CN101227911A - Use of spirostenols to treat mitochondrial disorders - Google Patents

Abstract

The present invention relates to methods and compositions 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

The application of spirostenols to treat mitochondrial disorders

Related application

The application requires the priority of U.S. Provisional Application sequence number of submitting on April 1st, 2,005 60/667,229 and the U.S. Provisional Application sequence number of submitting on July 8th, 2,005 60/697,518, and described application is incorporated into this paper by reference.

Background of invention

Mitochondrion is to be present in the chamber that removes the uniqueness in exo-erythrocytic each somatic cell, and is responsible for producing by oxidative phosphorylation and surpasses 90% the human body that is used for and earn a bare living and support to grow required energy.When mitochondrial respiratory chain depletion, the energy level of cell reduces rapidly, causes the infringement of cell death and organ dysfunction.Depend on related organ, the infringement of mitochondrion physiological function relates to neurodegenerative diseases and disorder.These diseases and disorderly disease and the disorder that comprises the central nervous system, as Alzheimer and parkinson, and those encroach on peripheroneural disease and disorder, such as muscle disorder (myopathy) and multiple kidney, liver and disordered breathing.

According to the order of severity difference of mitochondrial disorders, the order of severity of disease can be between slight and death.No matter whether mitochondrion depletion is the reason or the result of the disease that experienced, the protection respiratory function is vital to recover impaired physiological function.In addition, the preservation of the tissue successfully transplanted for needs of the maintenance of mitochondrial respiratory function also is important.

For the also readily good curing method of the mitochondriopathy on the gene basis, but treatment can help mitigation symptoms or delay or prophylactic development (M.Zeviani etc., Brain; 127:2153 (2004)).Treatment is personalized for each patient, and different and different according to the disorderly character and the order of severity continue the infringement that exists already though these treatments that take stopgap measures will not reverse.Yet current some experimental strategies of assessing comprise the intervention of gene therapy and medicine.

Strategy comprises via protein reader (import machinery) (G.Manfredi etc. in the gene modified or the gene prod lead-in wire plastochondria, Natur.Genet, 30:394 (2002)), and (the R.W.Taylor etc. that duplicate by sequence-specific anti-genomic (antigenomic) peptide-nucleic acid mutation inhibiting body mtDNA, Natur.Genet, 15:212 (1997)).These means also are not used in clinical.

Through selecting, in the isolated myopathy case, by using muscle poison (myotoxic) medicine, the control meat fiber damages and by there not being the satellite cell regeneration of sudden change, obtains the minimizing (W.Irwin etc., J Biol.Chem.277:2221 (2002)) of heterogeneous mutant load.Yet this therapy is invalid for improving the ptosis in five patients that suffer from PEO (PEO).

Creatine is the substrate of synthetic phosphagen and is the storage compounds of abundant energy the most in muscle, heart and the brain.81 open trials of suffering from patient's (comprising that 17 are suffered from mitochondriopathy) of multiple neuromuscular disorder show that ischemic isometric contraction grip (isometrichandgrip strength) and the isometric dorsiflex torsion of non-ischemic (dorsiflexion torque) significantly improve.Yet, another is in 16 placebo of suffering from the patient of chronic PEO or mitochondrial myopathy, double blinding, at random in the cross matching, athletic performance, eye motion or activities of daily living are not found significant effect (P.F.Chinnery etc., Am.J.Med.Genet, 106:94 (2001)).In a word, the prompting of these data is at some but in the not every mitochondriopathy, creatine may be effective.

Though in the relevant mitochondriopathy of mtDNA-, coenzyme Q10 is invalid (N.Bresolin etc., J.Neurol.Sci, 100:70 (1990)), it causes the tangible improvement in " main " CoQ10 shortage.As if idebenone than the analog of the coenzyme Q10 of short chain, to improving hypertrophic neuropathy effectively (P.Rustin etc., Lancet, the 354:477 (1999) in the friedreich's ataxia; C.Mariotti etc., Neurology, 60:1676 (2003)).

Therefore, there is the lasting demand that the treatment mitochondrial disorders is comprised the method for mitochondriopathy.

The invention summary

The invention provides for example people's method of mammal that treatment suffers from the mitochondrial disorders of the neuropathy that is not the central nervous system or be subjected to its threat, described method comprises formula (I) chemical compound or its pharmaceutically acceptable salt that gives described mammal effective dose:

R wherein ₁, R ₂, R ₄, R ₇, R ₁₁, R ₁₂And R ₁₅Independent separately be hydrogen, choose wantonly insertion-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)-(C ₁-C ₈) alkyl; Hydroxyl, N (R ') ₂, carboxyl, oxo or sulfonic acid, R ₃Be hydroxyl, (C ₁-C ₈) alkoxyl, (C ₁-C ₂₂) alkyl CO ₂Or R ' O ₂C (CH ₂) _2-8CO ₂-, (C wherein ₁-C ₂₂) alkyl or (CH ₂) _2-8Can choose wantonly and comprise 1-2 CH=CH unit, a 1-2 OH and/or 1-2 epoxy substituent group, toluene-4-sulfonyl oxygen base or benzoyloxy; R ₆, R ₈, R ₉, R ₁₀, R ₁₃, R ₁₄, R ₁₆And R ₁₇Independent separately is hydrogen, (C ₁-C ₈) alkyl, hydroxyl (C ₁-C ₈) alkyl, (C ₁-C ₈) alkoxyl or hydroxyl; With X be O, S, S (O) or N (R '), N (Ac) or N (toluene-4-sulfonyl oxygen base),

Wherein each R ' independence is H, (C ₁-C ₈) alkyl, phenyl or benzyl.

R ₁₆And/or R ₁₇Be preferably CH ₃

Alkyl is preferably (C ₂-C ₈) alkyl.

Therefore, the present invention includes the Therapeutic Method that treatment is suffered from mitochondrial disorders or is subjected to the described disorderly patient who threatens, wherein said disorder is not central nervous system's a neuropathy, and described method comprises formula (II) chemical compound or its pharmaceutically acceptable salt that gives patient's effective dose:

R wherein ₁, R ₂, R ₄, R ₇, R ₁₁, R ₁₂And R ₁₅Independent separately is hydrogen, (C ₁-C ₈) alkyl, hydroxyl, amino, carboxyl, oxo, sulfonic acid, or optional insertion-NH-,-N ((C ₁-C ₈) alkyl)-,-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)-(C ₁-C ₈) alkyl, wherein R ' is H or (C ₁-C ₈) alkyl; R ₃Be hydroxyl, (C ₁-C ₆) alkyl CO ₂-, HO ₂C (CH ₂) ₂CO ₂-, toluene-4-sulfonyl oxygen base or benzoyloxy; R ₆, R ₈, R ₉, R ₁₀, R ₁₃And R ₁₄Independent separately is hydrogen, (C ₁-C ₈) alkyl, hydroxyl (C ₁-C ₈) alkyl, (C ₁-C ₈) alkoxyl or hydroxyl; With X be O, N (H), N (Ac) or N (toluene-4-sulfonyl oxygen base).

As discussing in the following discussion, spiral shell stenol (spirostenols), particularly 5,6 unsubstituted spiral shell stenols are as (22S, 25S)-(20S)-spiral shell steroid-5-alkene-3 beta-yl alkyl caproate (spirost-5-en-3 β-yl hexanoate), be the natural 22R-hydroxycholesterol oxycholesterol derivant that is present in the Radix gynurae segeti (Gynura japonica) (Compositae (asteraceae)), it can provide new therapeutic strategy, promptly by direct targeting mitochondrial respiratory chain with and targeting the virose A β of mitochondrion is worked.(22S, 25S)-(20S)-spiral shell steroid-5-alkene-3 beta-yl alkyl caproate also eliminates the inductive uncoupling of oxidative phosphorylation and strengthens the effect of cyclosporin A (effective blocker in mitochondrial membrane permeability conversion hole), and prompting is worked to membrane permeability conversion hole.

Mitochondrial disorders comprises mitochondriopathy, is not considered to the CNS neuropathy and can adopts existing method treatment, and described mitochondrial disorders comprises: eyes: retinitis pigmentosa, optic nerve atrophy; Muscle: extraocular muscles dysfunction (ptosis, acquired stravismus, ophthalmoplegia), muscle injury syndrome (osteofascial compartment syndrome (compartmentsyndrome)); Mitochondrial function due to the drug side effect complete (that is, and the high activity antiretroviral therapy, HAART); Myopathy; Heart: any cardiod plastochondria functional defect (that is, HAART, mega-HAART, cancer therapy drug such as anthracene nucleus class (anthracyclins)) due to heart attack (heart stroke), heart attack (heart attack), cardiac disorder (heart conditions), heart disease, cardiomyopathy and the drug side effect; Liver: hepatocyte function is incomplete; Kidney and hormonal system: renal tubules pathological changes, diabetes; And disordered breathing.

The present invention also provides new formula I or II chemical compound and compositions, such as comprising and the formula I of the effective dose of pharmaceutically acceptable carrier combinations and/or the Pharmaceutical composition of II chemical compound.

The accompanying drawing summary

Fig. 1 is by the differentiation of the coefficienting respiration in the monitoring rat brain mitochondria, and the SP-233 (10 to 100pM) of assessment progressive concentration is to the effect of mitochondrial respiratory chain.This mitochondrial respiratory coefficient (MRC) is V ₃/ V ₄V ₄Be basic O ₂Consume V ₃For adding the O behind the ADP (ATP product) ₂Consume.Regulate mitochondrion concentration to 0.4mg protein/ml.When concentration that it should be noted that SP-233 is low to moderate 10pM, with the contrast resistance relatively, cause that MRC reduces by 50% (p＜0.001).The result represents with mean value SD; By ANOVA, pass through subsequently Dunnett ' s check organize between relatively.

The effect of the oxidative phosphorylation uncoupling that Fig. 2 (a): SP-233 causes CCCP-in the rat brain mitochondria.In order to assess the effect of the oxidative phosphorylation uncoupling that SP-233 causes CCCP-, add before malate/glutamate, Glu, the mitochondrion fragment is hatched 3min in 37 ℃ in the presence of SP-233, then add CCCP behind the 1min.Be exposed to uncoupling agents CCCP (1 μ M) and increase O ₂Be consumed to 150% (p＜0.01) of basic value, this changes the increase of reflection oxygen consumption.In all tested concentration (or even 1pM), SP-233 eliminates the metabolism (p＜0.001) of CCCP.This SP-233 is to the inhibitory action and reduction O of " uncoupling " ₂It is relevant and be non-concentration-dependence to be consumed to the 60-65% of foundation level, (b): the anoxybiotic effect that SP-233 causes CsA-.Anoxia causes to bathing in bubble (bathing) mitochondrial culture medium by adding CsA (1 μ M).This test is by adding ADP and compare having CsA and lack under the SP-233.At first add SP-233 or its solvent, and make to hatch and continue 1.5min, light and slow afterwards adding CsA.Then, add ADP behind adding substrate malate/glutamate, Glu and the 2.5min behind the 1.5min.Be exposed to the decline that SP-233 does not suppress the MRC that caused by CsA, and opposite, the work of CsA strengthens in order to the mode of concentration-dependence.The result represents with mean value SD; By ANOVA, pass through subsequently Dunnett ' s check organize between relatively.

Fig. 3 fresh with aged A β _1-42Effect to the mitochondrial respiratory coefficient of rat brain mitochondria.Add before substrate malate/glutamate, Glu, at A β _1-42Existence under mitochondrion is hatched 1.5min in 37 ℃.Add ADP after adding malate/glutamate, Glu 1min.This experiment compares through omitting ADP.Even under the low concentration of 0.1pM, A β _1-42Significantly reduce MRC.Fresh 4 amyloid is to more remarkable than ageing form of the effect of MCR, and it reduces MRC separately is 55-63% and 43-53%.The result represents with mean value SD; By ANOVA, pass through subsequently Dunnett ' s check organize between relatively.

Fig. 4 SP-233 is to by fresh or aged A β _1-42The change effect of the mitochondrial respiratory coefficient in the rat brain mitochondria that causes.Under 37 ℃, in the presence of SP-233, mitochondrion hatched 3min and at (a) or aged (b) of prepared fresh A β _1-42Existence under hatch 1.5min, add malate/glutamate, Glu subsequently.Add ADP after adding substrate malate/glutamate, Glu 1min.Do not compare having to carry out same experiment under the ADP, understand SP-233 A β _1-42Effect.(a): compare fresh A β with matched group _1-42MRC reduction by 71% and this effect are partly suppressed by SP-233.Observe the effective function with the SP-233 of least concentration, it makes MRC return to 39% (38.56 ± 0.34 pairs 28.93 ± 1.75, p＜0.01) of control value.(b): compare aged A β with control value _1-42Make MRC reduce by 51%, but SP-233 does not prevent this effect.The result represents with mean value SD; By ANOVA, pass through subsequently Dunnett ' s check organize between relatively.

Fig. 5 is SP-233 antagonism A β in the human neuroblastoma cell _1-42The assessment of-toxic the protective effect that causes.Use A β _1-42(0.1,1 and 10 μ M) or solvent and be exist or lack hatch SK-N-AS neuroblastoma cell 72h under the SP-233 (1 μ M) after, adopt the cytotoxicity of MTT analysis and evaluation A β.SP-233 (1 μ M) prevention is by three A β _1-42The neurotoxicity that concentration causes.The result represents with mean value SD; By ANOVA, pass through subsequently Dunnett ' s check organize between relatively.

Use A β _1-42The processing neuroblastoma cell demonstrates strong immunoreactivity, and (itself and mitochondrial respiratory chain complex II locate (co-localized) altogether, expression A β _1-42Be present in the mitochondrion).Handle elimination A β with SP-233 _1-42Immunoreactivity, represent that it may stop A β _1-42Enter mitochondrion.

Fig. 6 SP-233 is to the effect of mitochondrial respiratory.Spiral shell stenol derivant (22R, 25R)-chemical formula of 20 α-spiral shell steroid-5-alkene-3 beta-yl alkyl caproate (SP-233) is shown in (a).The differentiation of the coefficienting respiration by monitoring isolating rat brain mitochondria, the SP-233 (1aM to 100pM) of assessment progressive concentration is to the effect of mitochondrial respiratory chain, and it is shown in (b).This mitochondrial respiratory coefficient (MRC) is defined as V ₃/ V ₄, V wherein ₄Be basic O ₂Consume and V ₃Be the O behind the adding ADP (ATP generation) ₂Consume.Regulate mitochondrion concentration to 0.4mg protein/ml.When the concentration of attention SP-233 is low to moderate 1fM, compare, cause that MRC reduces by 40% (p＜0.001) with the contrast resistance.The result represents with the mean value SD of three parts of repeated experiments of independently carrying out.

Oxidative phosphorylation uncoupling that Fig. 7 SP-233 causes CCCP-and the anoxybiotic effect that CsA-is caused, (a) in order to assess the effect of the oxidative phosphorylation uncoupling that SP-233 causes CCCP-, add before malate/glutamate, Glu, the mitochondrion fragment is hatched 3min in 37 ℃ in the presence of SP-233.Add CCCP behind the 1min.Be exposed to uncoupling agents 1 μ MCCCP and increase O ₂Be consumed to 150% (p＜0.01) of basic value.In all tested concentration, SP-233 eliminates the metabolism (p＜0.001) of CCCP.SP-233 is to the inhibitory action and reduction O of " uncoupling " ₂It is relevant and be non-concentration-dependence to be consumed to the 60-65% of foundation level.(b) anoxia causes to the culture medium of bathing the tailors tack plastochondria by adding 1 μ M CsA.In contrast, exist CsA to add ADP with lacking under the SP-233.At first add SP-233 or its solvent, and make to hatch and continue 1.5min, light and slow afterwards adding CsA.1.5min the back adds ADP after adding substrate malate/glutamate, Glu and 2.5min.Be exposed to the decline that SP-233 does not suppress the MRC that caused by CsA.On the contrary, the work of CsA strengthens in order to the mode of concentration-dependence.The result represents with the mean value SD of three parts of experiments of independently carrying out.

Fresh or the aged A β of Fig. 8 _1-42Effect to the mitochondrial respiratory coefficient in the rat brain mitochondria.Before adding substrate malate/glutamate, Glu, under 37 ℃, at A β _1-42Existence under mitochondrion is hatched 1.5min.Add ADP after adding malate/glutamate, Glu 1min.In the mixtures incubated of these experiments, omitted ADP.Even under the concentration of 0.1pM, A β _1-42Significantly reduce MRC.Fresh 4 amyloid is more remarkable than the effect of ageing form to the effect of MCR, makes MRC decline 55-63% and 43-53% separately.The result represents with the mean value SD of three parts of repeated experiments of independently carrying out.

Fig. 9 SP-233 is to by fresh or aged A β _1-42The effect of the variation of the MRC that causes.Before adding malate/glutamate, Glu, under 37 ℃, in the presence of SP-233, mitochondrion hatched 3min and at (a) or aged (b) of prepared fresh A β _1-42Existence under hatch 1.5min.Add ADP after adding malate/glutamate, Glu 1min.Control reaction is carried out under shortage ADP.Compare fresh A β with matched group _1-42MRC reduction by 71% and this effect are partly suppressed by SP-233.

Observe with the SP-233 of least concentration test and have effective function, it makes MRC return to 39% (38.56 ± 0.34 pairs 28.93 ± 1.75, p＜0.001) of control value.Compare aged A β with control value _1-42Make MRC reduce by 51%, but SP-233 does not prevent this effect.The result represents with the mean value SD of three parts of repeated experiments of independently carrying out.

Figure 10 is in the human neuroblastoma cell, and SP-233 resists A β _1-42The assessment of-toxic the protective effect that causes.Use A β _1-42(0.1,1 and 10 μ M) or solvent and be after existing or lack under the SP-233 (1 μ M), hatching SK-N-AS culture 72h adopt the cytotoxicity of MTT analysis and evaluation A β.The result represents with the mean value SD of three parts of repeated experiments of independently carrying out.

Figure 11 SP-233 is to A β _1-42The burnt microscopic analysis of copolymerization of the effect of the mitochondrion picked-up in SK-N-AS human neuroblastoma cell.Take the photograph from typical image healthy, unscathed (contrast) cell and be shown in (a1-4).These cultures are not accepted any A β _1-42Or SP-233.Take the photograph personal A β _1-42The typical image that 10 μ M handle 3 hours SK-N-AS cell is shown in (b1-4).Take from and use A β _1-42The typical image that 10 μ M and SP-233 1 μ M handle 3 hours SK-N-AS cell is shown in (c1-4).Cell is with DAPI nuclear after stain dyeing (a1, b1 and c1) and to A β _1-42Labelling (FITC; A2, b2 and c2) and complex II 70-kDa subunit (Dallas Pink; A3, b3 and c3).The image that merges is shown in a4, b4 and c4.Use A β _1-42What the demonstration of processing neuroblastoma cell was strong is total to localized immunoreactivity (b2) with mitochondrial respiratory chain (b4) complex II, and handles elimination A β with SP-233 _1-42Immunoreactivity (c2).

Figure 12 in the SK-N-AS cell, the neuroprotective of SP-233 confrontation line plastochondria Complex Inhibition agent.After adding SP-233 or its solvent 1 hour, five kinds of different mitochondrion toxin are added in the SK-N-AS cultures.Before carrying out the cell viability detection,, rotenone, malonate and glutinous thiazole mykol (myxothiazol) were hatched 24 hours lacking or existing under the SP-233.Incubation period for KCN and oligomycin is 6 hours.The result represents with the mean value SD of three parts of repeated experiments of independently carrying out.Compare with the culture of handling without SP-233, ^*P＜0.05, ^*P＜0.01, ^{* *}P＜0.001.

Figure 13 SP-233 is to the neuroprotective of SK-N-AS neuronal cell antagonism PAO.In culture medium, add SP-233 or its solvent after 1 hour, add PAO.Carrying out before cell viability detects, culture medium was hatched in PAO 24 hours lacking or exist under the SP-233.The result represents with the mean value SD of three parts of repeated experiments of independently carrying out.Compare with the cell of handling without SP-233, ^*P＜0.01.

The effect of the uncoupling of the mitochondrial respiratory chain in the SK-N-AS cell that Figure 14 SP-233 causes FCCP-.In the SK-N-AS culture, add SP-233 or its solvent after 1 hour, add FCCP.Carrying out before cell viability detects, culture was hatched in FCCP 24 hours lacking or exist under the SP-233.The result represents with the mean value SD of three parts of repeated experiments of independently carrying out.Compare with the cell of handling without SP-233, ^*P＜0.01.

Figure 15 is schematically shown by the mitochondrion site of SP-233 targeting.Point out key element with green arrow by the respiratory chain of SP-233 targeting.Neuroprotective and solid line green arrow a little less than dotted arrow is represented are represented strong protective effect.Observe the effect of the most significant antagonism KCN (inhibitor of complex IV), oligomycin (inhibitor of complex V) and PAO (promoter of MPT).

Detailed Description Of The Invention

As hereinafter discussing, the infringement of mitochondrial respiratory chain has been described relevant with Alzheimer's (AD) widely, and existing report A β_1-42Make mitochondrial respiratory chain uncoupling and impel film permeability conversion (MPT) hole of mitochondria to open, cause cell dead. Existing report spirostenols (22S, 25S)-(20S)-spiral shell steroid-5-alkene-3 beta-yl capronate (SP-233) is by being combined with peptide and making peptide inactivation neuroprotective cell resist A β_1-42Toxicity. The present invention is based on following discovery, i.e. the protective effect of SP-233 also causes the protection to mitochondrial function. Found the A β of skin mole (slightly mole) concentration_1-42By the protective effect that is partly reversed by SP-233, the mitochondrial respiratory coefficient in the mitochondria that the reduction rat forebrain separates. The protective effect of this SP-233 be likely by its direct target respiratory chain with and target A β_1-42Due to.

SP-233 also eliminates by cyaniding carbonyl 3-chlorophenyl hydrazone (carbonyl cyanide 3-chlorophenylhydrazone, the effect of the phosphorous oxide acidifying uncoupling that CCCP) causes and enhancing cyclosporin A (effective retarding agent in mitochondria MPT hole), prompting has effect to MPT. These characteristics of SP-233 can have contribution to its neuroprotective effect because the unlatching in the uncoupling of respiratory chain and mitochondria MPT hole be two can be by A β_1-42The harmful event that triggers.

End user SK-N-AS neuroblastoma cell adopts the burnt microscopic analysis of copolymerization, also observes A β_1-42Gather with SP-233 A β in the online mitochondrial matrix_1-42From matrix, remove fully. These results show, with A β_1-42Directly effect and SP-233 (when being present in the mixtures incubated with 4 amyloid) can protect these cells with the toxicity of antagonism A β-cause to the performance of the mitochondrial function of human nerve cell with SP-233. Jointly, these results show A β_1-42Can pass through its direct target A β; mitochondrial function is brought into play direct toxic action and SP-233 its neuroprotective effects of performance, thus protection respiratory chain and the compound that provides a group to be used for the treatment of mitochondria sick and disorderly (rather than AD and the neural pathology of other CNS).

As comprising art-recognized mitochondria disease and illness at term mitochondrial disorders used herein, wherein work in mitochondrial disorders or insufficiency symptom, process and/or the consequence when playing disease. Latter's illness comprises nerve degenerative diseases such as Alzheimer's, Parkinson's disease, multiple sclerosis or ALS and the neuronal death that is caused by apoplexy, cerebral ischemia and other brain and chorda dorsalis injury. Existing report comprises (22S, 25S)-(20S)-spirostenols of spiral shell steroid-5-alkene-3-base capronate is used for neural toxicity that antagonism A-causes and they are effective to AD treatment (L.Lecanu etc., Steroids, and be the theme of the PCT application WO/03/077869 that announces 69:1 (2004)). Unknown (22S, 25S)-(20S)-spiral shell steroid-5-alkene-3-base capronate resists application and the application in treatment multiple line plastochondria disease thereof of multiple stressor for the protective wire plastochondria before.

Mitochondria disease or mitochondria myopathy are the diseases of one group of invasion and attack mitochondria, also disturb muscle function. This group disease comprises that Kearns-Sayre comprehensively levies, Leigh ' s comprehensively levies, mitochondrial DNA deletion is comprehensively levied (MDS), mitochondria brain myopathy, dlactic acid is poisoned and palsy sample outbreak (MELAS), the myoclonus epilepsy disease (MERFF) of companion's ragged-red fiber, the neural intestines and stomach brain myopathies (MNGIE) of mitochondria, neural pathology, incoordination and retinitis pigmentosa (NARP) and carrying out property external eyes myoparalysis (PEO).

The symptom of mitochondria myopathy comprises that muscle weakness or motion are lost anti-(exercise intolerance), (cardiac muscle is sick) in heart failure or the rhythm and pace of moving things disorder, dementia, dyskinesia, the outbreak of palsy sample, deafness, lost one's sight, ptosis, eye motion are limited, vomiting and epileptic attack. The serious degree of the prognosis that these are disorderly is weak to dead scope in carrying out property. Thereby mitochondrial disorders can comprise the disorder of eyes, such as optic nerve atrophy, upper ptosis, acquired stabimus and eye muscle paralysis. The side effect that mitochondrial function can comprehensively not levied by muscle injury the treatment of (or osteofascial compartment syndrome (compartment syndrome)), medicine entirely such as HAART and relate to the medicine (such as the anthracene nucleus class) that damage heart anti--cancer therapy, hepatocyte function not entirely, incretion renal tubule pathology and disordered breathing cause.

Most of mitochondria myopathies were occuring before 20 years old and are usually losing anti-or muscle weakness with motion when beginning. Between active stage, it is tired or weak easily that muscle can become at health. Though muscle cramp is rare, can occur. It is also relevant with these disorders with expiratory dyspnea to feel sick, have a headache.

As referring to disorder relevant with relating to mitochondrial disease or disorder in the patient or any treatment of disease at term used herein " treatment " or " processing ", it comprises, but be not limited to easily to suffer from described disorder or disease, this disorder or disease occur in prevention among the patient of described disorder or disease but not yet be diagnosed with; Suppress described disorder or disease, for example, this disorder or advancing of disease are detained; Alleviate described disorder or disease, for example, this disorder or disease are disappeared; Or eliminate by described disease or the disorderly illness that causes, for example, this disease or disorderly symptom are stopped. As used herein, " mitochondrial disorders " or " mitochondria is sick " means to comprise that all are in disorder disclosed herein.

This method also can be used to prevent or ameliorate body is outer as the organ or tissue in waiting or transplanting in mitochondrial disorders.

Relate to term " prevention " or " preventing " relevant with mitochondrial disease or disorder in the patient, if refer to not occur described disease or disorder, then there are not disease or disorderly development, or if described disease or disorder occured, and should disorder or disease do not have further development, or the inaction symptom of the sign of visible disease in logic.

Preferred stereoisomer is 3S, and 10R and 13S, also has 20S, 22S and 25S, and wherein the numbering of carbon skeleton is consistent with numbering in spiral shell stone alkene-3-alcohol. Thereby preferred formula (I or II) compound is (22S, 25S)-(20S)-spiral shell steroid-5-alkene-3 beta-yl capronate (SP233). Note, unless refer else, be shown in formula (I) or (II) in carbon atom saturated by hydrogen.

Unless otherwise defined, each term " alkyl ", prefix " alkane " (as in alkoxyl) and suffix " alkyl " (as in hydroxy alkyl) refer to straight chain (such as, butyl) or the side chain C of (such as, isobutyl group)_1-8The hydrocarbon chain. Alkylidene, inferior alkene base, inferior alkynes base refer to respectively divalence C_1-8Alkyl (such as ethylidene), alkene base and alkynes base. Preferably, alkyl is (C₁-C ₆) alkyl, such as butyl, hexyl, methyl, ethyl, propyl group or isopropyl. Term " alkyl " comprises cycloalkyl, (cycloalkyl) alkyl and alkyl (cycloalkyl) alkyl. Term " alkene base " comprises the alkyl that contains 1-2 CH=CH unit equally, and corresponding cycloalkenyl group part.

Particularly, (C ₁-C ₈) alkyl can be methyl, ethyl, propyl group, isopropyl, butyl, isobutyl group, tert-butyl, amyl group, 3-amyl group, heptyl or octyl group; (C ₃-C ₈) cycloalkyl can be monocycle, dicyclo or trinucleated and comprise cyclopropyl, cyclobutyl, cyclopenta, cyclohexyl, dicyclo [2.2.2] octyl or norborny (norbornyl), and multiple terpene hydrocarbon and terpenoid structure.(C ₃-C ₆) cycloalkyl (C ₁-C ₂) alkyl comprises aforesaid cycloalkyl and can be cyclopropyl methyl, cyclobutylmethyl, cyclopentyl-methyl, cyclohexyl methyl, 2-cyclopropyl ethyl, 2-cyclobutyl ethyl, 2-cyclopenta ethyl or 2-cyclohexyl ethyl.Heterocyclylalkyl comprises such cycloalkyl, and wherein the cycloalkyl ring system is monocycle, dicyclo or trinucleated and optional 1-2 S, non-snperoxiaized O or N (R ') and 4-8 ring carbon atom of comprising; Such as morpholinyl, piperidyl, piperazinyl, indanyl, 1,3-dithiane-2-base etc.Any cycloalkyl or heterocycloalkyl ring system option comprise 1-3 two keys or epoxy moieties and optional by 1-3 OH, (C ₁-C ₆) alkanoyl oxygen base, (CO), (C ₁-C ₆) alkyl or (C ₂-C ₆) alkynyl substituted.(C ₁-C ₈) alkoxyl can be methoxyl group, ethyoxyl, propoxyl group, isopropoxy, butoxy, isobutoxy, the second month in a season-butoxy, amoxy, 3-amoxy or hexyloxy; (C ₂-C ₆) thiazolinyl can be vinyl, pi-allyl, 1-acrylic, 2-acrylic, 1-butylene base, crotyl, 3-cyclobutenyl, 1-pentenyl, pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl or 5-hexenyl; Hydroxyl (C ₁-C ₈) alkyl or hydroxyl (C ₁-C ₈) alkoxyl can be the alkyl that is replaced by 1 or 2 OH group, such as alkyl such as the hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl, 1-hydroxybutyl, the 4-hydroxybutyl, 3 that are replaced by 1 or 2 OH group, 4-dihydroxy butyl, 1-hydroxyl amyl group, 5-hydroxyl amyl group, 1-hydroxyl hexyl or 6-hydroxyl hexyl; (C ₁-C ₂₂) alkyl CO ₂-can be acetate, propionyloxy, butyryl acyloxy, isobutyl acyloxy, penta acyloxy or hexylyloxy, or (C ₈-C ₂₂) CO ₂-can show as the residue of naturally occurring fatty acid.

In one embodiment of the invention, R ₁₀And/or R ₁₂Be CH ₃

In another embodiment of the invention, R ₁₆And R ₁₇Be CH ₃

In another embodiment of the present invention, R ₁, R ₂And/or R ₁₂Can be H or OH.

In another embodiment, R ₁, R ₂, R ₄, R ₆, R ₇, R ₈, R ₉, R ₁₁, R ₁₂, R ₁₄And R ₁₅Be H.

R ₂Can be (C ₁-C ₂₂) alkyl CO ₂-, comprise (C ₈-C ₂₂) alkyl CO ₂-or (C ₁-C ₆) alkyl CO ₂-, or can be OH or O (C ₁-C ₈) alkyl or HO ₂C (CH ₂) ₂CO ₂-.

X can be O or NR ', comprises NH or NAc.

R ₃Also can be OR ₂₃, R wherein ₂₃Be removable hydroxyl-blocking group such as tosyl, mesyl, three (C ₁-C ₄) alkyl silicyl, THP, Eto (Et), benzyl, benzoyloxy carbonyl etc.The C of chemical compound ₃-(C ₈-C ₂₂) fatty acid ester (C wherein ₃For hydroxyl replaces) be also included within the present invention, wherein fatty acid is preferably natural generation.

Can be used for implementing several above-mentioned formulas of the present invention (I) and (II) chemical compound be shown in the following table 1:

Table 1.

Title	Chemical name	The source
			SP224	(20 α)-25 ξ-methyl-(22R, 26)-azacyclo-	(Solanum?asperum)

	Furan steroid-5-alkene-3 ξ-alcohol (azacyclofurost-5-en-3 ξ-ol)	(Solanaceae)
			SP226	(20 ξ)-25 ξ-methyl-N-acetyl group-(22R, 26)-azacyclo-furan steroid-5-alkene-3 ξ-alcohol	(Solanum asperum) (Solanaceae)
SP227	(22R, 25 ξ)-(20 α)-spiral shell steroid-5-alkene-(2 α, 3 ξ)-glycol	Radix gynurae segeti (Gynurajaponica) (Compositae)
			SP229	(20 α)-25 ξ-methyl-N-p-toluenesulfonyl-(22R, 26)-azacyclo-furan steroid-5-alkene-3 ξ-Ji p-toluenesulfonic esters	Australian eggplant (Solanumaviculare) (Solanaceae)
SP230	(22R, 25 ξ)-(20 α)-(14a, 20 α)-spiral shell steroid-5-alkene-(3 β, 12 β)-glycol	Radix gynurae segeti (Gynurajaponica) (Compositae)
			SP231	(22R, 25S)-(20 ξ)-spiral shell steroid-5-alkene-3 ξ-alcohol	Radix gynurae segeti (Gynurajaponica) (Compositae)
SP232	(22R, 25 ξ)-(20 α)-spiral shell steroid-5-alkene-3 beta-yl benzoate	Cymbidium ensifolium (L.) Sw. Radix gynurae segeti (Gynurasp.) (Compositae)
			SP233	(22S, 25S)-(20S)-spiral shell steroid-5-alkene-3 beta-yl alkyl caproate	Cymbidium ensifolium (L.) Sw. Radix gynurae segeti (Gynurasp.) (Compositae)
SP234	(22R, 25 ξ)-(20 α)-spiral shell steroid-5-alkene-(1 ξ, 3 ξ)-glycol	Radix gynurae segeti (Gynurajaponica) (Compositae)
			SP235	(22R, 25S)-(20 α)-spiral shell steroid-5-alkene-3 β-alcohol	Radix gynurae segeti (Gynurajaponica) (Compositae)
SP236	(22R, 25S)-(20 α)-spiral shell steroid-5-alkene-3 beta-yl succinate	Cymbidium ensifolium (L.) Sw. Radix gynurae segeti (Gynurasp.) (Compositae)
			SP238	(20 α)-25S-methyl-N-acetyl group-(22S, 26)-azacyclo-furan steroid-5-alkene-3 beta-yl propionic ester	(Solanum asperum) (Solanaceae)

Before the drug treatment disease relevant, the active compound of the present invention and the preparation of pharmaceutically acceptable carrier of effective dose can be formed Pharmaceutical composition with impaired mitochondrial function." effective dose " or " effective dose on the pharmacology " refers to need be to the amount of being given the chemical compound of therapeutical effect by the treatment patient.Animal and human's dosage mutual relation (based on every square metre of body surface area milligram number) is by Freireich etc., Cancer Chemother.Rep. 50, 219 (1966) describe.Body surface area can be by patient's height and body weight approximate test.Referring to, as, ScientificTables, Geigy Pharmaceuticals, Ardley, New York, 1970,537.Effective dose can be based on finding the effective external concentration of this chemical compound of the toxicity that suppresses A β or other protective wire plastochondria.The dosage that openly is used for the treatment of this chemical compound of neuronal cell line model below.The professional is confessed as this area, and effective dose is also according to different and different with optional other therapeutic agent that gives jointly of using of route of administration, excipient.

Method of pharmacy that can be by standard as, be used to measure LD ₅₀(colony's 50% fatal dose) and ED ₅₀(colony's 50% dose therapeutically effective), the toxicity and the therapeutic efficiency of mensuration active component.Dosage rate between toxicity and the therapeutical effect is therapeutic index and ratios available LD ₅₀/ ED ₅₀Express.The chemical compound that shows big therapeutic index is preferred.In the time can using the chemical compound that shows toxic side effects, must careful design make such targeting compounds be attacked the transmission system of tissue, so that the potential damage of non-infected cell is reduced to minimum, thus reduce its side effect.

Be included in the described method kit of the present invention, composition of medicine and Pharmaceutical composition by crystal formation (as, polymorph), enantiomeric form, isomeric forms and the tautomer of description chemical compound and pharmaceutically acceptable salt thereof.Be equipped with from following processed with acid as illustrative pharmaceutically acceptable salt: formic acid, acetic acid, propanoic acid, succinic acid, hydroxyacetic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, fumaric acid, acetone acid, aspartic acid, glutamic acid, benzoic acid, ortho-aminobenzoic acid, methanesulfonic acid, stearic acid, salicylic acid, right-hydroxy benzoic acid, phenylacetic acid, mandelic acid, pounce on acid, Loprazolam, ethane sulfonic acid, benzenesulfonic acid, pantothenic acid, toluenesulfonic acid, the 2-hydroxyethanesulfonic acid, p-anilinesulfonic acid., the cyclohexyl sulfamic acid, agaric acid (algenic), b-hydroxyl butyric acid, galactosaccharic acid and galacturonic acid.

Term " prodrug " refers to that the metabolic process conversion produces the medicine or the chemical compound (active part) of pharmacological action by passing through in vivo.Prodrug is considered to prodrug usually, is giving the patient and after absorption subsequently, via some process, is being converted into activity or has more active kind such as metabolic process.Other product that is got by conversion process is easy to be disposed by health.Prodrug has to be present in usually causes it still less active and/or give the chemical group of medicine dissolution or some other characteristic on the prodrug, such as ester group or acyl group.In case described chemical group comes out from the prodrug cracking, can produce and have more active medicine.Prodrug can be designed to reversible medicaments derivative and be used as trim to strengthen the tissue of transport of drug to the tool specific site.The design of prodrug has at present effectively increased the water solublity of treatment chemical compound, and described chemical compound is used for the zone that targeting water is primary solvent.For example, Fedorak, etc., Am.J.Physiol, 269, G210-218 (1995) has described dexamethasone-β-D-glucuronide.McLoed, etc., Gastroenterol. 106, 405-413 (1994) has described dexamethasone-succinate-dextran.Hochhaus, etc., Biomed.Chrom. 6, 283-286 (1992) has described dexamethasone-21-sodium sulfo benzoate and Dexamethasone-21-isonicotinate.Also have, J.Larsen and H.Bundgaard, Int.J.Pharmaceutics, 37, 87 (1987) describe N-acyl group sulfanilamide as the effectively evaluation of prodrug derivant.J.Larsen etc., Int.J.Pharmaceutics, 47, 103 (1988) describe N-methyl sulfanilamide as the effectively evaluation of prodrug derivant.Also for example, Sinkula etc., J.Pharm.Sci, 64, prodrug is described among the 181-210 (1975).Prodrug is also preparing other formula (I) or (II) is being used as synthetic intermediate in the chemical compound by synthetic mutual transformation approach known in the art.For example, referring to, I.T.Harrison, Compendium of Organic Synthetic Methods, Wiley-Interscience (1971) is used for changing mutually the substituent method of spiral shell stenol.

Term " derivant " refers to by atom, molecule or a group by another atom, molecule or group displacement or replacement, and the chemical compound that produces from the chemical compound of another similar structures.For example, the hydrogen atom of chemical compound can be replaced the derivant that produces this chemical compound by alkyl, acyl group, amino etc.

" plasma concentration " refers to the concentration of material in blood plasma or serum.

" drug absorption " or " absorption " refers to from medicine-feeding part to the body circulation for example enter the motor process of patient's blood flow.

" bioavailability " refers to that active part (medicine or metabolite) absorbs in the body circulation and the available degree that becomes of drug effect position in vivo." metabolism " refers to medicine chemical conversion process in vivo.

" pharmacodynamics " refers to determine the factor of the observed biologically relevant with the drug level of site of action.

" pharmacokinetics " refers to determine to reach and keep the factor of the drug level of site of action.

" plasma half-life " refers to that plasma drug level decayed for 50% required time from its maximum concentration.

Term " about " used in the disclosure means " being similar to " and comprise variable in the parameter that will occur in the practice of association area.Use as illustrative term " about " refers to dosage outside institute's how, and also effective and safe, and such dosage is also included within the scope of this claim.

Term " detectable serum-concentration " means the serum-concentration (specifically detecting with every ml, every dl or contained mg, μ g or the ng therapeutic agent of per 1 serum) that the administration post-absorption enters the therapeutic agent of blood flow.

Term " pharmaceutically acceptable " is used to describe this paper, means the noun of being modified and is suitable in the drug products.Pharmaceutically acceptable salt comprises metal ion and organic ion.Preferred metal ion includes, but are not limited to suitable alkali metal (Ia family) salt ion, alkaline-earth metal (IIa family) salt ion and other physiology and goes up acceptable metal ion.Ion as an example comprises aluminum, calcium, lithium, magnesium, potassium, sodium and the zinc of common valency.Preferred organic ion comprises protonated tertiary amine and quaternary ammonium cation, and part comprises trimethylamine, diethylamine, N, N '-dibenzyl-ethylenediamin, chloro procaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucosamine) and procaine.Pharmaceutically acceptable acid as an example comprises and is not limited to hydrochloric acid, hydrobromic acid, phosphoric acid, sulphuric acid, Loprazolam, acetic acid, formic acid, tartaric acid, maleic acid, malic acid, citric acid, 1-Hydroxy-1,2,3-propanetricarboxylic acid., succinic acid, lactic acid, gluconic acid, glucuronic acid, acetone acid, oxalacetic acid, fumaric acid, propanoic acid, aspartic acid, glutamic acid, benzoic acid etc.

Usually the form with Pharmaceutical composition gives the present composition.Can give these compositionss by any suitable approach, described approach comprises, but be not limited to oral, nasal feeding (nasogastric), rectum, transdermal, parenteral (for example, in subcutaneous, intramuscular, intravenous, the bone marrow and intradermal injection or give by infusion techniques), intranasal, saturating mucosa, implantation, intravaginal, part, mouthful cheek and Sublingual.Such preparation can conventional comprise buffer agent, antiseptic, penetration enhancer, can be adaptive carrier and other therapeutic or non--therapeutic ingredient.

The present invention also comprises the method contain with the Pharmaceutical composition of one or more formula I of pharmaceutically acceptable carrier or mixed with excipients or II chemical compound of using.As comprising any at term used herein " pharmaceutically acceptable carrier " or " pharmaceutically acceptable excipient " and all solvents, disperse medium, coating material, antibacterial agent and antifungal, isotonic agent and absorption delay agent etc.The field that bases on practicality for can not absorbing material of such medium and reagent is familiar with.Except reaching the not adaptive degree of any conventional media and reagent and compositions, its application is expected.Additional active component also can mix in the compositions.In the preparation present composition, compositions can with pharmaceutically acceptable mixed with excipients, through excipient dilution or with the carrier encapsulation of such formed capsule, pouch or other container.The carrier material that can be used for preparing the present composition is those any conventional excipient that use and should be based on selecting with the release characteristics of the suitability of active medicine and the dosage form wanted in pharmacy.

Below select illustrative pharmaceutical excipient as an example:

(a) binding agent is such as Radix Acaciae senegalis, alginic acid and salt thereof, cellulose derivative, methylcellulose, hydroxy ethyl cellulose, hydroxy propyl cellulose, Magnesiumaluminumsilicate, Polyethylene Glycol, natural gum, poly saccharic acid (polysaccharide acids), bentonite, HYDROXY PROPYL METHYLCELLULOSE, gelatin, polyvinylpyrrolidone, polyvinylpyrrolidone/vinyl acetate copolymer, polyvinylpolypyrrolidone, polyvidone, polymethacrylates, HYDROXY PROPYL METHYLCELLULOSE, hydroxy propyl cellulose, starch, pregelatinized Starch, ethyl cellulose, Tragacanth, dextrin, cyclodextrin, microcrystalline Cellulose, sucrose or glucose etc.

(b) complex, clay, alginate, natural gum or the Sodium Carboxymethyl Starch of disintegrating agent such as starch, pregelatinized corn starch, pregelatinized Starch, cellulose, cross-linked carboxymethyl cellulose, Sodium Carboxymethyl Starch, polyvinylpolypyrrolidone, crospolyvinylpyrrolidone, cross-linked carboxymethyl cellulose sodium (croscarmellose), microcrystalline Cellulose, calcium alginate, sodium alginate and be used to prepare any disintegrating agent of tablet.

(c) filler such as lactose, calcium carbonate, calcium phosphate, calcium hydrogen phosphate, calcium sulfate, microcrystalline Cellulose, cellulose powder, glucose, dextrates (dextrates), dextran, starch, pregelatinized Starch, sucrose, xylitol, lactose, mannitol, sorbitol, sodium chloride, Polyethylene Glycol etc.

(d) surfactant such as sodium lauryl sulphate, sorbitan mono-oleic acid ester, polyoxyethylene sorbitan monooleate dehydration, polysorbate, polaxomers, bile salts, glyceryl monostearate, pluronic (Pluronic ^TM) series (BASF) etc.

(e) solubilizing agent such as citric acid, succinic acid, fumaric acid, malic acid, tartaric acid, maleic acid, 1,3-propanedicarboxylic acid, sodium bicarbonate and sodium carbonate etc.

(f) stabilizing agent is such as any antioxidant, buffer agent, or also can use acid etc.

(g) lubricant such as magnesium stearate, calcium hydroxide, Pulvis Talci, stearoyl fumaric acid sodium, hydrogenated vegetable oil, stearic acid, behapate glyceride, magnesium stearate, calcium stearate and sodium stearate, stearic acid, Pulvis Talci, paraffin, boric acid, sodium benzoate, sodium acetate, sodium chloride, DL-leucine, Polyethylene Glycol, enuatrol or sodium lauryl sulphate etc.

(h) wetting agent such as oleic acid, glyceryl monostearate, sorbitan mono-oleic acid ester, sorbitol anhydride list dodecanoate, Emulphor FM, polyoxyethylenesorbitan sorbitan monooleate, polyoxyethylene sorbitol acid anhydride list dodecanoate, enuatrol or sodium lauryl sulphate etc.

(i) diluent such as lactose, starch, mannitol, sorbitol, glucose, microcrystalline Cellulose, calcium hydrogen phosphate, sucrose-substrate diluent, Icing Sugar, monobasic calcium sulfate monohydrate, calcium sulfate dihydrate, calcium lactate trihydrate, dextrates, inositol, hydrolyzed cereal solids, amylose, cellulose powder, calcium carbonate, glycine or bentonite etc.

(j) anti--adhesive agent or fluidizer such as Pulvis Talci, corn starch, DL-leucine, sodium lauryl sulphate and magnesium stearate, calcium stearate or sodium stearate etc.

(k) pharmaceutically acceptable carrier comprises Radix Acaciae senegalis, gelatin, silica sol, glycerol calcium phosphate, calcium lactate, maltodextrin, glycerol, magnesium silicate, sodium caseinate, soybean lecithin, sodium chloride, tricalcium phosphate, dikalium phosphate, sodium stearoyl lactate, carrageenin, monoglyceride, diglyceride or pregelatinized Starch etc.

Also have, pharmaceutical preparation is for example being discussed among the Remington ' s The Science and Practice ofPharmacy (2000).Other discussion to pharmaceutical preparation can be at Liberman, H.A. and Lachman, and L.Eds.Pharmaceutical Dosage Forms, Marcel Decker, New York finds among the N.Y.1980.Contain the tablet of the present composition or granule and can be film coating or enteric coating.

Except the treatment that is used for the people, the present invention also is used for other patient and comprises veterinary animal, reptile class animal, birds, external animal and domestic animal, comprises mammal, rodent etc.Mammal comprises primate, for example, and monkey or mongoose lemur, horse, Canis familiaris L., pig or cat.Rodent comprises rat, mice, Sciurus vulgaris or Cavia porcellus.

When appointment gave the mitochondrial toxicity inhibitor, Pharmaceutical composition of the present invention was useful.Believe that these compositionss will be effective to the treatment of mitochondriopathy especially.

In order to treat neurological sexual disorder/mitochondrial disorders, available Pharmaceutical composition of the present invention, with enough cause therapeutic response as, reduce the amount of the mitochondrial toxicity of medicine-cause, the The compounds of this invention of doses is provided, for example, about 5ng is to about 1000mg or about 100ng to about 600mg or about 1mg about 500mg or about 20mg dosage of about 400mg extremely extremely.The effective dose scope of concrete administration will be at about 0.0001mg/kg to 1500mg/kg, more preferably from 1 to 1000mg/kg, more preferably from about 1 to 150mg/kg body weight and 50 to 100mg/kg body weight most preferably from about.Can every day one to about four dosed administrations or with multidose administration every day to cause therapeutical effect.Dosage unit as the illustrative present composition specifically can comprise, for example, the The compounds of this invention of about 5ng, 50ng, 100ng, 500ng, 1mg, 10mg, 20mg, 40mg, 80mg, 100mg, 125mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 700mg, 800mg, 900mg or 1000mg.The administration frequency that can select dosage form to provide to want is in order to reach specific dosage.The amount and the dosage regimen of the unit dosage forms of treatment disease or the disorderly compositions that is given, comprise patient's age, body weight, sex and the state of an illness, disease or the disorderly order of severity, the approach and the frequency of administration according to multiple factor, and as is well known can be widely different.

In one embodiment of the invention, give the patient compositions with effective dose, promptly the amount that reaches the dose therapeutically effective that produces the therapeutical effect want with The compounds of this invention a period of time in the patients serum gives compositions.As illustration, in be grown up on an empty stomach (general at least 10 hours on an empty stomach), give compositions about dose therapeutically effective that in the patients serum, reached The compounds of this invention in 5 minutes after giving compositions.In another embodiment of the invention, giving patient's compositions reaches The compounds of this invention in the patients serum in the time of about 10 minutes dose therapeutically effective.In another embodiment of the invention, giving patient's compositions reaches The compounds of this invention in the patients serum in the time of about 20 minutes dose therapeutically effective certainly.

Go back in the embodiment of the present invention, giving patient's compositions reaches The compounds of this invention in the patients serum in the time of about 30 minutes dose therapeutically effective certainly.In another embodiment of the present invention, giving patient's compositions reaches The compounds of this invention in the patients serum in the time of about 40 minutes dose therapeutically effective certainly.In one embodiment of the invention, since giving patient's compositions about 20 minutes to about 12 hours, in the patients serum, reach the dose therapeutically effective of The compounds of this invention.In another embodiment of the invention, giving patient's compositions certainly about 20 minutes to about 6 hours, in the patients serum, reach the dose therapeutically effective of The compounds of this invention.In another embodiment of the present invention, giving patient's compositions certainly about 20 minutes to about 2 hours, in the patients serum, reach the dose therapeutically effective of The compounds of this invention.

Go back in the embodiment of the present invention, giving patient's compositions certainly about 40 minutes to about 2 hours, in the patients serum, reach the dose therapeutically effective of The compounds of this invention.With in another embodiment of the present invention, giving patient's compositions certainly about 40 minutes to about 1 hour, in the patients serum, reach the dose therapeutically effective of The compounds of this invention.

In one embodiment of the invention, the dosage with the serum-concentration that is fit to provide half maximal dose of The compounds of this invention gives the present composition.As illustration, after giving the present composition, in the patient, reach about 0.01 to about 1000nM, or about 0.1 to about 750nM, or about 1 to about 500nM, or about 20 to about 1000nM, or about 100 to about 500nM, or about serum-concentration of 200 to about 400nM.

The compositions of the present invention expection provided therapeutical effect to about 24 hours interval in about 5 minutes after administration, if want, and can be once a day or twice administration in a day.In one embodiment of the invention, The compounds of this invention with the average serum concentration that is fit to provide the dosage with following half maximal dose gives compositions, promptly after giving patient's compositions about 10,20,30 or 40 minutes, reach in patient's body at least about 1 μ g/ml, or at least about 5 μ g/ml, or at least about 10 μ g/ml, or at least about 50 μ g/ml, or at least about 100 μ g/ml, or at least about 500 μ g/ml or at least about 1000 μ g/ml.

Produce the amount of the essential medicine of therapeutical effect, can be based on for example, it is definite through testing that medicine enters the effectiveness of absorbance, bioavailability of medicament and the described disorder of treatment of serum.Yet, should be understood that, the given dose level at any concrete patient of medicine of the present invention, depending on activity, the patient that multiple factor comprises employed specific compound (comprises, for example, no matter the patient is the state that is on an empty stomach or has taken food) age, body weight, health status, sex and diet, administration number of times, discharge rate, drug regimen and the order of severity and the form of medication of the concrete disorder of being treated.Generally can make it reach best safety and effect through the titration determination therapeutic dose.Typically, derive from the body and/or the dose-effect relationship of in vitro tests at first can to give the suitable dosage of patient provide usefulness guidance.The Animal Model Study can be used for instructing relevant effective dose for the treatment of gastrointestinal dysfunction or disease according to the present invention usually.Should be appreciated that according to therapeutic scheme, the dosage that gives will depend on multiple factor, comprise the concrete medicine that gives, route of administration, concrete patient's the state of an illness etc.Generally speaking, wish to give effectively to reach the amount of finding effectively to produce the chemical compound of the suitable serum levels of the concentration of therapeutical effect with external a period of time.Therefore, finding that the chemical compound proof is for example, when having external activity during half maximum effective dose 200nM, people give expectation the amount of the medicine of the half maximum effective dose that a period of time in vivo effectively provides about 200nM concentration, the therapeutical effect that this volume production life is wanted, for example, the treatment disorder relevant with the inductive neurotoxicity of high β-amyloplaste with other as being selected from indication by this area professional's suitable detection.Determining of these parameters will be within the professional skill of this area.These situations will be familiar with and description arranged in standard textbook by this area.

In order to detect and determine to enter the The compounds of this invention of patient's effective dose, can use standard analytical techniques to detect the The compounds of this invention serum-concentration.

The compositions of the present invention design provides therapeutical effect to about 24 hours interval giving about 30 minutes of patient.In one embodiment, compositions provided such therapeutical effect after about 30 minutes.In another embodiment, compositions provides therapeutical effect to surpass about 24 hours, makes administration once a day to improve patient's compliance.

Also can be the medicine of treatment or prevention mitochondriopathy with another indication, for example, kreatinin and this method, kit and compositions use in conjunction (" conjoint therapy ").When with use in conjunction of the present invention, promptly in conjoint therapy, can realize addition or synergism, can reduce or eliminate many (if not all) unnecessary side effect like this.The characteristic of the minimizing side effect of these medicines usually owing to, for example, need reach the therapeutical effect of administering drug combinations and the dosage that reduces.

Phrase " conjoint therapy " comprises and gives the present composition and unite to give the medicine of another indication for treatment or prevention patient mitochondrial disorders, as the part of particular treatment, being intended to provides useful effect from these are used for the treatment of the combined effect of medicine of nervus retrogression and/or mitochondrial disorders.The beneficial effect of described use in conjunction includes, but are not limited to the pharmacokinetics that caused by uniting of medicine or the combined effect of pharmacodynamics.Uniting of these medicines gives to carry out in the time period of a qualification (usually according to selected associating, basic while, a few minutes, several hours, several days, a few week, several months or several years) usually." conjoint therapy " generally is not intended to comprise and gives two or more these medicines as the monotherapy scheme of separating, and these medicines cause associating of the present invention by accident and at random." conjoint therapy " be intended to comprise in sequential mode and give these medicines, promptly give each therapeutic agent at different time, and give these medicines or at least two medicines in simultaneously mode almost.For example, by giving single tablet or capsule or each medicine in a plurality of single capsule agent or tablet of each medicine that the patient has the regulation ratio, can realize almost administration simultaneously.Sequential or almost give each medicine simultaneously and can realize by any suitable approach.Can give the present composition by oral or nasal feeding, and the therapeutic agent of other use in conjunction can give by any approach that is suitable for concrete medicine, the approach that includes, but are not limited to oral route, percutaneous approach, intravenous route, intramuscular approach or directly absorb by mucosal tissue.For example, can give the present composition by oral or nasal feeding, and can be by therapeutic agent oral or the transdermal administration use in conjunction.The order that gives medicine is very not strict." conjoint therapy " can comprise that also giving as described above medicine unites and give other bioactive ingredients, for example, but is not limited to analgesic, and the associating non-drug therapy, for example, but is not limited to operative treatment.

The treatment chemical compound that constitutes conjoint therapy can be combination dosage forms that is intended to give substantially simultaneously or the dosage form of separating.The treatment chemical compound that constitutes conjoint therapy also can with give by the arbitrary treatment chemical compounds that require two step dosage regimens sequentially.Thereby dosage regimen must be sought medical advice and be treated chemical compound and separate active medicine sequential administration that a period of time gives, that separate.Interval between the multiple dosing step can from, for example a few minutes to several hours were to several days, this depends on the dynamics of characteristic such as usefulness, dissolubility, bioavailability, plasma half-life and the treatment chemical compound of each treatment chemical compound, and the effect and the patient's age and the patient's condition that depend on food intake.24 hours circadian differences of concentration of target molecules also can determine best dosage interval.

No matter the treatment chemical compound of conjoint therapy is while, simultaneously basic or sequential giving, can relate to such dosage regimen, described scheme requires by oral route to give a kind of treatment chemical compound and by for example, oral route, percutaneous approach, intravenous route, intramuscular routes or give another kind of treatment chemical compound by the approach that mucosal tissue directly absorbs.No matter give the treatment chemical compound of conjoint therapy by oral, suction spraying, rectum, part, mouthful cheek, Sublingual or parenteral approach (for example is, subcutaneous, intramuscular, vein and intradermal injection), separately or give together, each such treatment chemical compound will be included in the suitable pharmaceutical formulation that pharmaceutically acceptable excipient, diluent or other preparation composition are arranged.

For oral administration, Pharmaceutical composition can contain the formula (I) of requirement or (II) chemical compound and for for example, and tablet, hard or soft capsule, lozenge, cachet, buccal tablet (troche), dispersible powder, granule, suspending agent, elixir, solution or any other quite are suitable for the form of oral administration.As illustration, such Pharmaceutical composition can be prepared to the form of the discrete doses unit of the reactive compound that contains scheduled volume, such as tablet or capsule.Such peroral dosage form also can comprise, for example, and buffer agent.Available in addition enteric coating prepares tablet, pill etc.

The Pharmaceutical composition that is suitable for mouthful cheek or sublingual administration comprises lozenge (lozenges) and pastille (pastilles), the former is contained in the reactive compound in fragrant substrate such as sucrose and Radix Acaciae senegalis or the Tragacanth, and the latter is contained in inert base such as such as the reactive compound in gelatin and glycerol or sucrose and the Radix Acaciae senegalis.

The liquid dosage form that is used for oral administration can comprise pharmaceutically acceptable Emulsion, solution, suspending agent, syrup and elixir, its contain be usually used in this area diluent such as water.Such compositions also can comprise, for example, and wetting agent, emulsifying agent and suspending agent and sweeting agent, correctives and aromatic.

The example of the liquid dosage form that is fit to comprises, but be not limited to aqueous pharmaceutical, it comprises the soluble derivative of reactive compound and beta-schardinger dextrin-or beta-schardinger dextrin-such as sulfobutyl ether beta-schardinger dextrin-, heptyl-2,6-two-O-methyl-beta-schardinger dextrin-, hydroxypropyl-beta-schardinger dextrin-and DM-.

Also can give Pharmaceutical composition of the present invention by injection (vein, muscle, subcutaneous).Injectable compositions like this can be used, and for example, saline, glucose or water are as the carrier mass that is fit to.If desired, available suitable acid, alkali or buffer agent are regulated the pH value of compositions.The extender (bulking), dispersant, wetting agent or the suspending agent that are fit to comprise that mannitol and Polyethylene Glycol (as PEG400) also can be included within the compositions.Be suitable for parenteral compositions and also can be included in freeze dried reactive compound in the injection vials.Can before injection, add aqueous solution dissolving said composition.

Can suppository etc. form give Pharmaceutical composition.Such rectum for example preferably contains total amount with preparation, and about 0.075 to about 75%w/w or about 0.2 to about 40%w/w or about reactive compound of 0.4 to about 15%w/w.Carrier mass such as cocoa butter, cupu oil (theobromaoil) and other oil and Polyethylene Glycol suppository base can be used for such compositions.If desired, also can use other carrier mass such as coating material (for example, HYDROXY PROPYL METHYLCELLULOSE film coating) and disintegrating agent (for example, cross-linked carboxymethyl cellulose sodium and polyvinylpolypyrrolidone).

In colloid drug delivery system such as liposome, microemulsion and huge Emulsion, this chemical compound can be free shape or be encapsulated in the microcapsule.

Can prepare these Pharmaceutical compositions by any suitable pharmaceutical methods, described method comprises reactive compound of the present invention and the blended step of one or more carrier mass.In a word, compositions is that reactive compound and liquid or pulverizing solid carrier or both are mixed equably and nearly, then, if desired, is molded into product.For example, can choose wantonly and suppress or the molded tablet for preparing by powder or granule described chemical compound with one or more auxiliary elements.Can such as powder or graininess chemical compound, choose wantonly and mix by with free-pouring form, on the machine that is fit to, suppress, the preparation compressed tablets with binding agent, lubricant, inert diluent and/or surfactant/dispersant.Can be by will be with the moistening powder compounds of inert liquid diluent, mold pressing on the machine that is fit to, preparation molded tablet.

Also available conventional coating material is such as Opadry ^TMWhite YS-1-18027A (or another color) tablet of the present invention is carried out coating, and the wt part of coating can be the overall budget garment piece heavy about 3%.Can be by using methods known in the art, the preparation present composition is so that provide compositions quick, that continue or delay to discharge after giving the patient.

When excipient was used as diluent, it can be solid, semisolid or liquid substance, the solvent of its atom active component, carrier or medium.Thereby compositions can be the form of the powder of tablet, chewable tablet, pill, powder, lozenge, sachet (sachets), cachet cachets, elixir, suspending agent, Emulsion, solution, syrup, aerosol (as solid or in liquid medium), soft hard gelatin capsule and sterilization packaging.

In one embodiment of the invention, preparation process can adopt the combination of following a kind of method or several method, and described method comprises: (1) is done and is mixed; (2) directly compacting; (3) pulverizing, (4) dry method or non-water are granulated, (5) wet granulation or (6) fusings (fusion).Lachman etc.. The Theory and Practice of Industrial Pharmacy(1986).

In another embodiment of the invention, mix with pharmaceutical excipient by making therapeutic agent of the present invention, form the solid preformulation composite of the homogeneous mixture that contains medicine and excipient, preparation solid composite, for example tablet.When mentioning that these pre-preparation compositions are uniform, it means therapeutic agent dispersion equably in whole compositions, and compositions can be easy to be divided into effective unit dosage forms again like this, such as tablet, pill and capsule.Then this solid preformulation is subdivided into the unit dosage forms of type described herein.

Compressed tablets is for containing active component and selecting to help to process and improve the solid dosage forms that the prescription of the excipient of product property prepares by compacting.Term " compressed tablets " refers generally to be used for the plain sheet of orally ingestible, not coated tablet, gently presses after (pre-compaction tapping) again through final compacting preparation by single compacting or by preloading method.

Can carry out coating or carry out other and mix tablet of the present invention or pill, with the dosage form of advantage that the administered over prolonged effect is provided.For example, tablet or pill can comprise internal layer dosage and outer dose components, and the latter is the form of coating that the former is carried out.Multiple material can be used for such enteric layer or coating, comprises many polymeric acid and polymeric acid and such material such as the mixture of lac, hexadecanol and cellulose acetate.

Use long-term continuing and discharge implant, can be suitable in needs give the patient of the present composition continuously, treating mitochondrial disorders.As discharging, be meant that implant is through making up and arrange with the active component that transmits treatment level at least 30 days, preferred 60 days at " for a long time " used herein.The long-term implant that continues to discharge is familiar with by those of ordinary skills, and comprises delivery systems more described above.

In another embodiment of the invention, the chemical compound that is used for the treatment of mitochondrial disorders is to contain one or more the present invention and treat the kit of chemical compound or the form of packing material presents.These the present invention treat chemical compound can kit or the packaged of package, wherein per hour, every day, weekly or the dosage in every month (or other cycle) be arranged for suitable sequential ground or administration simultaneously.The present invention also provides and contains a large amount of kit or the packing materials of dosage unit packing, that be suitable for continuous administration every day respectively, and wherein each dosage unit comprises that at least a the present invention treats chemical compound.This drug delivery system can be used for making that to give the embodiment that any multiple the present invention treats chemical compound easy.In one embodiment, this system contains a large amount of every days or the dosage of administration weekly.Described kit or packing material also can contain and be used for conjoint therapy so that the suitable administration of dosage form is easy to medicine.Kit or packing material also comprise the cover operation instructions at the patient.

The present invention will wherein use following abbreviation by being further described with reference to following detailed embodiment:

AChEI, acetylcholinesteraseinhibitors inhibitors; A β, amyloid-beta peptide; ADDLs, the dispersivity part of amyloid derivation; ANT, acenine nucleotide translocase; APP, amyloid precursor protein; CCCP, 3-chlorophenyl hydrazone carbonyl cyanide; FCCP is right-Trifluoromethoxyphen-l hydrazone carbonyl cyanide; CsA, cyclosporin A; MPT, the membrane permeability conversion; MRC, the mitochondrial respiratory coefficient; PAO, phenyl arsenoxide; SP-233, (22S, 25S)-(20S)-spiral shell steroid-5-alkene-3 beta-yl alkyl caproate; SP-233, (22R, 25R)-(20 α)-spiral shell steroid-5-alkene-3 beta-yl alkyl caproate.

Mitochondrion permeability conversion (MPT) can be further defined as the permeability that non-specific increase occurs in the mitochondrial inner membrane under the unfavorable conditions, the mitochondrion Ca during such as increase mitochondrial matrix or oxidative stress ^{+ 2}Content and cause the assembling (unlatching) in the non-specific hole (" large channel ") in the mitochondrial inner membrane causes mitochondrial membrane potential disappearance, mitochondrial respiratory uncoupling and cellular energy depletion, and all these can cause cell death.

Embodiment 1. uses SP-233 protection mitochondrial function

A. material and method

1. material

A β _1-42Peptide available from American Peptide Company (Sunnyvale, CA, USA) and be stored in-80 ℃.(Eugene, OR is USA) with anti-A β available from Molecular Probes for anti--complex II antibody _1-42Antibody from Signet Laboratories (Dedham, MA, USA).The spiral shell stenol, (22S, 25S)-(20S)-spiral shell steroid-5-alkene-3 beta-yl alkyl caproate (SP-233) available from Interbioscreen (Moscow, Russia).Cyclosporin A (CsA) and 3-chlorophenyl hydrazone carbonyl cyanide (CCCP) derive from Sigma (St.Louis, MO, USA).Dulbecco ' s minimum essential medium (DMEM), hyclone (FBS) and SK-N-AS human neuroblastoma cell available from ATCC (Manassas, VA, USA).

2. mitochondrion separation method

Use the male Long-Evans rat of body weight 230-280g.After Mus beheaded, take out brain rapidly and on ice contain the 6ml dissociating buffer (under 4 ℃, TRIS 20mM, sucrose 250mM, KCl 40mM, EGTA 2mM, bovine serum albumin 1mg/ml; PH7.2) Potter-Elvejhem homogenizer (Eurostar, IKA-Verke, Staufen, Germany) middle homogenate.With homogenate in the centrifugal 8min of 2000xg, to remove cell debris and nucleus.To contain mitochondrial supernatant in the centrifugal 10min of 12000xg.Precipitate is suspended in breathing buffer (D-mannitol 300mM, KCl 10mM, the KH of 300 μ l ₂PO ₄10mM, MgCl ₂5mM; PH7.2 is in 37 ℃) in.The highly enriched mitochondrion of this precipitate (Zini etc. 1996).Measure protein concentration in the mitochondrial suspension with the method for Lowry etc. 1951.

3. the preparation of different solutions

Prepare A β again with fresh distilled water _1-42Solution is to provide the concentration of 500 μ M.A β _1-42As the fresh liquid of preparation again or in 4 ℃ of aggregated forms of hatching after solution 48h carries out " wearing out ".With preceding two kinds of solution are diluted with breathing buffer (seeing above-mentioned), directly add in the mitochondrion with the concentration of wanting then.In experiment, carry out (stating as follows), only use the fresh A β of preparation again with neuroblastoma cell _1-42Solution.At first SP-233, CCCP and CsA are dissolved in N, dinethylformamide (DMF) is used fresh distilled water diluting then.

4. monitoring mitochondrial respiratory

Adopt the Oxytherm (Hansatech, Norfolk, UK) the research mitochondrial function that connect PC (Compaq, PIII 400MHz).37 ℃ of constant temperature camera incubatas of Oxytherm are connected to platinum-Yin Clarck electrode are used for O ₂Detect.Exist or lacking under CCCP (uncoupling agents of oxidative phosphorylation) or the CsA (inhibitor in MPT hole), monitoring in the presence of ADP (precursor of ATP) as the O of the labelling of mitochondrial function and survival ability ₂Consume.O ₂The detection that consumes allows to calculate mitochondrial respiratory coefficient (MRC), and it is V ₃/ V ₄V ₄Be basic O ₂Consume V ₃Be the O behind the adding ADP (ATP generation) ₂Consume.By the differentiation of monitoring MRC, the SP-233 (1pM to 1nM) of assessment progressive concentration is to the effect of mitochondrial respiratory chain.Use to breathe buffer agent with the mitochondrion concentration adjustment to 0.4mg protein/ml.By adding the substrate activation mitochondrion of malate/glutamate, Glu as the complex I of respiratory chain.State 2 is the baseline rate of mitochondrion oxygen consumption.When synthesizing by adding ADP startup ATP, the speed of oxygen consumption increases the state of reaching 3 significantly.When the supply of ADP lacks, O ₂The speed that consumes is got back to baseline (state 2), promptly is called as state 4 (Chance and Williams 1956).Base respiration under the contrast of each experiment exists corresponding to ADP.

5.SP-233 the effect of the oxidative phosphorylation uncoupling that CCCP-is caused

Adding CCCP (1 μ M) makes respiratory chain reaction uncoupling and increases O ₂Consume.In order to assess the uncoupling that SP-233 causes CCCP-, under 37 ℃, the mitochondrion fragment is hatched 3min in the presence of SP-233, add malate/glutamate, Glu afterwards, then add CCCP behind the 1min.

6.SP-233 the assessment of the anoxia functions that CsA-is caused

Cause anoxia by adding CsA (1 μ M) to bathing foster mitochondrial hatching in the culture medium (Zini etc. 1996).By adding ADP under the SP-233 and carry out this controlled trial having CsA and lack.As described above, at first add SP-233 or its solvent and hatch 1.5min, add CsA subsequently.Then, add substrate malate/glutamate, Glu behind the 1.5min, add ADP behind the 2.5min.

7.SP-233 antagonism A β _1-42The assessment of the protective effect that-mitochondrial function that causes is incomplete

In 37 ℃, make SP-233 be present in 3min and A β in the mixtures incubated _1-42Middle 1.5min adds substrate malate/glutamate, Glu subsequently.Add ADP after adding malate/glutamate, Glu 1min.SP-233 is to A β _1-42The control experiment of effect the same with the experiment of when lacking ADP, implementing.

8.SP-233 antagonism A β in the human neuroblastoma cell _1-42The assessment of-toxic the protective effect that causes

With human neuroblastoma SK-N-AS cell inoculation in the 96-of the DMEM that contains 10%FBS orifice plate (7 * 10 ⁴Individual cells/well) in, and in 5%CO ₂With cultivate under 95% humidity.Add A β then _1-42(0.1,1 and 10 μ M) or its solvent and making is incubated in to exist or lack SP-233 (1 μ M) and continues 72h down.Use bromination 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium  (MTT) analyzes (Trevigen, Gaithersburg, MD) cytotoxicity of assessment A β.

9. the human neuroblastoma cell mitochondrial absorbs A β _1-42Immunocytochemical assay

Human neuroblastoma SK-N-AS cell inoculation is gone up and in 37 ℃ 5%CO in 13-mm diameter coverslip (20,000 cell/coverslipes) ₂, overnight incubation in the DMEM that contains 10%FBS.Add A β then _1-42(10 μ M) or its solvent, and under existence or shortage SP-233 (1 μ M), continue to hatch 3h.For the evaluation line plastochondria to A β _1-42Picked-up, use anti--oxidative phosphorylation (OxPhos) complex II 70-kDa subunit mouse monoclonal antibody that the complex II of antagonism mitochondrial respiratory chain produces (Molecular Probes, Eugene, OR, USA) and antagonism A β _1-42The rabbit polyclonal antibody that produces of amino acid residue 33-42 (MA USA), carries out common position property (co-localization) research for SignetLaboratories, Dedham.Neuroblastoma cell with phosphoric acid-buffer saline (PBS) 1X washing, is fixed and uses PBS 1 X rinsing in-20 ℃ with methanol.With the PBS 1 X closing cell 30min that contains 10% donkey serum, in the PBS 1 X solution that contains 1.5% donkey serum and 0.01% trinitrotoluene (Triton) X100, hatch 24h in 4 ℃ of first primary antibodies that produce with antagonism complex II (1/200) then then.After washing this prepared product 3 times with PBS 1 X, the donkey that adds 1/200 times of dilution is anti--and the secondary antibodies of mice brilliant pink b (rhodamine) labelling (JacksonImmunoResearch, West Grove, PA, USA) and under room temperature, continue to hatch 2h.Use the antagonism A β of 1/500 times of dilution then according to identical scheme _1-42The primary antibody that produces of aminoacid 33-42.Use is with fluorescent marker Alexa Fluor  488 (Molecular Probes, Eugene, OR, USA) donkey of labelling-anti--rabbit secondary antibodies demonstration positive staining.Then with contain 4 ', (VectorLaboratories.Burlingame, CA is USA) with the coverslip mounting for the aqueous mountant of 6-diamidino-2-phenylindone-2-hydrochlorate (DAPI).Use OlympusFluoview BX61 laser scanning microscope to take confocal images.

10. statistical analysis

For each experiment, its result represents with mean value SD.By do behind the ANOVA Dunnett ' s check organize between relatively.

B. result

1.SP-233 effect to mitochondrial respiratory control

In 10,30 and the concentration studies SP-233 of 100pM.Compare with matched group, under the concentration that is low to moderate 10pM, SP-233 causes that 50% of the remarkable meaning of rat brain mitochondria MRC tool reduces (p＜0.001) (Fig. 1).Though not remarkable by the difference between the effects that the SP-233 of multiple concentration produces, the result also is not inclined to display density-effect relation, because MRC is the reduction of carrying out property in the 10-100pM concentration range.

2.SP-233 the effect of the mitochondrial respiratory coefficient correction that CCCP-or CsA-are caused

Uncoupling agents CCCP increases rat brain mitochondria O in 1 μ M ₂(Fig. 2 a) to be consumed to 150% (p＜0.01) of basic value.The SP-233 of all test concentrations (1-1000pM) suppresses this metabolism (p＜0.001) of CCCP fully.Uncoupling that the inhibition CCCP-that is produced by SP-233 causes and reduction O ₂Consumption figures is relevant to and be non-concentration-dependence the 60-65% of foundation level.SP-233 does not offset the MRC that is caused by CsA and reduces, and opposite, it strengthens this effect (Fig. 2 b) of CsA in the mode of concentration-dependence.

3. fresh and aged A β _1-42Effect to the mitochondrial respiratory coefficient

A β _1-42Significance ground reduces the MRC of rat brain mitochondria, even the concentration of its existence is low to moderate 0.1pM (Fig. 3).Adopt this effect of fresh 4 amyloid more remarkable than aged form, its inhibitory action is respectively 55-63% and 43-53%.This difference can show the A β of non--aggregated forms _1-42Peptide than aged aggregated forms penetrates into mitochondrion to a greater degree.

4.SP-233 to fresh and aged A β _1-42The effect of the correction of the mitochondrial respiratory coefficient that causes

Compare fresh A β with matched group _1-42Make MRC reduce by 71% and this effect suppressed partly by SP-233 that (Fig. 4 a).In the SP-233 (1pM) of least concentration of test, MRC returns to the about 40% of control value, and with single A β that uses _1-42The value of gained compares, and MRC significance ground increases (28.93 ± 1.75 pairs 38.56 ± 0.34, p＜0.001).Compare aged A β with matched group _1-42Make MRC reduce by 51%, but not significance ground prevention this effect (Fig. 4 b) of SP-233.

5.SP-233 to A β _1-42-the toxic effect that causes to SK-N-AS human neuroblastoma cell

Fig. 5 shows that SP-233 is to A β _1-42The effect that the SK-N-AS cell viability that causes reduces.SP-2331 μ M prevention is by used A β _1-42Three neurotoxicityes that concentration causes, it reduces by 19%, 26% and 28% (p＜0.01) respectively.

6.SP-233 to A β _1-42Enter the effect of SK-N-AS human neuroblastoma cell mitochondrial

Use A β _1-42The neuroblastoma cell of handling shows complex II strong and mitochondrial respiratory chain localized immunoreactivity altogether, and A is provided β _1-42Enter mitochondrion and be present in Intramitochondrial evidence.Handle elimination A β with SP-233 _1-42Immunoreactivity shows its blocking-up A β _1-42Enter cell and mitochondrion.

C. discuss

Having known AD is Clinical symptoms with the damage of carrying out property and the loss of memory of cognitive process.Very early, " memory (mnesic) " aspect of this disease has related to the degeneration of central cholinergic system path, is reflected as the reduction of synapse acetylcholine (ACh) concentration.Thereby drug development mainly concentrates on the exploitation that makes ACh level in the brain recover and cause acetylcholinesteraseinhibitors inhibitors (AChEIs), and tacrine is the prototype of this compounds.Yet, no matter how clinical data is hopeful, the beneficial effect appropriateness of tacrine, no matter and in order to the exploitation of bright (rivastigmine) that cut down this, galantamine and donepezil (donepezil) for the AChEIs of new generation of representative, compare with tacrine, the time that they postpone the generation of AD patient's symptom does not shorten.(1 year or 2 years) of this weak point postpones (Tariot and Winblad, 2001 in the AD of faster progress symptom; Waldemar etc. 2001; Grossberg etc. 2004), although for the patient with and relatives very precious, probably since cholinergic nerve carry out sexual involution, thereby limited the application of AChEIs.Yet, in the recent period 565 live in community slightly implement to the moderate AD patients studies show that donepezil is not to be cost-effective, its interests are lower than MIN respective threshold (Courtney etc. 2004).Because the AD drug development does not make substantial progress, even the Memantine hydrochloride (memantine) of N-methyl-D-aspartate salt (NMDA) receptor antagonist gets permission to be used for the treatment of moderate in the recent period to severe AD (Livingston and Katona 2004), still have the eager demand of exploitation than the effective more medicine of AChEIs.

Only carried out sufficient demonstration at the cause of disease of the AD of the early onset thereof form of familial disease, this disease relates to APP, the sudden change of old and feeble albumen (presenilin)-1 or old and feeble albumen-2 gene.On the contrary, the reason of representing the tardy property of AD of about 95% case of all AD to distribute form waits to determine.The energy decline of mitochondrion infringement and oxidative damage many theories occur and wherein has proposing to think, owing to may be that (Schulz etc. 1997 for the cause of AD; Beal, 2000).

It is incomplete to evidence show that the AD etiology relates to mitochondrial function, and but associated is to find SP-233 (a kind of naturally occurring spiral shell stenol) in the recent period the neuroprotective PC12 of unit cell antagonism A β _1-42-the neurotoxicity (Lecanu etc. 2004) that causes, this causes the inventor to check that SP-233 recovers because of being exposed to A β _1-42The ability of the isolating mitochondrial function that is weakened.In a word, this discovery points out that SP-233 has the potentiality of the influence fundamental mechanism relevant with A β, and it has related to the pathogeny of AD.

In past 10 years, A β is to the existing extensive studies of the effect of mitochondrial function, and the data of these research generations has been pointed out the incomplete multiple effect of caused mitochondrial function of this peptide.Be presented at A β in the neuron linear plastochondria _1-42With Segment A β _25-35Suppress to breathe and activate key enzyme, (Kaneko etc. 1995 such as succinate dehydrogenase, ketoglurate dehydrogenase, pyruvic dehydrogenase and cytochrome oxidase; 2002a such as Casley).Report is also arranged, be exposed to A β _25-35The neuron linear plastochondria in, the inhibitory action of different composite body of respiratory chain takes place, and (Pereira etc. 1998; Canevari etc. 1999; 2002b such as Casley) and this identical fragments of peptides promote the hole-unlatching relevant (Moreira etc. 2001 with MPT; Moreira etc. 2002; Bachurin etc. 2003).

The The above results that is presented on herein also shows, as shown in reducing as MRC, and A β _1-42The mitochondrial respiratory that suppresses rat brain mitochondria, and with the A β that is low to moderate 0.1pM _1-42The prepared fresh of concentration with peptide ageing form this inhibitory action (see figure 3) takes place all.Have more significant inhibitory effect with fresh form, this may be because A β _1-42The poly form, such as ADDLs, will cross over mitochondrial membrane in less degree ground compared with monomeric form or oligomeric form, so these monomers or oligomeric form more may be brought into play its illeffects and in fact have more toxicity to respiratory chain.Also have, lack SP-233 aged to standing/the A β of aggregated forms _1-42Mitochondrial protective effect (see figure 4) may be because SP-233 can not be with in conjunction with the same manner of monomeric form aggregated forms in conjunction with 4 amyloid.

SP-233 energy partial prophylaxis is by the A β of prepared fresh _1-42The reduction of the MRC that causes and protection mitochondrial function, the data before making it are confirmed that this data data shows that SP-233 is to being exposed to A β _1-42The synthetic restitution of ATP (Lecanu etc. 2004) of PC12 cell.Because SP-233 has activity in low-down concentration (picomole scope), its mechanism of action possibility anhydride relative specificity.This data is also confirmed result recently, and described result shows that SP-233 passes through the formation of the monomeric form and the inhibition neurotoxicity oligomer ADDLs of binding peptide, and neuroprotective unit cell is with antagonism A β _1-42Neurotoxicity (Lecanu etc. 2004).

Showing A β _1-42In locating altogether with the complex II of respiratory chain, A β is pointed out in the burnt microscopic analysis of the copolymerization of implementing on the human neuroblastoma cell _1-42Can cross over the film of cell and mitochondrion.As SP-233 and A β _1-42All be present in when hatching in the culture medium, corresponding to the immunochemistry of 4 amyloid dyeing be suppressed fully and this " removings " effect of SP-233 with the part recovery of MRC.In addition, though SP-233 is partly, prevention A β _1-42Neurotoxic effect to identical human neuroblastoma cell.This result has special meaning, because recent result points out, amyloid deposition is present in the mitochondrion of the taper cortical neuron of AD brain after death (Fernandez-Vizarra etc. 2004).These histological characteristics have been described to incident very early stage in the progression of disease, and it neuronal death occurred and caused before Double helix fiber (filaments) forms.

SP-233 is to A β _1-42Combination and " removings " effect definite after, studied SP-233 and can cause extra antagonism A β mitochondrial direct effect _1-42The probability of protective effect.In this, obtained proof, A β promotes the unlatching in the MPT hole in the brain mitochondria before, causes the mitochondrial function infringement, and the effect of being somebody's turn to do is eliminated by CsA, and (Moreira etc. 2001; Bachurin etc. 2003; Abramov etc. 2004).This experiment shows that as CsA SP-233 reduces MRC.SP-233 also strengthens the effect of CsA, point out its have CsA-sample characteristic and thereby suppress the unlatching in MPT hole, can help its antagonism A β _1-42-infull the protective effect of mitochondrial function that causes.

SP-233 can also eliminate the oxidative phosphorylation uncoupling that is caused by CCCP and (see Fig. 2 a).Show A β _1-42(Kremer etc. 2001 for failure line plastochondria membrane structure; Rodrigues etc. 2001) and in the AD brain flowability of mitochondrial membrane can change (Mecocci etc., 1996), two phenomenons all cause the mitochondrion uncoupling.Therefore, although SP-233 " coupling again (recoupling) " effect still fails to illustrate fully, it can be made contributions to the recovery of mitochondrial function described herein.In addition, the scavenging action of SP-233 (supposition relates to its modulation to the MPT hole) and again coupled action unite with soluble why SP-233 offsets A β under the concentration that is low to moderate 1pM _1-42Effect.

Embodiment 2. spiral shell stenols (22R, 25R)-20 α-spiral shell steroid-5-alkene-3 beta-yl alkyl caproate blocking-up The mitochondrial function infringement of mitochondrion picked-up A β and prevention A β in the neuronal cell-cause

A. material and method

1. material

A β _1-42Peptide available from American Peptide Company (Sunnyvale, CA).(Eugene is OR) with anti--A β available from Molecular Probes for anti--complex II antibody _1-42Antibody from Signet Laboratories (Dedham, MA).Spiral shell stenol (SP-233) available from Interbioscreen (Moscow, Russia).Cyclosporin A (CsA), 3-chlorophenyl hydrazone (CCCP) carbonyl cyanide, right-trifluoromethoxy-phenyl hydrazones carbonyl cyanide (FCCP), rotenone, malonate, sticking thiazole (myxothiazol), potassium cyanide (KCN), oligomycin and phenyl arsenoxide (PAO) derive from Sigma (St Louis, MO).Dulbecco ' s minimum essential medium (DMEM), hyclone (FBS) and SK-N-AS human neuroblastoma cell available from ATCC (Manassas, VA).

2. the segmental separation of mitochondrion

For the separate mitochondria fragment, the rat of body weight 230-350g is beheaded, take out brain rapidly and containing the ice-cold dissociating buffer of 6ml (TRIS 20mM, sucrose 250mM, KCl 40mM, EGTA 2mM, bovine serum albumin 1mg/ml on ice; PH7.2) Potter-Elvejhem homogenizer (Eurostar, IKA-Verke, Staufen, Germany) middle homogenate.In 4 ℃ with homogenate in the centrifugal 8min of 2000xg, to remove cell debris and nucleus.To contain mitochondrial supernatant in the centrifugal 10min of 12000xg in 4 ℃.Precipitation is suspended in breathing buffer (D-mannitol 300mM, KCl 10mM, the KH of 300 μ l ₂PO ₄10mM, MgCl ₂5mM; PH7.2 is in 37 ℃) in.This precipitates highly enriched mitochondrion (Zini etc. 1996).With the protein concentration in the method mensuration mitochondrial suspension of Lowry.

3. mensuration mitochondrial respiratory

(Hansatech, Norfolk UK) measure mitochondrial function to adopt the Oxytherm that connects PC (Compaq, PIII 400MHz).37 ℃ of constant temperature camera incubatas of Oxytherm are connected to platinum-Yin Clarck electrode are used for O ₂Detect.Use O ₂Consume as in the presence of ADP (precursor of ATP), and have or lack CCCP (uncoupling agents of oxidative phosphorylation) or CsA (inhibitor in the membrane permeability conversion hole) labelling of mitochondrial respiratory down.O ₂The detection that consumes allows to calculate mitochondrial respiratory coefficient (MRC), is defined as V ₃/ V ₄, V wherein ₄Be basic O ₂Consume V ₃Be the O behind the adding ADP (ATP generation) ₂Consume.Use the breathing buffer agent that the mitochondrion final concentration is adjusted to 0.4mg protein/ml.Malate/glutamate, Glu of the substrate of complex I by adding respiratory chain activates mitochondrion.Definition status 2 is the baseline rate of mitochondrion oxygen consumption.When synthesizing by adding ADP startup ATP, the speed of oxygen consumption increases the state of reaching 3 significantly.When the supply of ADP lacks, O ₂The speed that consumes is got back to baseline (state 2), promptly is called as state 4 (Chance and Williams 1956).All are all breathed with baseline the influence of the experimental processing of breathing or only exist the mitochondrial respiratory under the ADP to make comparisons.

In order to test the mitochondrial function incomplete effect of SP-233 to causing by CCCP, in 37 ℃, isolating mitochondrion is hatched 3min in SP-233, add malate/glutamate, Glu afterwards.After one minute, add CCCP.In order to test the mitochondrial function incomplete effect of SP-233 to causing by CsA, before adding CsA, isolating mitochondrion is hatched 1.5min in SP-233, add ADP after adding malate/glutamate, Glu and 2.5min behind the 1.5min.At last, in order to test SP-233 to A β _1-42-infull the influence of mitochondrial function that causes in 37 ℃, is hatched 3min with the mitochondrion fragment and in A β in SP-233 _1-42In hatch 1.5min, add malate/glutamate, Glu subsequently.After adding malate/glutamate, Glu 1min, add ADP.In all experiments, SP-233, CCCP and CsA at first are dissolved in N, in the dinethylformamide (DMF), use fresh distilled water diluting then.The A β that in these experiments, uses _1-42Through be formulated as concentration 500 μ M again with fresh distilled water.A β _1-42Prepare liquid again or " wear out " and afterwards exist making this solution hatch 48h as fresh with aggregated forms in 4 ℃.Dilute this two kinds of solution with the breathing buffer agent before using, directly be added to the concentration that reaches required in the mitochondrion then.

4. cell culture

With human neuroblastoma SK-N-AS cell inoculation in the 96-of the DMEM that contains 10%FBS orifice plate (7 * 10 ⁴Individual cells/well) in, and in 5%CO ₂With cultivate in 95% humidity.There is or do not exist a processing culture down with the SP-233 of following variable concentrations: A β _1-42(0.1,1 and 10 μ M), rotenone (50nM), malonate (100mM), sticking thiazole (0.3 μ M), KCN (12mM), oligomycin (0.5 μ g/ml), FCCP (3 μ M) and PAO (0.25 μ M).(description MD) is used bromination 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium  (MTT) analyzing and testing cytoactive for Trevigen, Gaithersburg according to the preparation merchant.In order to relate to A β _1-42Experiment, with 1 μ M A β _1-42Or without A β _1-42Handle (insult) and measure cell viability after 72 hours.Only use the fresh A β of preparation again _1-42Solution.In order to relate to the experiment of mitochondrion toxin (rotenone, malonate, sticking thiazole, KCN and oligomycin), there are or lacking 1,3,10,30 or 100 μ M A β _1-42Under handle 6 or detect cell viability after 24 hours.Add mitochondrion toxin to culture and added SP-233 in 1 hour before.In order to comprise the experiment of FCCP and PAO, adding FCCP or PAO previous hour, with or need not 1 to 100 μ M scope in the SP-233 of progressive concentration hatch parallel cell culture.Assess cell viability after 24 hours.At first with FCCP, rotenone, malonate, sticking thiazole, KCN, oligomycin and PAO all are dissolved in the ethanol as stock solution, reach specified final concentration with the culture medium dilution afterwards.Ethanol percentage rate in the culture medium is 0.09%.

5.A β _1-42Immunochemical analyses

Human neuroblastoma SK-N-AS cell inoculation is gone up and in 37 ℃ 5%CO in 13-mm diameter coverslip (20,000 cell/coverslipes) ₂, overnight incubation in the DMEM that contains 10%FBS.Existing or lacking under the SP-233 (1 μ M), use A β then _1-42(10 μ M) or its solvent are hatched culture 3h.For the evaluation line plastochondria to A β _1-42Picked-up, use anti--oxidative phosphorylation (OxPhos) complex II 70-kDa subunit mouse monoclonal antibody that the complex II by the antagonism mitochondrial respiratory chain produces (Molecular Probes, Eugene, OR) and antagonism A β _1-42The rabbit polyclonal antibody that produces of amino acid residue 33-42 (SignetLaboratories, Dedham MA), carry out common position Journal of Sex Research.Neuroblastoma cell with phosphoric acid-buffer saline (PBS) washing, is fixed and uses the PBS rinsing in-20 ℃ with methanol.Then with the PBS closing cell 30min that contains 10% donkey serum, then in 4 ℃ of antibody incubation 24h that produce with antagonism complex II (1: 200 in the PBS that contains 1.5% donkey serum and 0.01%Triton X100).Use the PBS washed cell then, with donkey anti--secondary antibodies of mice brilliant pink b labelling (1: 200; Jackson ImmunoResearch, West Grove PA) is hatched 2h under room temperature.Use antagonism A β then _1-42The antibody that produces of aminoacid 33-42 (1: 500) and with fluorescent marker Alexa Fluor  488 (OR) donkey of labelling-anti--rabbit secondary antibodies is then carried out identical sequence step for Molecular Probes, Eugene.With contain 4 ', 6-diamidino-2-phenylindone-2-hydrochlorate (DAPI; Vector Laboratories, Burlingame, aqueous mountant CA) is with the coverslip mounting.Use Olympus Fluoview BX61 laser scanning microscope picked-up confocal images.

6. statistical analysis

All results represent with mean value SD.Adopt variation analysis (ANOVA), compare between organizing with Dunnett ' s check subsequently.P value＜0.05 is considered to have significance.

B. result

1.SP-233 the effect that the mitochondrial respiratory that CCCP-or CsA-are caused changes

At first, by the differentiation of monitoring MRC, SP-233 is in the effect (Fig. 6) of concentration range 1fM to 100pM to mitochondrial respiratory in assessment.The SP-233 of progressive concentration causes the reduction of carrying out property of MRC.Compare with matched group, when concentration is low to moderate 1nM, remarkable reduction (p＜0.001 of the MRC of the rat brain mitochondria that SP-233 causes; Fig. 6 b).SP-233 concentration causes that in 10pM or when being higher than 10pM MRC reduces by 50%.

CCCP makes the oxidative phosphorylation uncoupling and increases O ₂Consume.Shown in Fig. 7 a, 1 μ MCCCP increases rat brain mitochondria O ₂Be consumed to 150% (p＜0.01) of basic value.The SP-233 of 1-1000pM scope concentration eliminates this effect (all being p＜0.001) fully, and in the presence of the SP-233 of all these concentration, the inhibitory action of the uncoupling that CCCP-causes and O ₂It is relevant that consumption figures is reduced to the 60-65% of foundation level.

Hypoxia causes (Zini etc. 1996) by adding 1 μ M CsA to bathing foster mitochondrial culture medium.SP-233 does not offset the MRC that is caused by CsA and reduces.On the contrary, its mode with concentration-dependence strengthens this effect (Fig. 7 b).

2.SP-233 to by fresh and aged A β _1-42The effect that the mitochondrial respiratory that causes changes

With A β _1-42Be added in the isolating mitochondrion, even the A β of 0.1pM concentration _1-42Significance ground reduces MRC (Fig. 8).Adopt fresh 4 amyloid than the peptide with ageing form, this effect is more remarkable; The former inhibitory action is than the latter big approximately 10%.Can infer the A β of non--aggregated forms _1-42Penetrate in the mitochondrion to a greater degree than peptide aged, aggregated forms.Shown in Fig. 9 a, with fresh A β _1-42Be added to isolating mitochondrion and cause MRC to be reduced to 71% of control value, and this reduction is partly reversed by SP-233.The SP-233 of least concentration (1pM) after tested, MRC returns to about 39% of control value.With single A β that uses _1-42The value of gained compares, and MRC significance ground increases (28.93 ± 1.75 pairs 38.56 ± 0.34, p＜0.001).Compare aged A β with matched group _1-42Make MRC reduce by 51%, but SP-233 does not influence this aged A β in significance ground _1-42The variation of the MRC that causes (Fig. 9 b).

3.SP-233 to A β _1-42-the toxic effect that causes to SK-N-AS human neuroblastoma cell

Figure 10 shows that SP-233 is to A β in the SK-N-AS cell _1-42-neurovirulent the effect that causes.At all three A β _1-42Under the existence of test concentrations, add 1 μ M SP-233 and cause cell viability significantly to increase.SP-233 reduces respectively by 0.1,1 and 10 μ M A β _1-42Cause neurovirulent 19% (p＜0.01 is with single A β that uses _1-42Relatively), 26% (p＜0.01) and 28% (p＜0.01).

4.SP-233 to A β _1-42Enter the effect of SK-N-AS cell mitochondrial

Figure 11 shows DAPI dyeing (a1, b1 and c1), A β in the SK-N-AS human neuroblastoma cell _1-42Immunofluorescence label (a2, b2 and c2) and the typical image of complex II 70-kDa subunit immunofluorescence label (a3, b3 and c3).(Merged) image that merges is shown in figure a4, b4 and c4.Use A β _1-42The neuroblastoma cell of handling shows strong A β _1-42Immunoreactivity (Figure 11 b2), the labelling of itself and mitochondrial respiratory chain complex II is located (Figure 11 b4) altogether, and A is provided β _1-42Enter mitochondrion and be present in Intramitochondrial evidence.A β _1-42Immunoreactivity is eliminated (Figure 11 c2) in the presence of SP-233, prompting SP-233 blocking-up A β _1-42Enter in cell and the mitochondrion.

5. the neuroprotective of SP-233 confrontation line plastochondria Complex Inhibition agent in the SK-N-AS cell

SP-233 does not show the neuroprotective (Figure 12 a, b and c) of complex I, II and the III inhibitor of any antagonism mitochondrial respiratory chain.SP-233 resists rotenone and 100 μ M SP-233 significance ground to anti-stick thiazole (myxoyhiazol) (both p＜0.05) in 1 μ M concentration significance ground.Although this protective effect is significant, the value of the effect in these two examples is little.In contrast, SP-233 shows the toxic beneficial effect (Figure 12 d and 12e) that significant antagonism is caused by complex IV inhibitor KCN and complex V inhibitor oligomycin.The SP-233 of low concentration (1,3 and 10 μ M) effectively resists KCN; And high dose (30 and 100 μ M) does not provide beneficial effect.The protective effect of SP-233 antagonism oligomycin is dosage-dependence, though 10 μ M SP-233 do not have proof to have the neuroprotective of remarkable meaning on the statistics.The inhibitory action of these digital proofs SP-233 protection SK-N-AS cell antagonism complex IV and V.

6.SP-233 neuroprotective to PAO in the SK-N-AS cell and FCCP

Acenine nucleotide translocase (ANT) is for being positioned at the channel protein of mitochondrial inner membrane, under physiological condition, with 1: 1 ratio output ATP and input ADP.PAO suppresses ANT, promotes the unlatching in permeability conversion hole, causes apoptosis.One micromole SP-233 reduces 25% (Figure 13, p＜0.01) of PAO to the toxic action of SK-N-AS neuronal cell.SP-233 and any neuroprotective greater than 1 μ M concentration are irrelevant.In addition, when the concentration that gives was 100 μ M, the uncoupling of the respiratory chain that SP-233 causes FCCP-showed little, but significant (p＜0.01) effect (Figure 14).

C. discuss

AD is a Clinical symptoms with the damage of carrying out property and the loss of memory of cognitive process.Very early, " memory (mnesic) " aspect of this disease is relevant with the degeneration of central cholinergic system path, is reflected as the reduction of synapse acetylcholine concentration (ACh).Thereby drug research concentrates on and makes ACh level recovery in the brain.And therefore cause the exploitation of acetylcholinesteraseinhibitors inhibitors (AChEIs).Though these medicines are nugatory for patient AD and theys' nursing staff, AchEIs only slows down disease process and does not stop symptomatic treatment medicine (Tariot and Winblad, 2001 of its development in limited one period; Waldemar etc. 2001; Grossberg etc. 2004).Even N-methyl-D-aspartate salt (NMDA) receptor antagonist Memantine hydrochloride has got permission to treat moderate in the recent period to severe AD (Livingston and Katona 2004), still press at the new medicine of the AD cause of disease and new therapeutic strategy.

The etiology of the family of AD disease, early onset thereof form is proved that fully it relates to the sudden change of APP (old and feeble albumen-1 or old and feeble albumen-2) gene.On the contrary, the reason of representing the tardy property of the AD of about 95% case of all AD to distribute form waits to determine.Many theories and some of them theory occurring, to have proposed energy decline owing to mitochondrion infringement and oxidative damage may be that (Schulz etc. 1997 for the cause of AD; Beal, 2000).The theory that this mitochondrial function is incomplete, with recent discovery be spiral shell stenol derivant, SP-233, but the neuroprotective PC12 of unit cell antagonism A β _1-42-the neurotoxicity (Lecanu etc. 2004) that causes causes checking whether SP-233 can recover to be exposed to A β together _1-42Isolating mitochondrial function.Result provided herein proves that in isolating rat brain mitochondria and in the human neuroblastoma cell, SP-233 all can protect mitochondrial function antagonism A β _1-42Toxic action.These observe the probability that prompting SP-233 has the relevant fundamental mechanism of the neurotoxicity of influence and A β-cause, and it has related to the pathogeny of AD.

In past 10 years, A β is to the existing extensive studies of the effect of mitochondrial function, and the data that these researchs produce points out that this peptide can cause the incomplete multiple effect of mitochondrial function.Be presented at A β in the neuron linear plastochondria _1-42With Segment A β _25-35Suppress to breathe and activate key enzyme, (Kaneko etc. 1995 such as succinate dehydrogenase, ketoglurate dehydrogenase, pyruvic dehydrogenase and cytochrome oxidase; 2002a such as Casley).Report is also arranged, be exposed to A β _25-35The neuron linear plastochondria in, (Pereira etc. 1999 for the inhibitory action of respiratory chain complex; Canevari etc. 1999; 2002b such as Casley), and this identical fragments of peptides promote the hole-unlatching relevant (Moreira etc. 2001 with MPT; Moreira etc. 2002; Bachurin etc. 2003).In addition, be presented on result's demonstration of this paper, shown as the MRC reduction, A β _1-42The mitochondrial respiratory that suppresses rat brain mitochondria.And this inhibitory action betides with the A β that is low to moderate 0.1pM prepared fresh and peptide ageing form _1-42In the reaction of concentration.May be, have more significant inhibitory effect, because A is β with fresh form _1-42The polymer form, such as ADDLs, will cross over mitochondrial membrane in less degree ground compared with monomeric form or oligomeric form, so these monomers or oligomeric form more may be brought into play its illeffects and in fact have more toxicity to respiratory chain.Also have, lack by SP-233 aged to being exposed to/the A β of aggregated forms _1-42Mitochondrial protection may be because SP-233 can not be with in conjunction with the same manner of monomeric form aggregated forms in conjunction with 4 amyloid.

Meaningfully, SP-233 energy partial prophylaxis is by the A β of prepared fresh _1-42The reduction of the MRC that causes, the demonstration SP-233 before making it is to being exposed to A β _1-42The result of the synthetic restitution of ATP (Lecanu etc. 2004) of PC12 cell be confirmed.The results suggest that SP-233 works in low-down concentration, its mechanism of action may be relative specificities.Data described herein is also confirmed result recently, and described result shows that SP-233 comes neuroprotective cell antagonism A β by the monomeric form of binding peptide and the formation of inhibition neurotoxicity oligomer ADDLs _1-42Neurotoxicity (Lecanu etc. 2004).

Showing A β _1-42In locating altogether with the complex II of respiratory chain, A β is pointed out in the burnt microscopic analysis of the copolymerization of implementing on the human neuroblastoma cell _1-42Can cross over epicyte and mitochondrial membrane.As SP-233 and A β _1-42All be present in when hatching in the culture medium, the 4 amyloid labelling is eliminated, and SP-233 this " removing " effect recovers with the part of MRC.In addition, SP-233 partial prophylaxis A β _1-42Neurotoxic effect to identical human neuroblastoma cell.This result has special meaning, because recent result shows, amyloid deposition is present in the mitochondrion of the taper cortical neuron of AD brain after death (Fernandez-vizarra etc. 2004).These histological characteristics have been described to incident very early stage in the progression of disease, and it neuronal death occurred and caused before the Double helix fiber forms.On this basis, as described herein, SP-233 makes A β _1-42Inactivation can make SP-233 be used for the reason more abundant (Lecanu etc. 2004) of AD treatment.

SP-233 is to A β _1-42Combination and " removings " effect definite after, checked SP-233 can cause extra antagonism A β to mitochondrial direct effect _1-42The probability of protective effect.In this, show that A β promotes the unlatching in the MPT hole in the brain mitochondria, cause the mitochondrial function infringement, and the effect of being somebody's turn to do is eliminated by CsA, and (Moreira etc. 2001; Bachurin etc. 2003; Abramov etc. 2004).This experiment discloses, and as CsA, SP-233 reduces MRC.SP-233 also strengthens the effect of CsA, points out it may have CsA-sample characteristic.Be its unlatching that can suppress the MPT hole, it can help to resist A β _1-42-infull the protective effect of mitochondrial function that causes.This hypothesis (Korge etc., 2001) is supported in the protective effect of the antagonism PAO of SP-233 (protein tyrosine phosphatase inhibitors of a kind of known promotion MPT).Thereby SP-233 makes the neuronal cell vigor recover 25% under the concentration of 1 μ M; Yet the SP-233 of higher concentration and protective effect are irrelevant.The effect that SP-233 opens the hole of MPT-association can via direct and/or indirect action (as, insert allosteric modification behind mitochondrial membrane by it) take place.

In isolating mitochondrion and in neuroblastoma cell, SP-233 can also eliminate the oxidative phosphorylation uncoupling that is caused by CCCP.Show A β _1-42(Kremer etc. 2001 for failure line plastochondria membrane structure; Rodrigues etc. 2001) and the AD brain in the flowability of mitochondrial membrane can change (Mecocci etc., 1996); Two phenomenons all cause the mitochondrion uncoupling.Therefore, although SP-233 " coupling again " effect still fails to illustrate fully, it can be made contributions to the recovery of mitochondrial function described herein.In addition, the scavenging action of SP-233 (supposition relates to its adjusting to the MPT hole) and again coupled action unite with soluble why SP-233 can offset A β under the concentration that is low to moderate 1pM _1-42Effect.

In order to attempt to determine other the target of SP-233 in mitochondrial respiratory chain, verified, SP-233 can neuroprotective blastoma cell antagonism complex I, the inhibitor of III, IV and V, but the inhibitor of non-confrontational complex II (Figure 15).In complex III and IV, observe the most significant effect, though SP-233 also shows the toxic little significant neuroprotective of resisting rotenone and sticking thiazole-cause respectively under the concentration of 1 μ M and 100 μ M.Antagonism KCN and oligomycin recover the value and antagonism A β of cell survival _1-42The relatively prompting of value, complex IV and V are the main target of the mitochondrial function infringement of 4 amyloid-cause.Discovery before these results have confirmed promptly shows Segment A β _25-35Suppress the complex IV (Canevari etc., 1999) in the isolating brain mitochondria.Yet, be identical authors report A β with described data forms contrast _25-35Not effect of other complex to respiratory chain.This notable difference can be interpreted as the difference of employed amyloid kind, and produces the β about A thus _25-35With A β _1-42The problem of the effect in the neuro pathology.

Because AD is the neurological sexual disorder that can not cure and because existing treatment (wherein great majority are alone based on suppressing AChE) has critical limitations, presses for more effectively medicine of exploitation.The micromolecule SP-233 of this paper report has shown that before it suppresses A β by the formation that stops ADDLs _1-42-the neurotoxicity that causes, the antagonism of protection brain mitochondria is exposed to A β _1-42Illeffects.The potential molecular mechanism of the neuroprotective of SP-233 may be, to small part be because its " removing " A β _1-42, its " anti--uncoupling " effect, the ability that it is opened at protective effect and the inhibition MPT hole thereof of mitochondrion complex IV and V.In a word, data provided herein verified and expanded before discovery, show that promptly SP-233 has anti--amyloid characteristic, and they mitochondrion and respiratory chain as A β _1-42Direct target with SP-233.

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All publications, patent and patent application are incorporated into this paper by reference. Although the present invention contacts its some preferred embodiment and is described in aforementioned specification, and in order to illustrate that purpose carried out detailed description, those skilled in the art should be appreciated that, do not deviating under the basic principle of the present invention, the present invention allows can have suitable variation to the modification of other embodiment and some details described herein.

Claims (31)

1. one kind comprises the mammiferous Therapeutic Method of suffering from mitochondrial disorders or being subjected to the mitochondrial disorders threat, described mitochondrial disorders is not central nervous system's a neuropathy, and described method comprises formula (I) chemical compound or its pharmaceutically acceptable salt that gives described mammal effective dose:

R wherein ₁, R ₂, R ₄, R ₇, R ₁₁, R ₁₂And R ₁₅Independent separately be hydrogen, choose wantonly insertion-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)-(C ₁-C ₈) alkyl; Hydroxyl, N (R ') ₂, carboxyl, oxo or sulfonic acid, R ₃Be hydroxyl, (C ₁-C ₈) alkoxyl, (C ₁-C ₂₂) alkyl CO ₂Or R ' O ₂C (CH ₂) _2-8CO ₂-, (C wherein ₁-C ₂₂) alkyl or (CH ₂) _2-8Can choose wantonly and comprise 1-2 CH=CH unit, a 1-2 OH and/or 1-2 epoxy substituent group, toluene-4-sulfonyl oxygen base or benzoyloxy; R ₆, R ₈, R ₉, R ₁₀, R ₁₃, R ₁₄, R ₁₆And R ₁₇Independent separately is hydrogen, (C ₁-C ₈) alkyl, hydroxyl (C ₁-C ₈) alkyl, (C ₁-C ₈) alkoxyl or hydroxyl; With X be O, S, S (O) or N (R '), N (Ac) or N (toluene-4-sulfonyl oxygen base);

Wherein each R ' independence is H, (C ₁-C ₈) alkyl, phenyl or benzyl.

2. the Therapeutic Method of claim 1, wherein R ₁₀And R ₁₃Be CH ₃

3. claim 1 or 2 Therapeutic Method, wherein R ₁₆And R ₁₇One or both of be CH ₃

4. the Therapeutic Method of any one, wherein R among the claim 1-3 ₁, R ₂Or R ₁₂Be H or OH.

5. the Therapeutic Method of any one, wherein R among the claim 1-4 ₁, R ₂, R ₄, R ₆, R ₇, R ₈, R ₉, R ₁₁, R ₁₂, R ₁₄And R ₁₅Be H.

6. the Therapeutic Method of any one, wherein R among the claim 1-5 ₃Be (C ₁-C ₂₂) alkyl CO ₂-, (C ₁-C ₆) alkyl CO ₂-or be OH.

7. the Therapeutic Method of any one among the claim 1-6, wherein X is O.

8. the Therapeutic Method of any one among the claim 1-6, wherein X is NH.

9. suffering from mitochondrial disorders or be subjected to the Therapeutic Method of treatment mitochondrial disorders among the patient that mitochondrial disorders threatens for one kind, wherein said disorder is not central nervous system's a neuropathy, and described method comprises formula (II) chemical compound or its pharmaceutically acceptable salt that gives described patient's effective dose:

R wherein ₁, R ₂, R ₄, R ₇, R ₁₁, R ₁₂And R ₁₅Independent separately is hydrogen, (C ₁-C ₈) alkyl, hydroxyl, amino, carboxyl, oxo, sulfonic acid, or optional insertion-NH-,-N ((C ₁-C ₈) alkyl)-,-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)-(C ₁-C ₈) alkyl, wherein R ' is H or (C ₁-C ₈) alkyl; R ₃Be hydroxyl, (C ₁-C ₆) alkyl CO ₂-, HO ₂C (CH ₂) ₂CO ₂-, toluene-4-sulfonyl oxygen base or benzoyloxy; R ₆, R ₈, R ₉, R ₁₀, R ₁₃And R ₁₄Independent separately is hydrogen, (C ₁-C ₈) alkyl, hydroxyl (C ₁-C ₈) alkyl, (C ₁-C ₈) alkoxyl or hydroxyl; With X be O, N (H), N (Ac) or N (toluene-4-sulfonyl oxygen base).

10. the method for claim 9, wherein R ₃Be OH or (C ₁-C ₆) alkyl CO ₂-.

11. the method for claim 9 or 10, wherein X is O.

12. the method for claim 9 or 10, wherein X is NH.

13. the method for any one, wherein R among the claim 9-12 ₁₀And R ₁₃Be CH ₃

14. the method for any one, wherein R among the claim 9-13 ₁, R ₂Or R ₁₂Be OH.

15. the method for any one, wherein R among the claim 9-15 ₁, R ₂, R ₄, R ₆, R ₇, R ₈, R ₉, R ₁₁, R ₁₂, R ₁₄And R ₁₅Be H.

16. the method for any one in claim 1 or 9; wherein said chemical compound is selected from (20 α)-25 ξ-methyl-(22R; 26)-azacyclo-furan steroid-5-alkene-3 ξ-alcohol; (20 ξ)-25 ξ-methyl-N-acetyl group-(22R; 26)-azacyclo-furan steroid-5-alkene-3 ξ-alcohol; (22R; 25 ξ)-(20 α)-spiral shell steroid-5-alkene-(2 α; 3 ξ)-glycol; p-methyl benzenesulfonic acid (20 α)-25 ξ-methyl-N-p-toluenesulfonyl-(22R; 26)-azacyclo-furan steroid-5-alkene-3 ξ-Ji ester; (22R; 25 ξ)-(20 α)-(14 α; 20 α)-spiral shell steroid-5-alkene-(3 β; 12 β)-glycol; (22R; 25S)-(20 ξ)-spiral shell steroid-5-alkene-3 ξ-alcohol; benzoic acid (22R; 25 ξ)-(20 α)-spiral shell steroid-5-alkene-3 beta-yl ester; caproic acid (22S; 25S)-(20S)-spiral shell steroid-5-alkene-3 beta-yl ester; (22R; 25 ξ)-(20 α)-spiral shell steroid-5-alkene-(1 ξ; 3 ξ)-glycol; (22R; 25S)-(20 α)-spiral shell steroid-5-alkene-3 β-alcohol; succinic acid (22R; 25S)-(20 α)-spiral shell steroid-5-alkene-3 beta-yl ester and propanoic acid (20 α)-25S-methyl-N-acetyl group-(22S, 26)-azacyclo-furan steroid-5-alkene-3 beta-yl ester.

17. the method for claim 16, wherein said chemical compound be caproic acid (22S, 25S)-(20S)-spiral shell steroid-5-alkene-3 beta-yl ester.

18. the method for claim 1 or 9, wherein said chemical compound gives with the dosage form of this chemical compound of containing effective therapeutic dose.

19. the method for claim 18, wherein said dosage form are selected from tablet, Perle, hard gelatin capsule, suspension tablet, effervescent tablet, powder, effervescent powder, chewable tablet, solution, suspending agent, Emulsion, ointment, gel, patch and suppository.

20. the method for claim 19, wherein said dosage form also comprises pharmaceutically acceptable excipient.

21. the method for claim 20, wherein pharmaceutically acceptable excipient comprise binding agent, disintegrating agent, filler, surfactant, solubilizing agent, stabilizing agent, lubricant, wetting agent, diluent, anti--adhesive agent, fluidizer or pharmaceutically acceptable carrier.

22. the method for any one among the claim 1-21, wherein said mitochondrial disorders are mitochondriopathy.

23. the method for any one among the claim 1-21, wherein said mitochondrial disorders are myopathy.

24. the method for claim 23, wherein said myopathy are cardiomyopathy.

25. the method for any one among the claim 1-21, wherein said mitochondrial disorders are the renal tubules pathological changes.

26. the method for any one among the claim 1-21, wherein said mitochondrial disorders is caused by osteofascial compartment syndrome or muscle injury syndrome.

27. the method for any one among the claim 1-21, wherein central nervous system's the neuropathy neural cell injury, epilepsy, anxiety neurosis, spinal cord lesion, ALS, Alzheimer, Huntington and the parkinson that are selected from neural cell injury that full brain and focal ischemia's property or hemorrhagic apoplexy, head trauma, spinal cord injury, hypoxia cause, cause by sudden cardiac arrest or transient respiratory distress of the newborn.

28. a Pharmaceutical composition, it comprises and at least a formula (I) of effective therapeutic dose of pharmaceutically acceptable excipient composition or (II) chemical compound.

29. formula (I) chemical compound or its pharmaceutically acceptable salt are used for the treatment of application in the medicine of mitochondriopathy or mitochondrial disorders in preparation, described mitochondriopathy or mitochondrial disorders are not central nervous system's neuropathy:

R wherein ₁, R ₂, R ₄, R ₇, R ₁₁, R ₁₂And R ₁₅Independent separately be hydrogen, choose wantonly insertion-MR '-,-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)-(C ₁-C ₈) alkyl; Hydroxyl, N (R ') ₂, carboxyl, oxo or sulfonic acid, R ₃Be hydroxyl, (C ₁-C ₈) alkoxyl, (C ₁-C ₂₂) alkyl CO ₂Or R ' O ₂C (CH ₂) _2-8CO ₂-, (C wherein ₁-C ₂₂) alkyl or (CH ₂) _2-8Can choose wantonly and comprise 1-2 CH=CH unit, a 1-2 OH and/or 1-2 epoxy substituent group, toluene-4-sulfonyl oxygen base or benzoyloxy; R ₆, R ₈, R ₉, R ₁₀, R ₁₃, R ₁₄, R ₁₆And R ₁₇Independent separately is hydrogen, (C ₁-C ₈) alkyl, hydroxyl (C ₁-C ₈) alkyl, (C ₁-C ₈) alkoxyl or hydroxyl; With X be O, S, S (O) or N (R '), N (Ac) or N (toluene-4-sulfonyl oxygen base);

Wherein each R ' independence is H, (C ₁-C ₈) alkyl, phenyl or benzyl.

30. formula (II) chemical compound or its pharmaceutically acceptable salt are used for the treatment of application in the medicine of mitochondriopathy or mitochondrial disorders in preparation, described mitochondriopathy or mitochondrial disorders are not central nervous system's neuropathy:

R wherein ₁, R ₂, R ₄, R ₇, R ₁₁, R ₁₂And R ₁₅Independent separately is hydrogen, (C ₁-C ₈) alkyl, hydroxyl, amino, carboxyl, oxo, sulfonic acid, or optional insertion-NH-,-N ((C ₁-C ₈) alkyl)-,-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)-(C ₁-C ₈) alkyl, wherein R ' is H or (C ₁-C ₈) alkyl; R ₃Be hydroxyl, (C ₁-C ₆) alkyl CO ₂-, HO ₂C (CH ₂) ₂CO ₂-, toluene-4-sulfonyl oxygen base or benzoyloxy; R ₆, R ₈, R ₉, R ₁₀, R ₁₃And R ₁₄Independent separately is hydrogen, (C ₁-C ₈) alkyl, hydroxyl (C ₁-C ₈) alkyl, (C ₁-C ₈) alkoxyl or hydroxyl; With X be O, N (H), N (Ac) or N (toluene-4-sulfonyl oxygen base).

31. comprising the physiology, the application of claim 29 or 30, wherein said medicine go up acceptable carrier.

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