 niM, sucrose 250 mM, KCl 40 mM, EGTA 2 mM, bovine serum albumin 1 mg/ml; pH 7.2 at 4°C). The homogenate was centrifuged at 2000 *g for 8 min to remove cell debris and nuclei. The mitochondria-containing supernatant was centrifuged at 12000xg for 10 min. The pellet was resuspended in 300 μl of respiratory buffer (D-mannitol 300 mM, KCl 10 mM, KH2PO4 10 mM, MgC12 5 mM; pH 7.2 at 37°C). This pellet is highly enriched in mitochondria (Zini et al. 1996). The protein concentration of the mitochondrial suspension was determined by the method of Lowry et al. 1951.
3. Preparation of the different solutions Ap1-^2 solution was reconstituted with fresh distilled water to provide a concentration of 500 μM. Ap1^2 was used as freshly reconstituted or in an aggregated form after having been "aged" by incubating the solution for 48 h at 40C. Both solutions were diluted with the respiratory buffer (see above) before use and then added directly to the mitochondria at the desired concentration. In experiments conducted with neuroblastoma cells (see below), only the freshly reconstituted Aβi_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. Monitoring of mitochondrial respiration
Mitochondrial functions were studied using an Oxytherm (Hansatech, Norfolk, UK) linked to a PC (Compaq, PIII 400 MHz). The 37°C thermostated incubation chamber of the Oxytherm was connected to a platinum-silver Clarck electrode for O2 detection. O2 consumption was monitored as a marker of mitochondrial function and viability in the presence of ADP (precursor for ATP) in the presence or absence of CCCP (an uncoupling agent for oxidative phosphorylation), or CsA (an inhibitor of the MPT pore). Measurement of O2 consumption permitted calculation of the mitochondrial respiratory coefficient (MRC) which is V3/V4. V4 is the basal O2 consumption, V3 is the O2 consumption after adding ADP (ATP production). The effect of increasing concentrations of SP-233 (1 pM to 1 nM) on the mitochondrial respiratory chain was assessed by monitoring the evolution of MRC. The concentration of mitochondria was adjusted to 0.4 mg protein/ml using respiratory buffer. Mitochondria were activated by addition of malate/glutamate as substrates of complex I of the respiratory chain. State 2 is the baseline rate of mitochondrial oxygen consumption. When ATP synthesis was initiated by addition of ADP, the rate of oxygen consumption increased dramatically to reach state 3. When the supply of ADP had been depleted, the rate of O2 consumption returned to baseline (state 2) which was called state 4 (Chance and Williams 1956). Controls for each experiment corresponded to basal respiration in the presence of ADP.
5. Effect of SP-233 on CCCP-induced uncoupling of oxidative phosphorylation Addition of CCCP (1 μM) uncouples the respiratory chain reaction and increases O2 consumption. To assess the effect of SP-233 on CCCP-induced uncoupling, the mitochondrial fraction was incubated for 3 min at 37°C in presence of SP-233 before adding malate/glutamate, followed 1 min later by the addition of CCCP. 6. Assessment of the effect of SP-233 on CsA-induced hypoxia
Hypoxia was induced by adding CsA (lμM) to the incubation medium bathing the mitochondria (Zini et al. 1996). This test was controlled by adding ADP in the presence of CsA and absence of SP-233. As described above, SP- 233 or its vehicle was added first and incubation was carried out for 1.5 min before addition of CsA. Then, the substrate malate/glutamate was added 1.5 min later, and ADP was added 2.5 min later.
7. Assessment of the protective effect of SP-233 against Aβi-.β-induced mitochondrial dysfunction
SP-233 was present in the incubation mixture for 3 min and Ap1-^2 for 1.5 min at 37°C before addition of the substrates malate/glutamate. ADP was added 1 min after addition of malate/glutamate. Control for the effect of SP-233 versus Ap1^2 was the same as for the experiment conducted in the absence of ADP.
8. Assessment of the protective effect of SP-233 against A/31-42-induced toxicity in human neuroblastoma cells Human neuroblastoma SK-N-AS cells were seeded in 96-well plates
(7x104 cells/well) in DMEM containing 10% FBS and cultured in 5% CO2 and 95% humidity. Ap1^2 (0.1, 1 and 10 μM) or its vehicle was then added and incubation was continued for 72 h in the presence or absence of SP-233 (1 μM). The cellular toxicity of A/3 was assessed using the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT) assay (Trevigen, Gaithersburg, MD).
9. Immunocytochemical
Figure imgf000034_0001
uptake by mitochondria of human neuroblastoma cells Human neuroblastoma SK-N-AS cells were seeded on 13 -mm diameter coverslips (20,000 cells/coverslip) and incubated overnight at 370C, 5% CO2, in DMEM containing 10% FBS. Aβl-42 (10 μM) or its vehicle was then added and incubation was continued for 3 h in the presence or absence of SP-233 (1 μM). To assess mitochondrial uptake of Ap1^2, a co-localization study was performed using a mouse monoclonal antibody Anti-OxPhos Complex II 70-kDa subunit raised against complex II of the mitochondrial respiratory chain (Molecular Probes, Eugene, OR, USA) and a rabbit polyclonal antibody raised against amino acid residues 33-42 of Aβ1--42 (Signet Laboratories, Dedham, MA, USA). Neuroblastoma cells were washed with phosphate-buffered saline (PBS) IX, fixed with methanol at -200C and rinsed with PBS IX. Cells were then blocked with PBS IX containing 10 % donkey serum for 30 min and then incubated for 24 h at 4°C with the first primary antibody raised against complex II (1/200) in PBS IX solution containing 1.5% donkey serum and 0.01% Triton XlOO. After washing the preparation 3 times with PBS IX, a donkey anti-mouse rhodamine-labeled secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) was added at a 1/200 dilution and incubation was continued for 2 h at room temperature. The primary antibody raised against amino acids 33-42 of Aβi_42 was then used following the same protocol at a 1/500 dilution. Positive staining was revealed using a donkey-anti-rabbit secondary antibody labeled with the fluorescent marker Alexa Fluor® 488 (Molecular Probes, Eugene, OR, USA). Coverslips were then mounted with an aqueous mounting medium containing 4',6-diamidino-2-phenylindole-2-hydrochloride (DAPI) (Vector Laboratories. Burlingame, CA, USA). Confocal images were taken using an Olympus Fluoview BX61 Laser Scanning microscope.
10. Statistical analysis
For each experiment, the results are expressed as means ± SD. Comparisons between groups were made by an ANOVA followed by a Dunnett's test.
B. Results
1. Effect of SP-233 on mitochondrial respiratory control
SP-233 was studied at concentrations of 10, 30 and 100 pM. At a concentration as low as 10 pM, SP-233 induced a significant 50% decrease in the MRC of rat brain mitochondria, as compared to the control group (pO.OOl) (Fig. 1). Although the differences in the effects induced by various concentrations of SP-233 are not significant, the results do tend to display a concentration-effect relationship since the MRC was progressively decreased over the concentration range of 10-100 pM.
2. Effect of SP-233 on CCCP- or CsA-induced modification of the mitochondrial respiratory coefficient
The uncoupling agent CCCP at 1 μM increased the O2 consumption of rat brain mitochondria to 150% of the basal value (p<0.01) (Fig. 2a). All concentrations of SP-233 that were tested (1-1000 pM) completely inhibited this metabolic effect of CCCP (p<0.001). The inhibition of CCCP-induced uncoupling by SP-233 was associated with decreases in O2 consumption values to 60-65% of the basal level, and was concentration-independent. SP-233 did not counteract the decrease in the MRC induced by CsA, but on the contrary, it amplified this effect of CsA in a concentration-dependent manner (Fig. 2b).
3. Effects of fresh and aged AjSi-42 on the mitochondrial respiratory coefficient
AjS1-42 significantly decreased the MRC of rat brain mitochondria even when it was present at a concentration as low as 0.1 pM (Fig. 3). This effect was more pronounced with the fresh amyloid peptide than with the aged form, the respective inhibitory effects being 55-63% and 43-53%. This difference may be revealing that the non-aggregated form of AjS1-42 penetrates into the mitochondria to a greater extent than the aged, aggregated form of the peptide.
4. Effects of SP-233 on modifications of the mitochondrial respiratory coefficient induced by fresh and aged AjSi-42
Fresh A/31-42 reduced the MRC by 71% compared to the control, and this effect was partially inhibited by SP-233 (Fig. 4a). At the lowest concentration of SP-233 that was tested (1 pM), the MRC was restored to about 40% of the control value, and the MRC was significantly increased in comparison with the value obtained with AjS1-42 alone (38.56 ± 0.34 versus 28.93 ± 1.75, pO.OOl). Aged AjS1-42 reduced the MRC by 51% compared to the control but SP-233 did not significantly prevent this effect (Fig. 4b). 5. Effect of SP-233 on A/3i.42-induced toxicity on SK-N-AS human neuroblastoma cells
Fig. 5 shows the effect of SP-233 on the AjS1 -42 induced decrease in SK- N-AS cell viability. SP-233 1 μM prevented the neurotoxicity induced by the three concentrations of A/31-42 used by 19%, 26% and 28%, respectively (pθ.01).
6. Effect of SP-233 on A/3i-42 entry into the mitochondria of SK-N-AS human neuroblastoma cells Neuroblastoma cells treated with AjS1-42 displayed strong immunoreactivity that co-localized with complex II of the mitochondrial respiratory chain, providing evidence that A/31-42 entered, and was present inside, the mitochondria. Treatment with SP-233 abolished AjS1-42 immunoreactivity, indicating that it blocked the entry of AjS1-42 into the cell and the mitochondria.
C. Discussion
It is well known that AD is clinically characterized by a progressive impairment of cognitive processes and memory loss. Since the early days, this "mnesic" aspect of the disease has been related to the degeneration of central cholinergic pathways, reflected as a decrease in synaptic concentrations of acetylcholine (ACh). Thus, drug development research has been focused mainly on restoring the ACh levels in the brain, and has led to the development of the acetylcholinesterase inhibitors (AChEIs), tacrine being the prototype of this class of compounds. However, despite promising clinical data, the beneficial effects of tacrine have been modest, and despite the development of a new generation of AChEIs, represented by rivastigmine, galantamine and donepezil, the delay of symptom onset in AD patients has not been shortened, as compared to tacrine. This short (1 to 2 years) delay in the further progression of AD symptoms (Tariot and Winblad, 2001; Waldemar et al. 2001; Grossberg et al. 2004), although precious for the patients and their relatives, is probably due to the progressive degeneration of cholinergic neurons and is a limitation to the use of the AChEIs. Moreover, a recent study performed on 565 community-resident patients with mild-to-moderate AD indicated that donepezil is not cost effective, with benefits below minimally relevant thresholds (Courtney et al. 2004). Since, no major advance has been made in AD drag development, even though memantine, an N-methyl-D-aspartate (NMDA) receptor antagonist has recently been approved for treating moderately to severe AD (Livingston and Katona 2004), there remains an urgent need to develop agents that are more efficient than AChEIs.
The etiology of AD is well documented only for the familial early-onset form of the disease, which involves a mutation of the APP, presenilin-1 or presenilin-2 gene, hi contrast, the cause of the late-onset sporadic form of AD, which represents about 95% of all AD cases, remains to be established. Many theories have emerged, and among these it has been proposed that an energetic failure due to mitochondrial impairment and oxidative damage may be at the origin of AD (Schulz et al. 1997; Beal, 2000).
Evidence that mitochondrial dysfunction is involved in AD etiology, together with the recent finding that SP -233 (a naturally occurring spirostenol) can protect neuronal PC 12 cells against A/31-42-induced neurotoxicity (Lecanu et al. 2004), that led us to examine the capacity of SP-233 to restore the function of isolated mitochondria that were impaired by exposure to A1Si-42. Taken together, the present findings indicate that SP-233 has the potential to influence basic mechanisms which are associated with A/3 and which have been implicated in the pathogenesis of AD.
The effect of Aβ on mitochondrial function has been extensively investigated during the past ten years and the data generated by these studies has indicated various effects of the peptide that could lead to mitochondria dysfunction. AjSi-42 and the fragment A/325-35 have been shown to inhibit respiration and the activities of key enzymes, such as succinate dehydrogenase, α-ketoglutarate dehydrogenase, pyruvate dehydrogenase and cytochrome oxidase, in neuronal mitochondria (Kaneko et al. 1995; Casley et al. 2002a). Inhibition of the different complexes of the respiratory chain has also been reported to occur in neuronal mitochondria exposed to AjS25-35 (Pereira et al. 1998; Canevari et al. 1999;Casley et al. 2002b) and this same peptide fragment promoted pore-opening that is associated with the MPT (Moreira et al. 2001; Moreira et al. 2002; Bachurin et al. 2003).
Results presented herein above show further that AjS1-42 inhibits mitochondrial respiration of rat brain mitochondria, as revealed by a decrease in the MRC, and that this inhibition occurs at AjS1-42 concentrations as low as 0.1 pM with both freshly prepared and aged forms of the peptide (see Fig. 3). The inhibitory effect is more pronounced with the fresh form probably because polymeric forms of A/31-42 would traverse mitochondrial membranes to a lesser extend than the monomeric form or oligomeric forms such as ADDLs, these latter forms therefore being more likely to exert their detrimental effects on the respiratory chain and being de facto more toxic. Also, the lack of protection by SP-233 of mitochondria submitted to the aged/aggregated form of AjS1-42 (see Fig. 4) might be due to an inability of SP-233 to bind the aggregated form of the amyloid peptide in the same way that it binds the monomeric form.
SP-233 was able to partially prevent the decrease in MRC induced by freshly prepared A1S1-42 and protect mitochondrial function, confirming previous data which showed a restoring effect of SP-233 on the ATP synthesis of PC 12 cells exposed to A1S1-42 (Lecanu et al. 2004). As SP-233 was active in very low concentrations (picomolar range), its mechanism of action might be relatively specific. The present data also confirms recent results which showed that SP-233 protected neuronal cells against A1S1-42 neurotoxicity by binding the monomeric form of the peptide and inhibiting the formation of the neurotoxic oligomeric ADDLs (Lecanu et al. 2004). The confocal microscopy analysis performed on human neuroblastoma cells, in showing that A1S1-42 co-localized with complex II of the respiratory chain, indicates that AjS1-42 can cross both cell and the mitochondrial membranes. When SP-233 and A1S1-42 were both present in the incubation medium immunocytochemical staining corresponding to the amyloid peptide was completely suppressed, and this "scavenging" effect of SP-233 was accompanied by a partial restoration of the MRC. In addition, SP-233 prevented, although partially, the neurotoxic effect OfA1S1-42 on the same human neuroblastoma cells. This finding is of particular interest in view of recent results which have indicated that amyloid-deposits are present in the mitochondria of cortical pyramidal neurons of post-mortem AD brain (Fernandez- Vizarra et al. 2004).
These histological features have been described as being a very early event in the progression of the disease, appearing before the formation of paired-helical filaments and leading to neuronal death. After the binding and "scavenging" effects of SP-233 on AjSi-42 had been established, the possibility that a direct effect of SP-233 on the mitochondria might result in an additive protective effect against AjQ1-42 was investigated. In this regard, it had been previously demonstrated that A/3 promotes the opening of MPT pores in brain mitochondria, leading to functional impairment, and that this effect is abolished by CsA (Moreira et al. 2001; Bachurin et al. 2003; Abramov et al 2004). The present experiments show that SP-233, like CsA, decreases the MRC. SP-233 also amplifies the effect of the CsA, suggesting that it has CsA- like properties and thereby inhibits MPT pore-opening, which could contribute to its protective effect against AjSi-42 -induced mitochondrial dysfunction.
SP-233 was also able to abolish the uncoupling of oxidative phosphorylation induced by CCCP (see Fig. 2a). It has been shown that AJSJ-42 disrupts mitochondrial membrane structure (Kremer et al. 2001; Rodrigues et al. 2001) and that mitochondrial membrane fluidity may be altered in AD brain (Mecocci et al, 1996), both phenomena leading to mitochondrial uncoupling. Therefore, even though the "recoupling" effect of SP-233 cannot yet be fully clarified, it might contribute to the restoration of mitochondrial function described herein. Moreover, a combination of the scavenging effect of SP-233 (presumably involving its modulation of MPT pores) and its re-coupling effect might explain why SP-233 counteracted the effect OfAjSi-42 at concentrations as low as 1 pM.
Example 2. The Spirostenol (22R,25RV20a-spirost-5-en-3fi-yI Hexanoate Blocks Mitochondrial Uptake of AB in Neuronal Cells and Prevents AB- Induced Impairment of Mitochondrial Function A. Materials and methods 1. Materials
ABi-42 peptide was purchased from American Peptide Company (Sunnyvale, CA). The anti-complex II antibody was purchased from Molecular Probes (Eugene, OR), and the antibody anti-AJ31-42 was from Signet Laboratories (Dedham, MA). Spirostenol (SP-233) was purchased from Interbioscreen (Moscow, Russia). Cyclosporin A (CsA), carbonyl cyanide 3- chlorophenylhydrazone (CCCP), carbonyl cyanide p-trifluoromethoxy- phenylhydrazone (FCCP), rotenone, malonate, myxothiazol, potassium cyanide (KCN), oligomycin, and phenylarsine oxide (PAO) were obtained from Sigma (St Louis, MO). Dulbecco's Minimum Essential Medium (DMEM), fetal bovine serum (FBS), and SK-N-AS human neuroblastoma cells were purchased form ATCC (Manassas, VA).
2. Isolation of mitochondrial fractions
To isolate mitochondrial fractions, rats weighing 230-350 g were decapitated, and the brain was rapidly removed and homogenized on ice in a Potter-Elvejhem homogenizer (Eurostar, IKA-Verke, Staufen, Germany) containing 6 ml of ice-cold isolation buffer (TRIS 20 mM, sucrose 250 mM, KCl 40 mM, EGTA 2 mM, bovine serum albumin 1 mg/ml; pH 7.2). The homogenate was centrifuged at 2000xg for 8 min at 4°C to remove cell debris and nuclei. The mitochondria-containing supernatant was centrifuged at 12000xg for 10 min at 4°C. The pellet was resuspended in 300 μl of respiratory buffer (D-mannitol 300 mM, KCl 10 mM, KH2PO4 10 mM, MgCl2 5 mM; pH 7.2 at 37°C). This pellet is highly enriched in mitochondria (Zini et al. 1996). The protein concentration of the mitochondrial suspension was determined by the method of Lowry.
3. Measurement of mitochondrial respiration
Mitochondrial respiration was measured using an Oxytherm (Hansatech, Norfolk, UK) linked to a PC (Compaq, PIII 400 MHz). The 37°C incubation chamber of the Oxytherm was connected to a platinum-silver Clarck electrode for O2 detection. O2 consumption was employed as a marker of mitochondrial respiration in the presence of ADP (precursor for ATP) and in the presence or absence of the following: CCCP (an uncoupling agent for oxidative phosphorylation), CsA (an inhibitor of the membrane permeability transition pore), or AB1-42. Measurement of O2 consumption allowed for calculation of the mitochondrial respiratory coefficient (MRC)- defined as V3/V4 where V4 is the basal O2 consumption and V3 is the O2 consumption after adding ADP (ATP production). The final mitochondrial concentration was adjusted to 0.4 mg protein/niL using respiratory buffer. Mitochondria were activated by the addition of malate/glutamate, substrates of complex I of the respiratory chain. State 2 is defined as the baseline rate of mitochondrial oxygen consumption. When ATP synthesis was initiated by addition of ADP, the rate of oxygen consumption increased dramatically to reach state 3. When the supply of ADP had been depleted, the rate of O2 consumption returned to baseline (state 2), which was called state 4 (Chance and Williams 1956). The affects of all experimental treatments on respiration were compared to baseline respiration, or mitochondrial respiration in the presence of only ADP.
To test the effects of SP-233 on mitochondrial dysfunction induced by CCCP, isolated mitochondria were incubated in SP-233 for 3 min at 37°C before adding malate/glutamate. One minute later, CCCP was added. To test the effects of SP-233 on mitochondrial dysfunction induced by CsA, isolated mitochondria were incubated in SP-233 for 1.5 min before the addition of CsA. Malate/glutamate was added 1.5 min later, and ADP was added 2.5 min later. Finally, to test the affect of SP-233 on AB1_42-induced mitochondrial dysfunction, mitochondrial fractions were incubated in SP-233 for 3 min and AB1^t2 for 1.5 min at 37°C before addition of malate/glutamate. ADP was added 1 min after addition of malate/glutamate. hi all experiments, SP-233, CCCP, and CsA were first dissolved in N,N-dimethylformamide (DMF) and then diluted in fresh distilled water. AB1^2 employed in these experiments was reconstituted with fresh distilled water to a concentration of 500μM. AB1-42 was used as freshly reconstituted or in an aggregated form after having been "aged" by incubating the solution for 48 h at 4°C. Both solutions were diluted with the respiratory buffer before use and then added directly to the mitochondria to achieve the desired concentration.
4. Cell Culture
Human neuroblastoma SK-N-AS cells were seeded in 96-well plates (7xlO4 cells/well) in DMEM containing 10% FBS and were cultured in 5% CO .2 and 95% humidity. Cultures were treated with the following in the presence or absence of varying concentrations of SP-233: AB1-42 (0.1, 1, and 10 μM), rotenone (50 nM), malonate (100 mM), myxothiazol (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 manufacturer's instructions (Trevigen, Gaithersburg, MD). For experiments involving AB1-42, cell viability was measured 72 hours after the insult in the presence or absence of lμM AB1-42. Only the freshly reconstituted AB1-42 solutions were used. For experiments involving mitochondrial toxins (rotenone, malonate, myxothiazol, KCN, and oligomycin), cell viability was measured 6 or 24 hours after the insult in the presence or absence of 1, 3, 10, 30, or lOOμM AB1-42. SP-233 was added to cultures 1 hour prior to the addition of the mitochondrial toxins. For experiments involving FCCP and PAO, parallel cell cultures were incubated with or without increasing concentrations of SP-233, ranging from 1 to 100 μM, one hour prior to the addition of FCCP or PAO. Cell viability was assessed 24 hours later. FCCP, rotenone, malonate, myxothiazol, KCN, oligomycin, and PAO were all first dissolved as a stock solution in ethanol before being diluted in culture medium to reach the indicated final concentrations. The percentage of ethanol in the culture medium was 0.09%.
5. Immunocytochemical analysis of AB1-42
Human neuroblastoma SK-N-AS cells were seeded on 13 -mm diameter coverslips (20,000 cells/coverslip) and incubated overnight at 370C, at 5% CO2, in DMEM containing 10% FBS. Cultures were then incubated with AB1-42 (10 μM) or its vehicle for 3 h in the presence or absence of SP-233 (1 μM). To assess mitochondrial uptake OfAB1-42, a co-localization study was performed using an Anti-OxPhos Complex II 70-kDa subunit mouse monoclonal antibody raised against complex II of the mitochondrial respiratory chain (Molecular Probes, Eugene, OR) and a rabbit polyclonal antibody raised against amino acid residues 33-42
Figure imgf000043_0001
(Signet Laboratories, Dedham, MA). Neuroblastoma cells were washed with phosphate-buffered saline (PBS), fixed with methanol at -200C, and rinsed with PBS. Cells were then blocked with PBS containing 10% donkey serum for 30 min and then incubated for 24 h at 40C with the antibody raised against complex II (1:200 in PBS containing 1.5% donkey serum and 0.01% Triton XlOO). Cells were then washed with PBS and incubated with a donkey anti-mouse rhodamine-labeled secondary antibody (1 :200; Jackson ImmunoResearch, West Grove, PA) for 2 h at room temperature. The same sequential steps were then followed using the antibody raised against amino acids 33-42
Figure imgf000043_0002
(1:500) and a donkey-anti-rabbit secondary antibody labeled with the fluorescent marker Alexa Fluor® 488 (Molecular Probes, Eugene, OR). Coverslips were mounted with an aqueous mounting medium containing 4',6-diamidino-2-phenylindole-2-hydrochloride (DAPI; Vector Laboratories, Burlingame, CA). Confocal images were acquired using an Olympus Fluoview BX61 Laser Scanning microscope.
6. Statistical analysis
All results are expressed as mean ± SD. Comparisons between groups were carried out using an Analysis of Variance (ANOVA) followed by Dunnett's test. P values < 0.05 were accepted as significant.
B. Results
1. Effect of SP-233 on CCCP- or CsA-induced changes in mitochondrial respiration
First, the effects of SP-233 at concentrations ranging from 1 fM to 100 pM on mitochondrial respiration were assessed by monitoring the evolution of MRC (Fig. 6). Increasing concentrations of SP-233 resulted in a progressive decrease in the MRC. At concentrations as low as 1 nM, SP-233 induced a significant decrease in the MRC of rat brain mitochondria, as compared to the control group (pO.OOl; Fig. 6b). Concentrations of SP-233 at or above 10 pM, induced a 50% decrease in the MRC.
CCCP uncouples oxidative phosphorylation and increases O2 consumption. As shown in Figure 7a, 1 μM CCCP increases O2 consumption in rat brain mitochondria to 150% of the basal value (pO.Ol). Concentrations of SP-233 ranging from 1-1000 pM completely abolished this effect (pO.OOl for all), and the inhibition of CCCP-induced uncoupling in the presence of all of these SP-233 concentrations was associated with a decrease in O2 consumption values to 60-65% of the basal level.
Hypoxia was induced by adding 1 μM CsA to the incubation medium bathing the mitochondria (Zini et al. 1996). SP-233 did not counteract the decrease in the MRC induced by CsA. On the contrary, it amplified this effect in a concentration-dependent manner (Fig. 7b). 2. Effects of SP-233 on changes in mitochondrial respiration induced by fresh and aged ABi-42
Addition OfAB1-42 to isolated mitochondria significantly decreased the MRC, even when present at concentrations of 0.1 pM (Fig. 8). This effect was more pronounced with the fresh amyloid peptide than with the aged form; the inhibitory effect of the former was approximately 10% greater. It was hypothesize that the non-aggregated form OfAB1-42 penetrates into the mitochondria to a greater extent than the aged, aggregated form of the peptide. As shown in Figure 9a, addition of fresh AB1-42 to isolated mitochondria resulted in a decrease in the MRC to 71% of the control values, and this decrease was partially reversed by SP-233. At the lowest concentration of SP-233 tested (1 pM), the MRC was restored to approximately 39% of the control value. The MRC was significantly increased in comparison to the value obtained with AB1- 42 alone (38.56 ± 0.34 vs. 28.93 ± 1.75, ρ<0.001). Aged AB1-42 reduced the MRC by 51% compared to the control, but SP-233 did not significantly affect this aged ABi-42-induced change in the MRC (Fig. 9b).
3. Effect of SP-233 on Aβl-42-induced toxicity on SK-N-AS human neuroblastoma cells Figure 10 shows the effect of SP-233 on AB1-42-induced neurotoxicity in
SK-N-AS cells. In the presence of all three AB1-42 concentrations tested, addition of 1 μM SP-233 resulted in a significant increase in cell viability. SP- 233 decreased neurotoxicity induced by 0.1, 1, and lOμM AB1-42, by 19% (pO.Ol, compared to AB1-42 alone), 26% (pO.Ol) and 28% (pO.Ol), respectively.
4. Effect of SP-233 on ABi-42 entry into the mitochondria of SK-N-AS cells
Figure 11 shows representative images of the DAPI staining (al, bl and cl), the immunofluorescent labeling OfAB1-42 (a2, b2 and c2), and the immunofluorescent labeling of the complex II 70-kDa subunit (a3, b3 and c3) in SK-N-AS human neuroblastoma cells. Merged images are shown in Figure a4, b4, and c4. Neuroblastoma cells treated with AB1-42 displayed strong AB1-42 immunoreactivity (Fig. 1 Ib2) that co-localized with labeling of complex II of the mitochondrial respiratory chain (Fig. Ilb4), providing evidence that AB1-42 entered, and was present inside, the mitochondria. ABI-42 immunoreactivity was abrogated in the presence of SP-233 (Fig. 1 Ic2), indicating that SP-233 blocked the entry OfAB1-42 into the cell and the mitochondria.
5. Neuroprotective effect of SP-233 against inhibitors of the mitochondrial complexes in SK-N-AS cells
SP-233 did not display any neuroprotective effect against the inhibitors of complexes I, II, and III of the mitochondria respiratory chain (Fig. 12 a, b, and c). At concentrations of 1 μM, SP-233 significantly protected against rotenone and 100 μM SP-233 significantly protected against myxoyhiazol (p<0.05 for both). Although this protective effect was significant, in both cases the magnitude of the effect was small. In contrast, SP-233 exerted a pronounced beneficial effect against toxicity induced by KCN, a complex IV inhibitor, and oligomycin, a complex V inhibitor (Fig. 12d and 12e). Low concentrations of SP-233 (1, 3, and 10 μM) were active against KCN; whereas, high doses (30 and 100 μM) offered no beneficial effect. The protective effect of SP-233 against oligomycin was dose-dependent, although 10 μM SP-233 did not demonstrate statistically significant neuroprotection. These data demonstrate that SP-233 protects SK-N-AS cells against inhibition of complexes IV and V.
6. Neuroprotective effect of SP-233 on PAO and FCCP in SK-N-AS cells
Adenine nucleotide translocase (ANT) is a channel protein located in the inner membrane of the mitochondria that, under physiological conditions, exports ATP and imports ADP with a 1 : 1 ratio. The inhibition of ANT by PAO promotes the opening of the permeability transition pore, which leads to apoptosis. One micro molar SP-233 reduced the toxic effect of PAO on SK-N- AS neuronal cells by 25% (Fig. 13, pθ.01). Concentrations of SP-233 above lμM were not associated with any neuroprotective effects. In addition, SP-233 exerted a small, but significant (p<0.01), effect on FCCP-induced uncoupling of the respiratory chain when administered at the concentration of 100 μM (Fig. 14). C. Discussion
AD is clinically characterized by a progressive impairment of cognitive processes and memory loss. Early on, this "mnesic" aspect of the disease was related to the degeneration of central cholinergic pathways, as reflected by decreases in synaptic concentrations of acetylcholine (ACh). Thus, drug research has focused on restoring the ACh levels in the brain, and this has led to the development of the acetylcholinesterase inhibitors (AChEIs). Although these drugs are invaluable for AD patients and their caregivers, AchEIs are symptomatic drugs that only slow down the progression of the disease for a limited period of time without stopping it (Tariot and Winblad, 2001 ; Waldemar et al. 2001; Grossberg et al. 2004). Even though memantine, an N-methyl-D- aspartate (NMDA) receptor antagonist, has recently been approved for treating moderately to severe AD (Livingston and Katona 2004), there is an urgent need for new drugs and new therapeutic strategies that target the etiological causes of the AD.
The etiology of AD is well documented for the familial, early-onset form of the disease, which involves a mutation of the APP (presenilin-1 or presenilin- 2) gene, hi contrast, the cause of the late-onset sporadic form of AD, which represents about 95% of all AD cases, remains to be established. Many theories have emerged, and among these is the proposal that an energetic failure due to mitochondrial impairment and oxidative damage maybe at the origin of AD (Schulz et al. 1997; Beal, 2000). This mitochondrial dysfunction theory, together with the recent finding that the spirostenol derivative, SP-233, can protect neuronal PC 12 cells against AB1-42-induced neurotoxicity (Lecanu et al. 2004), led to the examination of whether SP-233 is capable of restoring the function of isolated mitochondria exposed to AB1-42. The results provided herein demonstrate that SP-233 can protect mitochondrial functions against the toxic effects OfAB1-42, both in isolated rat brain mitochondria and in human neuroblastoma cells. These observations suggest that SP-233 has the potential to influence basic mechanisms that are associated with AB-induced neurotoxicity and have been implicated in the pathogenesis of AD.
The effect of AB on mitochondrial function has been extensively investigated during the past ten years, and the data generated by these studies indicates that various effects of the peptide could lead to mitochondrial dysfunction. AB1-42 and the fragment AB25-35 have been shown to inhibit respiration and the activities of key enzymes such as succinate dehydrogenase, α-ketoglutarate dehydrogenase, pyruvate dehydrogenase, and cytochrome oxidase, in neuronal mitochondria (Kaneko et al. 1995; Casley et al. 2002a). Inhibition of the respiratory chain complexes has also been reported in neuronal mitochondria exposed to AB25-35 (Pereira et al. 1999; Canevari et al. 1999; Casley et .al. 2002b), and this same peptide fragment promoted pore-opening that is associated with the MPT (Moreira et al. 2001; Moreira et al. 2002; Bachurin et al. 2003). In addition, the results presented herein revealed that AB1-42 inhibited mitochondrial respiration of rat brain mitochondria, as revealed by a decrease in the MRC. Moreover, this inhibition occurred in response to AB1-42 concentrations as low as 0.1 pM, with both freshly prepared and aged forms of the peptide. It is likely that the inhibitory effect was more pronounced with the fresh form since the polymeric forms OfAB1-42 would traverse mitochondrial membranes to a lesser extent than the monomelic form or oligomeric forms such as ADDLs. These latter forms are more likely to exert their detrimental effects on the respiratory chain and are de facto more toxic. Also, the lack of protection imparted by SP-233 on mitochondria exposed to the aged/aggregated form of AB1-42 might be due to the inability of SP-233 to bind the aggregated form of the amyloid peptide in the same way that it binds the monomelic form.
Interestingly, SP-233 was able to partially prevent the decrease in MRC induced by freshly prepared AB1-42, confirming previous results showing a restorative effect of SP-233 on ATP synthesis in PC12 cells exposed to AB1-42 (Lecanu et al. 2004). The finding that SP-233 is active at very low concentrations suggests that its mechanism of action might be relatively specific. The data described herein also confirmed recent results showing that SP-233 protects neuronal cells against AB1-42 neurotoxicity by binding to the monomeric form of the peptide and inhibiting the formation of the neurotoxic oligomeric ADDLs (Lecanu et al. 2004). The confocal microscopy analysis performed on human neuroblastoma cells, in showing that AB1-42 co-localized with complex II of the respiratory chain, indicated that AB1-42 is capable of traversing both the outer cell membrane and the mitochondrial membrane. When SP-233 and AB1-42 were both present in the incubation medium, amyloid peptide labeling was abolished, and this "scavenging" effect of SP -233 was accompanied by a partial restoration of the MRC. In addition, SP-233 partially prevented the neurotoxic effects OfABi-42 on the same human neuroblastoma cells. This finding is of particular interest in view of recent results showing that amyloid-deposits are present in the mitochondria of cortical pyramidal neurons of post-mortem AD brain
(Fernandez- Vizarra et al. 2004). These histological features have been described as being a very early event in the progression of the disease, appearing before the formation of paired-helical filaments and leading to neuronal death. On this basis, the inactivation OfAB1-42 by SP-233, as described herein, may justify use of SP-233 in the treatment of AD (Lecanu et al. 2004).
After the binding and "scavenging" effects of SP-233 on ABi-42 had been established, the possibility that a direct effect of SP-233 on mitochondria might result in an additive protective effect against AB 1-42 was examined. In this regard, it has been shown that AB promotes the opening of MPT pores in brain mitochondria, leading to functional impairment, and this affect is abolished by CsA (Moreira et al. 2001; Bachurin et al. 2003; Abramov et al. 2004). The experiments revealed that SP-233, like CsA, decreased the MRC. SP-233 also amplified the CsA' s effects, suggesting that it might have CsA-like properties. That is, it might inhibit MPT pore opening, which could contribute to its protective effect against ABi-42 -induced mitochondrial dysfunction. This hypothesis is supported by SP-233 's protective effect against PAO, a protein tyrosine phosphatase inhibitor known to promote the MPT (Korge et al., 2001). Thus, SP-233, at concentrations of 1 μM, restored neuronal cell viability by 25%; whereas, higher concentrations of SP-233 were not associated with a protective effect. SP-233 's effect on MPT-linked pore opening may occur via direct and/or indirect action (e.g., by allosteric modification after its insertion into mitochondrial membranes).
SP-233 was also able to abolish CCCP-induced uncoupling of oxidative phosphorylation in isolated mitochondria and in neuroblastoma cells. It has been shown that ABi-42 disrupts mitochondrial membrane structure (Kremer et al. 2001; Rodrigues et al. 2001) and that mitochondrial membrane fluidity may be altered in AD brain (Mecocci et al, 1996); both phenomena lead to mitochondrial uncoupling. Therefore, even though the "re-coupling" effect of SP-233 has not been fully clarified, it might contribute to the restoration of mitochondrial function described herein. Moreover, a combination of the scavenging effect of SP-233 (presumably involving its modulation of MPT pores) and its re-coupling effect might account for SP-233 's ability to counteract the effects OfAB1-42, at concentrations as low as 1 pM. In an attempt to identify other targets of SP-233 in the mitochondrial respiratory chain, it was demonstrated that SP-233 is able to protect neuroblastoma cells against inhibitors of complexes I, III, IV, and V, but not against inhibitors of complex II (Fig. 15). The most pronounced effect was observed in complexes III and IV, although SP-233, at concentrations of 1 μM and 100 μM, also exerted a small but significant neuroprotective effect against rotenone and myxothiazol-induced toxicity, respectively. The magnitude of the cell viability rescue against KCN and oligomycin compared to that against AB]-42 suggests that complexes IV and V are the main targets for the amyloid peptide- induced impairment of mitochondrial functions. These findings confirm previous results showing that the fragment Aβ25-35 inhibits complex IV in isolated brain mitochondria (Canevari et al., 1999). However, in contrast to the data, the same authors reported that AB25-35 has no effect on the other complexes of the respiratory chain. This apparent discrepancy could be explained by differences in the amyloid species used, and it raises questions about the role of AJ325-35 and AB1-42 in neuropathology.
As AD is an incurable neurodegenerative disorder and as the existing treatments, most of which are based solely on the inhibition of the AChE, have serious limitations, there is an urgent need to develop more effective drugs. Here it is reported that the small molecule, SP-233, which has previously been shown inhibits AB1-42-induced neurotoxicity by blocking the formation of the ADDLs, protects brain mitochondria against the deleterious effects OfAB1-42 exposure. The molecular mechanisms underlying SP-233 's neuroprotective effects might be due, at least in part, to its "scavenging" OfAB1-42, its "anti- uncoupling" effect, its protective effect toward mitochondrial complexes IV and V, and its ability to inhibit the opening of MPT pores. In conclusion, the data provided herein have confirmed and extended the previous findings showing that SP-233 possesses anti-amyloid properties, and they identify the mitochondria and the respiratory chain as direct targets for AB1-42 and SP-233. Bibliography
The publications listed below are incorporated by reference herein as though set forth in full.
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All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.

Claims

STATEMENTS OF THE INVENTION
1. A therapeutic method comprising administering to a mammal afflicted with or threatened by, a mitochondrial disorder that is not a neuropathology of the central nervous system comprising administering to said mammal an effective amount of a compound of formula (I):
Figure imgf000055_0001
wherein each OfR1, R2, R4, R7, Rn, R12, and R15, independently, is hydrogen, (C1-C8)alkyl, that is optionally inserted with -NR'-, -O-, -S-, -SO-, - SO2-, -0-SO2-, -SO2-O-, -C(O)-, -C(O)-O-, -O-C(O)-, -C(O)-NR'- or -NR'- C(O)-; hydroxy, N(R')2, carboxyl, oxo, or sulfonic acid, R3 is hydroxy, (C1- C8)alkoxy, (CrC22)alkylCO2, or R'O2C(CH2)2-8CO2-, wherein (Q-C^alkyl or (CH2)2-8 can optionally comprise 1-2 CH=CH units, 1-2 OH, and/or 1-2 epoxy substituents, toluene-4-sulfonyloxy or benzoyloxy; each of R6, R8, R9, R10, R13, R14, R16 and R17, independently, is hydrogen, (C1-C8)alkyl, hydroxyl(Cr C8)atøyl, (C1-C8)alkoxy, or hydroxy; and X is O, S, S(O) or N(R'), N(Ac) or N(toluene-4-sulfonyloxy) ; wherein each R1 is individually H, (C1-C8MkVl, phenyl or benzyl, or a pharmaceutically acceptable salt thereof.
2. The therapeutic method of claim 1 wherein R10 and R13 are CH3.
3. The therapeutic method of claims 1 or 2 wherein one or both OfR16 and R17 are CH3.
4. The therapeutic method of any one of claims 1-3 wherein R1, R2 or R12 are H or OH.
5. The therapeutic method of any one of claims 1-4 wherein R1, R2, R4, R6, R7, R8, R9, Rn, R12, R14 and R15 are H.
6. The therapeutic method of any one of claims 1-5 wherein R3 is (C1- C22)alkylCO2-, (d-C6)alkylCO2-, or is OH.
7. The therapeutic method of any one of claims 1 -6 wherein X is O.
8. The therapeutic method of any one of claims 1-6 wherein X is NH.
9. A therapeutic method of treating a mitochondrial disorder in a subject afflicted with or threatened by said disorder, wherein said disorder is not a neuropathology of the central nervous system, comprising administering to the subject an effective amount of a compound of formula (II):
Figure imgf000056_0001
wherein each OfR1, R2, R4, R7, R11, R12, and R15, independently, is hydrogen, (C1-Cg)alkyl, hydroxy, amino, carboxyl, oxo, sulfonic acid, or (C1- C8)alkyl that is optionally inserted with -NH-, -N((C1-C8)alkyl)-, -O-, -S-, -SO-, -SO2-, -0-SO2-, -SO2-O-, -C(O)-, -C(O)-O-, -O-C(O)-, -C(O)-NR'- or-NR'- C(O)-, wherein R' is H or (C1-C8)alkyl; R3 is hydroxy, (d-C6)alkylCO2-,
HO2C(CH2)2CO2-, toluene-4-sulfonyloxy or benzoyloxy; each of R6, R8, R9, R10, R13, and R14, independently, is hydrogen, (C1-C8)alkyl, hydroxyl(C1-Cg)alkyl, (C1-C8)alkoxy or hydroxy; and X is O, N(H), N(Ac) or N(toluene-4- sulfonyloxy), or a pharmaceutically acceptable salt thereof.
10. The method of claim 9 wherein R3 is OH or (C1-C6)alkylCO2-.
11. The method of claim 9 or 10 wherein X is O.
12. The method of claim 9 or 10 wherein X is NH.
13. The method of any one of claims 9-12 wherein R10 and R13 are CH3.
14. The method of any one of claims 9-13 wherein R1, R2 Or R12 are OH.
15. The method of any one of claims 9-15 wherein R1, R2, R4, R6, R7, R8, R9, R11, R12, R14 and R15 are H.
16. The method of any one of claims 1 or 9 wherein the compound is selected from the group consisting of (20α)-25ξ-methyl-(22R,26)- azacyclofurost-5-en-3ξ-ol, (20ξ)-25ξ-methyl-N-acetyl-(22R,26)-azacyclofurost- 5-en-3ξ-ol, (22R,25ξ)-(20α)-spirost-5-en-(2α,3ξ)-diol, (20α)-25ξ-methyl-N- paratoluenesulfonyl-(22R,26)-azacyclofurost-5-en-3ξ-yl paratoluenesulfonate, (22#,25ξ)-(20α)-(14α,20α)-spirost-5-en-(3β,12β)-diol, (22i?,25S)-(20ξ)-spirost- 5-en-3ξ-ol, (22i?,25ξ)-(20α)-spirost-5-en-3β-yl benzoate, (225,25 S)-(20S)- spirost-5-en-3β-yl hexanoate, (22i?,25ξ)-(20α)-spirost-5-en-(lξ,3ξ)-diol, (22i?,255)-(20α)-sρirost-5-en-3 β-ol, (22i?,255)-(20α)-sρirost-5-en-3 β-yl succinate, and (20α)-25»S'-methyl-N-acetyl-(225',26)-azacyclofurost-5-en-3 β-yl propanoate.
17. The method of claim 16 wherein the compound is (22S,25S)-(20S)- spirost-5-en-3β-yl hexanoate.
18. The method of claims 1 or 9 wherein the compound is administered in a dosage form comprising a therapeutically effective amount of the compound.
19. The method of claim 18 wherein the dosage form is selected from the group consisting of a tablet, a soft gelatin capsule, a hard gelatin capsule, a suspension tablet, an effervescent tablet, a powder, an effervescent powder, a chewable tablet, a solution, a suspension, an emulsion, a cream, a gel, a patch, and a suppository.
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 comprises a binder, a disintegrant, a filler, a surfactant, a solubilizer, a stabilizer, a lubricant, a wetting agent, a diluent, an anti-adherent, a glidant, or a pharmaceutically compatible carrier.
22. The method of any one of claims 1-21 wherein the mitochondrial disorder is a mitochondrial disease.
23. The method of any one of claims 1-21 wherein the mitochondrial disorder is a myopathy.
24. The method of claim 23 wherein the myopathy is cardiomyopathy.
25. The method of any one of claims 1-21 wherein the mitochondrial disorder is a tubulopathy.
26. The method of any one of claims 1-21 wherein the mitochondrial disorder is due to compartment syndrome or crushed muscle syndrome.
27. The method of any one of claims 1-21 wherein the neuropathology of the central nervous system is selected from the group consisting of global and focal ischemic or hemorrhagic stroke, head trauma, spinal cord injury, hypoxia- induced nerve cell damage, nerve cell damage caused by cardiac arrest or neonatal distress, epilepsy, anxiety, spinal cord lesion, ALS, Alzheimer's Disease, Huntington's disease, and Parkinson's disease.
28. A pharmaceutical composition comprising a therapeutically effective amount of at least one compound of formulas (I) or (II) in combination with a pharmaceutically acceptable excipient.
29. Use of a compound of formula (I):
Figure imgf000059_0001
wherein each OfR1, R2, R4, R7, Rn, R12, and R15, independently, is hydrogen, (CrC^alkyl, that is optionally inserted with -NR'-, -O-, -S-, -SO-, - SO2-, -0-SO2-, -SO2-O-, -C(O)-, -C(O)-O-, -O-C(O)-, -C(O)-NR'- or -NR'- C(O)-; hydroxy, N(R')2, carboxyl, oxo, or sulfonic acid, R3 is hydroxy, (C1- C8)alkoxy, (d-C22)alkylCO2, or R'O2C(CH2)2-8CO2-, wherein (C1-C22)alkyl or (CH2)2-8 can optionally comprise 1-2 CH=CH units, 1-2 OH, and/or 1-2 epoxy substituents, toluene-4-sulfonyloxy or benzoyloxy; each of R6, R8, R9, R10, R13, R14, R16 and R17, independently, is hydrogen, (C1-C8)alkyl, hydroxyl(Ci- C8)alkyl, (C1-C8)alkoxy, or hydroxy; and X is O, S, S(O) or N(R'), N(Ac) or N(toluene-4-sulfonyloxy) ; wherein each R is individually H, (Ci-C8)alkyl, phenyl or benzyl, or a pharmaceutically acceptable salt thereof to prepare a medicament for treating a mitochondrial disease or disorder that is not a neuropathology of the central nervous system.
30. Use of a compound of formula (II) :
Figure imgf000059_0002
wherein each OfR1, R2, R4, R7, R11, R12, and R15, independently, is hydrogen, (C1-C8)alkyl, hydroxy, amino, carboxyl, oxo, sulfonic acid, or (C1- C8)alkyl that is optionally inserted with -NH-, -N((C1-C8)alkyl)-, -0-, -S-, -SO-, -SO2-, -0-SO2-, -SO2-O-, -C(O)-, -C(O)-O-, -O-C(O)-, -C(O)-NR'- or -NR'- C(O)-, wherein R' is H or (C1-C8)alkyl; R3 is hydroxy, (C1-C6)alkylCO2-, HO2C(CH2)2CO2-, toluene-4-sulfonyloxy or benzoyloxy; each of R6, R8, R9, R10, R13, and R14, independently, is hydrogen, (Ci-C8)alkyl, hydroxyl(C1-C8)alkyl, (C1-C8)alkoxy or hydroxy; and X is O, N(H), N(Ac) or N(toluene-4- sulfonyloxy), or a pharmaceutically acceptable salt thereof to prepare a medicament for treating a mitochondrial disease or disorder that is not a neuropathology of the central nervous system.
31. The use of claim 29 or 30, wherein the medicament includes a physiologically acceptable carrier.
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