CN1984670A

CN1984670A - Cyclosporins to treat alzheimer's disease

Info

Publication number: CN1984670A
Application number: CNA2005800237213A
Authority: CN
Inventors: D·科恩; L·A·该赛
Original assignee: Novartis AG
Current assignee: Novartis AG
Priority date: 2004-07-13
Filing date: 2005-07-12
Publication date: 2007-06-20
Also published as: BRPI0513317A; AU2005261838A1; CA2573400A1; EP1893226A1; RU2007105141A; KR20070036127A; AU2009210375A1; WO2006005580A1; MX2007000502A; JP2008512351A

Abstract

Non-immunosuppressive, cyclophilin-binding cyclosporins, are useful as neuroprotective agents, e.g. in the prevention or treatment of pathological conditions associated with A beta secretion and/or production.

Description

The cyclosporin of treatment Alzheimer

Technical field

The present invention relates to the new purposes of cyclosporin, is the new medicinal usage of non-immunosuppressant cyclophilin in conjunction with cyclosporin especially.

Background technology

Cyclosporin A (CsA) but with exempt from albumen for example cyclophilin (CyP) combine, but and be all the FK506 and the rapamycin of exempting from the protein binding chemical compound and combine with FK506 conjugated protein (FKBP).But although exempt from protein binding is essential, and its immunosuppressive activity to these medicines is insufficient.But observe the biological effect of medicine/when exempting from albumen composition and the 3rd effector protein interaction.For example, CyP-CsA and FKBP-FK506 complex suppress the serine/threonine phosphatase activity of calcineurin, block cytokine such as interleukin-2,4 generation thus.On the other hand, FKBP-rapamycin complex suppresses to be called the kinases of FRAP (being also referred to as RAFT or mTOR) 5, and this kinases participates in the receptor-mediated T cell proliferation of interleukin-2.

From streptomyces species (Streptomyces sp.) A92-308110, isolate the new class chemical compound that is called as sanglifehrin.In present isolating 20 kinds of different sanglifehrin, sanglifehrin A (SFA) is the abundantest chemical compound, and demonstrates effective immunosuppressive activity.But SFA role of delegate mode is other known all different neotype immunosuppressant of protein binding chemical compound (being CsA, FK506 and rapamycin) of exempting from all.Shown SFA mitochondrion change duct (MTP) but in the complex to exempt from another--albumen cyclophilin A (CpA) but different binding sites directly in conjunction with exempting from albumen--cyclophilin D (CyD) also to suppress MTP open.

The purposes of non-immunosuppressant cyclophilin in conjunction with cyclosporin and treatment and prevention AIDS and AIDS associated disorders described, comprising general description to cyclosporins, their nomenclature and model of action in European patent no.484281.With EP 0,484, the disclosure of 281 B, the description of general description that particularly above relates to and the other parts that hereinafter relate to is included in the application's the explanation as a reference.

Surprisingly, have now found that with cyclophilin in conjunction with but do not have the cyclosporin of inhibitive ability of immunity to can be used as neuroprotective to be used for the treatment of and to comprise (but being not limited only to) Alzheimer (" AD ") and pathological state that A β produces and/or secretion is relevant.The born of the same parents that AD is characterized by amyloid speckle in the brain (mainly being made up of 40 or 42 amino acid whose peptides of A β) pile up outward.Pile up in the extracellular of these peptides is the pathology sign (Selkoe 1999) of this disease.These A β peptides are by the I type transmembrane protein of endoproteolysis cutting wide expression--and (Selkoe 1999 in amyloid precursor protein (APP) generation; Sisodia 2000).Two kinds of enzymes that generate cutting APP in the path at starch are called as beta-secretase and gamma-secretase, and it is respectively from N-terminal and C-terminal cutting APP.In this path, beta-secretase (BACE1) is first enzyme of cutting APP, produces excretory sAPP β fragment and conjunctival C-terminal fragment (CTF, C99) (people 1999 such as Vassar, Bennett).The C99 fragment is the substrate of gamma-secretase complex (GACE), and described complex cutting C99 produces A β and AICD (APP born of the same parents' intracellular domain).AICD is in conjunction with the complex that contains Tip60 and Fe65, and this complex suppresses the gene KAI1 (four transmembrane proteins (tetraspanin) cell surface molecule) (people such as Baek, Ohgi, 2002) in NF κ-B path.The GACE complex is by four solvents: senilism albumen 1 (PS1), slow-witted albumen (NCSTN), Aph1 and Pen2 (people such as Edbauer, Winkler, 2003; People such as Kimberly, LaVoie 2003) forms.PS1 function homologue--senilism albumen 2 (PS2) facilitates in the cell～and 20% A β produces (people such as Kimberly, Xia, 2000).Although these four kinds of components are essential and are enough to rebuild the GACE activity evidence (people 2003 such as Tesco, Koh) of APP cutting the causing A β generation of non-GACE mediation is arranged, (people 2001 such as Nunan, Shearman).

Summary of the invention

Illustrate new gene that the APP path relates to and be the complete etiology of determining this disease and better understand the committed step of impelling the complex mechanism that A β produces.Regulate the new gene of A β and the new therapeutic strategy of determining to help to develop this progression of disease of treatment of path.Some genetic linkages and chromosomal region and the related specifically (people such as Ertekin-Taner, Graff-Radford of tardy property Alzheimer (LOAD) (morbidity in＞65 years old), 2000), (people such as Bertram, Blacker, 2000), (people such as Scott, Hauser, 2003).The hypotype and the new gene that participates in APP processing of LOAD associated gene have been described, comprising cloning the description of regulating the extensive functional screening of A β generation ability in the CHOK1 cell to being used for detecting cDNA in common unsettled USSN.With the disclosure of this USSN_, particularly identify to modify the excretory gene of A β and hereinafter the description of described other parts be included in the application's the explanation as a reference.

But the discovery of exempting from the albumen pathway gene that produces key instrumentality as A β in the cell provides suitable Alzheimer drug targets.But some cDNA that exempt from the albumen path that relate to that find in the functional screening comprise Map kinases 4, calmodulin, CaM, acid ceramidase and TOB3 (a kind of AAA-adenosine triphosphatase).

Mitogen-activated protein kinase (MAPK) claims extracellular signal-regulated kinase (ERK) to belong to serine/threonine protein kitase family again, and its phosphorylation is also just being regulated or the negative target substrate of regulating initial signal cascade amplification incident.ERK replys widely thorn and goads signal play an important role to nucleus and regulator gene expression, mitosis, propagation, mobility, metabolism and apoptosis (Wada and the Penninger 2004) that transduce from cell membrane into action.The active inhibitor of MEK and ERK shows inhibition APP catabolism.In addition, MAPK can activate JNK and trigger cell apoptosis--the process (people such as Tesco, Koh, 2003) that known raising A β produces.Although do not wish to be subjected to theory, MAPK4cDNA can work by APP catabolism or active cell apoptosis.

APP born of the same parents' intracellular domain (AICD) fragment also with the cJun N-terminal kinases (JNK) that is connected map kinase path and APP processing interact (people 2002 such as Scheinfeld, Roncarati).MAPK4 is the ERK that activates JNK, therefore crosses and expresses the superactivation that MAPK4 can cause JNK, and it can increase the JNK-AICD interaction and cause that increasing A β produces.Activation-inducing apoptosis that also may JNK.Perhaps, MAPK4 can change the phosphorylation state of APP, the phosphorylation state that has shown described APP influences it by beta-secretase and the preferential processing of alpha-secretase enzyme (Hung and Selkoe 1994) by influencing the APP transportation and destroying the interaction of PS1/ beta-catenin, (people such as Walter, Capell, 1997).In addition, also the phosphorylation state of showed cell is crucial (people 1997 such as Seeger, Nordstedt) to the PS1 activity.JNK activates and protein phosphorylation also is that MAPK4 can work to improve some other paths that A β produces.

Another gene of identifying in the screening is a calmodulin, CaM.Calmodulin, CaM is ring-spiral-ring Ca ²⁺Conjugated protein, its by with the specific target protein that comprises CaMKII, CaMKIV, calcineurin, spectrin A2, p21 and the neuron nitric oxide synthase transduction Ca that interacts ²⁺Signal (Means1981).Work as Ca ²⁺During in conjunction with calmodulin, CaM, its experience conformation change can combine it with target protein and stimulates or inhibition target protein activity.In cell-free preparation and intact cell, use calmodulin, CaM antagonist W-7 and trifluoperazine to show that calmodulin, CaM is regulated A β and produced.The active inhibitor of these known calmodulin, CaMs also can suppress A β and produce (people such as Desdouits, Buxbaum, 1996).Therefore, Ca in the born of the same parents ²⁺The target protein of concentration and/or calmodulin, CaM can influence APP processing.This synthetic data has been verified to have served as to observe when expressing calmodulin, CaM and has been improved the fact that A β produces.

Ca ²⁺Imbalance, particularly Ca in the Cytoplasm ²⁺(people such as Yoo, Cheng, 2000) take place in raising that the A β that the decline of level is relevant with PS1 FAD sudden change produces together.PS1 FAD sudden change can significantly slacken to flow (CCE) in the capacity calcium and store and consume activated electric current (depletion-activatedcurrents), and the CCE of prompting reduction can improve A β and produce (people such as Yoo, Cheng, 2000).Find that in screening expressing excessively of calmodulin, CaM gene also can cause the reduction of cellular calcium level, and can prevent enough CCE.Ca in the possible born of the same parents ²⁺Minimizing can directly or indirectly improve the PS1 activity, cause from the more A β of emiocytosis.Below the data of Xiang Shuing show that SFA suppresses A β 40 effectively and A β 42 produces and inhibition C99 and scarce albumen cutting at quarter, but illustrate that the gamma secretase activity is by exempting from PROTEIN C yD and Ca ²⁺Homeostasis is relevant.

Another gene--people's acid ceramidase catalysis ceramide is hydrolyzed to sphingol and fatty acid (people such as Ferlinz, Kopal, 2001).Ceramide is as the precursor of most of sphingolipid, and is the signaling molecule that (normally activates) cell death inducing in a large amount of different cell types by Caspase-3.The ceramide levels of accumulation is relevant with Alzheimer, and is considered to the part of oxidation neurotoxicity path in the aged AD brain.Crossing expression ceramide raising A β generation makes the ceramidase cutting be associated with A β.As if because ceramide works by apoptosis, possible acid ceramidase also promotes apoptosis.

The ceramide situation of showed cell is regulated the stability of BACE and the biology generation (people 2003 such as Puglielli, Ellis) of A β simultaneously.The previous high-caliber ceramide that studies have shown that can not increase A β secretion (people 2003 such as Puglielli, Ellis) by relying on apoptotic mode.Cross the expression ceramidase and will reduce ceramide levels, and may change membrane fluidity ground and improve sphingol and free fatty (FFA) level, be the method (Wilson and Binder 1997) of known change tau protein (tau) phosphorylation and A β polymerization.Although mechanism it be unclear that, possible ceramide endonuclease capable influences APP processing and more A β generation and/or the secretion that the FFA level causes change.

TOB3 is an AAA-adenosine triphosphatase of carrying out molecular chaperones sample function, help to comprise the protein complex of protein secreting assembling, operate or separate assembling (people such as Strausberg, Feingold, 2002).This proteinoid helps to keep the integrity of endoplasmic reticulum (ER) as the protein of being processed by composing type.When lacking the AAA-atpase activity, the excessive accumulation of misfolded protein matter causes that ER expands and cell death (people such as Kobayashi, Tanaka, 2002).Cross and express TOB3 to improve that the excretory most probable explanation of A β influences protein folding with it relevant with the ability of transporting in ER.If stimulate this process,, may APP processing will be improved as crossing expression by TOB3.

Our garbled data had shown that expressing the more A β of TOB3 change APP processing causing produced and still less C99 and C83 C-terminal fragment.Compare with HEK 293 cells, TOB3 expresses and also reduce APP and sAPP alpha levels (data not shown) more in the N2A cell.This prompting TOB3 influences one or more components in the APP processing machine, causes the APP and the C99 cutting of raising.Because TOB3 participates in proteinic transportation and processing, determining TOB3 cutter system and specificity really in APP processing at present.

Another cDNA--carboxypeptidase Z (CPZ) that identifies in the functional screening is the member of metallocarboxypeptidase gene family together with CPE and CPD, thinks to work in the processing in their born of the same parents before biologically active peptide and protein secreting.CPZ is rich in cysteine coiled structure territory owing to containing with Wnt and the bonded functional N-terminal of Wnt matter, is unique carboxypeptidase, the key component that described Wnt matter is the Wnt signal transduction (people such as Moeller, Swindell, 2003).

Another noticeable cDNA is that (CyD is also referred to as CyF to cyclophilin D.Nomenclature is with reference to Current Medicinal Chemistry, and 2003,10,1485-1506 1485 Cyclophilin Das a Drug Target, people such as Waldmeier).Find that CyD participates in A β and produces.Cyclophilin such as CyD relate to protein transportation and sophisticated peptidyl prolyl isomerase.Except that CyD was positioned in the mitochondrial matrix alone, cyclophilin mainly was positioned at (people 2003 such as Waldmeier, Zimmermann) in the Cytoplasm.When crossing expression CyD, it can stablize endogenous PS1 N-terminal fragment (NTF).The generally damage of AD brain is the tau protein that exists by unusual phosphorylation--neurofibrillary tangles (Johnson and Bailey 2002) in the born of the same parents that a kind of microtubule bindin matter is formed.When crossing expression APP, CyD can improve the activity of Caspase-3, and the excretory possibility mechanism of A β is regulated in indication.These results suggest have served as when expressing, and CyD is by gamma-secretase path cutting APP and the necessary important factor of C99.

On the one hand, CyD is the inherent component in mitochondrial permeability conversion duct, and it is considered to combine and regulate duct open (people such as Waldmeier, Zimmermann, 2003) with the adenylic acid translocator.The permeability of mitochondrial membrane is to cause the result (Waldmeier 2002) who consumes mitochondrial membrane potential, discharges the cellular stress that causes apoptotic proteins matter and come to an end with apoptotic cell death.

The depletion of indication wire plastochondria is played significant effect (Busciglio 2002) by accumulation in the cell that promotes unusual β-APP processing and A β in mongolism patient's AD neuro pathology development.Also the raising of the interior calcium level of showed cell causes the accumulation (LaFerla 2002) of A β in the cell.Astoundingly, total length APP is not only along the secretory pathway transportation, and the mitochondrion of the cortex neurocyte that targeting is cultivated in the brain of transgenic mice AD model.The incomplete transposition of β-APP on mitochondrial membrane and accumulation can cause the mitochondrial fuctionning obstacle, and (people 2003 such as Anandatheerthavarada, Biswas) can also play an important role in the pathogeny of AD.Known cross the expression mitochondrial protein can cause form in the substrate insoluble aggregation (Uncoupling Proteins-3 for example, UCP3).These aggregations can be upset mitochondrial normal function and improve the passive permeability of inner membrance.CyD can handle in excessive APP or the segmental processing of C-terminal at mitochondrion and play an important role.

CyD is for example known target of cyclosporin of immune suppressant drug.These chemical compound sealing mitochondrial permeability conversions (MPT) also prevent apoptosis (Samantha J.Clarke 2002; Waldmeier 2002).But also known FK506 chemical compound with exempt from protein binding but do not combine (Uchino 2003) with CyD.

Being combined in by Quesniaux of cyclophilin if cyclosporin and people recombinate in Eur.J.Immuno1.1987, 17, it is bonded 1/5 to account for ciclosporin (being called cyclosporin A again) in the competitiveness enzyme linked immunosorbent assay described in the 1359-1365 at least, thinks that then it combines with cyclophilin.In this test, between the incubation period of BSA-cyclosporin of cyclophilin and bag quilt, add cyclosporin to be measured, and calculate the control reaction that does not contain competitor and produce the required concentration (IC of 50% inhibition ₅₀).Results expression is binding ratio (BR), is the IC of test compounds ₅₀IC with the cyclosporin that in similar test, uses ciclosporin replacement test ₅₀The denary logarithm of ratio.Therefore 1.0 BR refers to that test compounds and combining of cyclophilin matter are 1/10th intensity of cyclosporin, and negative value is represented than cyclosporin in conjunction with stronger.

The cyclosporin that works as neuroprotective has and is lower than 0.7 BR (because log ₁₀5=about 0.7), preferably be equal to or less than zero.

To be less than cyclosporin active 5% when the activity of cyclosporin in mixed lymphocyte reaction (MLR), preferably is less than at 2% o'clock, thinks that it is non-immunosuppressant.Mixed lymphocyte reaction is described in " (Immunological Methods) " by T.Meo, and L.Lefkovits and B.Peris edit, AcademicPress, N.Y.227-239 page or leaf (1979).Will be from the Balb/c mice splenocyte (0.5 * 10 in (female, 8-10 week) ⁶) and from 0.5 * 10 of CBA mouse (female, 8-10 week) ⁶The splenocyte that radiation (2000 rad) or ametycin are handled was hatched 5 days altogether.Induced propagation to reply in the Balb/c splenocyte by radiating allogenic cells, described replying can participate in DNA by the precursor with labelling and obtain measuring.Because the stimulus object cell is by radiating (or ametycin is handled), they are not replied the Balb/c cell with propagation but keep their antigenicity.IC with test compounds foundation among the MLR ₅₀IC with ciclosporin foundation in the parallel laboratory test ₅₀Compare.

Having found to be judged as in above-mentioned MLR non-immunosuppressant chemical compound usually is non-activity in the IL-2 reporter gene is measured, therefore can use the IL-2 reporter gene to measure, as as elementary screening, select the non-immunosuppressant cyclophilin binding compounds of the present invention's use.

The non-immunosuppressant cyclophilin coupling collar spore rhzomorph compound that works as the active substance of treatment and the A β secretion related pathologies state inhibitor of amyloid plaque extracellular accumulation among the AD (for example as) is called as " reactive compound " hereinafter.

Therefore reactive compound can be used for treating the active relevant clinical disease of any gamma-secretase with the secretion of A β peptide, endogenous PS1 N-terminal fragment expression (NTF) or raising.Reactive compound also can be used for treating and improves the relevant disease of the excretory apoptosis of A β.A β peptide forms and the sign of AD is not only in gathering subsequently, and is that (Selkoe 1989 for the essential part of other neurological disorder such as parkinson disease, Huntington Chorea and other SA; People such as Price, Borchelt 1993; People such as Citron, Vigo-Pelfrey 1994).These results clearly show that apoptosis and A β produce intrinsic interrelating.

Reactive compound can also be regulated " the periphery A β instrumentality " of not expressing in brain.The periphery amyloidosis can cause all after one's own heart with such phenotype (people 1992 such as Yamaguchi, Yamazaki), (Selkoe 1989) of amyloidosis cutis.If find the instrumentality of periphery A β, also the therapy that may make new advances for example stops the target that penetrates the blood brain barrier needs.Reduce periphery A β level and show A β level in the reduction Transgenic Mice Brain, cause that plaque still less forms (people 1999 such as Bohrmann, Tjernberg; People such as DeMattos, Bales 2002).

Find many reactive compounds,, have the structure different with ciclosporin particularly at 4 and/or 5.Other position of reactive compound that may be different with ciclosporin is 6 and 7.

One group of reactive compound be wherein 4 MeLeu group by different N-methylate aminoacid, for example y-hydroxyl-MeLeu, MeIle, MeVal, MeThr, MeAla, MeTyr or MeTyr (O-PO (OH) ₂) or the cyclosporin that replaces of Pro.Except that MeIle and MeThr, also can use special-shaped MeaIle and MeaThr.In the abnormal shape, the spatial chemistry of β-position has the conformation opposite with natural amino acid, thus common form and a pair of diastereomer of special-shaped formation.

Another group reactive compound is that wherein 5 Val is by the N-alkyl amino acid, and preferred N-methylamino acid replaces.The preferred alkylating aminoacid of N-is Val or Leu.Preferably [Val] ⁵The hydrogen of imino group is by non-side chain C _1-6Alkyl, preferable methyl, ethyl or n-pro-pyl, particularly methyl substituted.The one group of preferred reactive compound in back is brand-new.

Extraly or alternatively, some reactive compound can be different with ciclosporin on 1,2,3 and/or 6.

The particular type that is used for reactive compound of the present invention is ciclosporin derivant and the officinal salt thereof of formula A:

Wherein B is the amino acid residue of formula B

Wherein a represents the key of the α Abu residue of link position 2;

B represents the key of the C residue of link position 4;

The Alk representative contains the straight or branched alkylidene of 2 to 6 carbon atoms or contains the ring alkylidene of 3 to 6 carbon atoms, and

The R representative

Carboxyl or alkoxy carbonyl;

-NR ₁R ₂Base, wherein R ₁And R ₂Be identical or different and represent hydrogen, alkyl, C _2-4Alkenyl, C _3-6Cycloalkyl, phenyl (randomly replacing) or benzyl or contain 5 or 6 annular atomses and 1 to 3 heteroatomic saturated or unsaturated heterocycle base, or R wherein by halogen, alkoxyl, alkoxy carbonyl, amino, alkyl amino or dialkyl amido ₁And R ₂Form saturated or undersaturated heterocycle with the nitrogen-atoms that they connected, described heterocycle contains 4 to 6 annular atomses and randomly contains other hetero atom that is selected from nitrogen, oxygen or sulfur and also randomly replaced by alkyl, phenyl or benzyl; The atomic group of following formula

R wherein ₁And R ₂As above definition, R ₃Represent hydrogen or alkyl and n to be from 2 to 4 integer, and wherein alkyl represent to contain the straight or branched alkyl of 1 to 4 carbon atom;

C is MeLeu or 4-hydroxyl-MeLeu.

In disclosed International Patent Application WO 98/28328, WO 98/28329 and WO 9828330, further describe such ciclosporin derivant.Particularly preferred this compounds is the chemical compound of formula A,

Wherein B is amino acid residue B '

And C is amino acid residue 4-hydroxyl-MeLeu.

Particularly preferred reactive compound group is made up of chemical compound and the officinal salt thereof of formula I:

Wherein W is MeBmt, dihydro-MeBmt or 8 '-hydroxyl-MeBmt;

X is α Abu, Val, Thr, Nva or O-methylthreonine (MeOThr);

R is Sar or (D)-MeAla;

Y is MeLeu, γ-hydroxyl-MeLeu, MeIle, MeVal, MeThr, MeAla, MeTyr, MeTyr (O-PO (OH) ₂), MeaIle or MeaThr or Pro;

Z is Val, Leu, N-Alk-Val or N-Alk-Leu,

Wherein Alk represents the Me of Me or following replacement:

By the Me of vinyl substituted, described vinyl is randomly by phenyl or contain N, the S of 6 annular atoms numbers or the assorted aromatic radical of O replaces, or

By the Me that phenyl replaces, described phenyl is randomly replaced by halogen; And

Q is MeLeu, γ-hydroxyl-MeLeu or MeAla.

Group W, X, Y, Z and Q have following preferred implication independently:

Preferably, W is W ', and wherein W ' is MeBmt or dihydro-MeBmt;

Preferably, X is X ', and wherein X ' is α Abu or Nva, more preferably X ", X wherein " be α Abu;

Preferably, Y is Y ', and wherein Y ' is γ-hydroxyl-MeLeu, MeVal, MeThr, MeAla or MeTyr (O-PO (OH) ₂);

Preferably, Z is Z ', and wherein Z ' is Val or MeVal; With

Preferably, Q is Q ', and wherein Q ' is MeLeu.

One group of particularly preferred reactive compound is the chemical compound of formula I, and wherein W is W ', and X is X ', and Y is Y ', and Z is that Z ' and Q are Q '.

The reactive compound of particularly preferred formula I is:

A) [dihydro-MeBmt] ¹-[γ-hydroxyl-MeLeu] ⁴-ciclosporin;

B) [MeVal] ⁴-ciclosporin;

C) [MeIle] ⁴-ciclosporin;

D) [MeThr] ⁴-ciclosporin;

E) [γ-hydroxyl-MeLeu] ⁴-ciclosporin;

F) [Nva] ²-[γ-hydroxyl-MeLeu] ⁴-ciclosporin;

G) [γ-hydroxyl-MeLeu] ⁴-[γ-hydroxyl-MeLeu] ⁶-ciclosporin;

H) [MeVal] ⁵-ciclosporin;

I) [MeOThr] ²-[(D) MeAla] ³-[MeVal] ⁵-ciclosporin, or

J) [8 '-hydroxyl-MeBmt] ¹-ciclosporin.

M) [N-benzyl-Val] ⁵-ciclosporin,

N) [N-5-fluoro-benzyl-Val] ⁵-ciclosporin,

O) [N-pi-allyl-Val] ⁵-ciclosporin,

P) [N-3-phenyl-pi-allyl-Val] ⁵-ciclosporin,

Q) [Pro] ⁴-ciclosporin

Particularly preferred reactive compound is [MeIle] ⁴-ciclosporin and [γ-hydroxyl-MeLeu] ⁴-ciclosporin, most preferably [MeIle] ⁴-ciclosporin.

Except that the chemical compound of formula I, preferably reactive compound also comprises, for example

R) [γ-hydroxyl-MeLeu] ⁹-ciclosporin.

The method that can obtain reactive compound comprises:

1) fermentation

2) biotransformation

3) derive

4) semi-synthetic

5) complete synthesis.

These methods are described at large, and have more specifically described these methods in the embodiment 1 to 10 of EP 0484281 B He among the United States Patent (USP) No.5767069.Introduce these general introductions and the instruction of these embodiment in this application as a reference.Immunosuppressant and close loops that the embodiment 11 of EP 0484281 B has described the representative reactive compound relevant with ciclosporin close active measurement, and the instruction of this embodiment also is included in the application's the disclosure.

Therefore the invention provides non-immunosuppressant cyclophilin in conjunction with cyclosporin preparation be used for the treatment of or the medicine of prevention and A β secretion related pathologies state in purposes, described pathological state comprises for example Alzheimer, parkinson disease, tau protein disease (tauopathies), prion disease, volume temporo type dementia, striatonigral degeneration, Lu Yi body dementia, Huntington Chorea, Pick disease, amyloidosis and other and the relevant neurodegenerative disorders of excessive A β generation.

The present invention also is provided for treating or the method for prevention and A β secretion related pathologies state, described pathological state comprises for example Alzheimer, parkinson disease, tau protein disease, prion disease, volume temporo type dementia, striatonigral degeneration, Lu Yi body dementia, Huntington Chorea, Pick disease, amyloidosis and other neurodegenerative disorders, and described method comprises the reactive compound of the present invention of described patient being used effective dose.

Can be by any conventional route, particularly through intestinal ground, for example with the oral ground of drinkable solutions, tablet or capsule form; Or parenterally, for example use reactive compound with injection or suspensions.Dose indicating every day of intravenous injection approach can be from 1 to 20mg/kg, and preferably from 3 to 10mg/kg, and dose indicating every day of oral route is from 1 to 50mg/kg, preferably from 10 to 30mg/kg.

The toxicity of reactive compound is considered to be lower than ciclosporin.Because reactive compound is non-immunosuppressant, has avoided ciclosporin some side effect relevant with immunosuppressant.The side effect that other is relevant with cyclosporin, particularly nephrotoxicity and the central nervous system's toxicity during life-time service all advantageously is lower than ciclosporin.

The preferred Galenic formula that is used for reactive compound comprises those based on the microemulsion described in UK Patent Application 2222 770A, and it comprises local and oral form; Also comprise the oral and injectable form that obtains from the solid solution that contains fatty acid sugar monoesters (for example sucrose monolaurate) as described in UK Patent Application 2 209 671A.Be applicable to that Orally administered unit dosage forms for example comprises every dose from 25 to 200mg reactive compounds.

Example of formulations A, the B, C and the D that introduce EP 0484281 B here be as a reference:

These preparations composition separately and the detailed description of their preparation method are arranged in UK Patent Application 2 222 770, introduce its content here as a reference.

Can in vivo or prove the purposes of reactive compound in the testing in vitro as neuroprotective, for example:

Reactive compound of the present invention can be provided separately, or with itself and other combinations of substances or sequential associating.For example, can be with reactive compound of the present invention and antiinflammatory, (but being not limited only to) glucocorticoid co-administered after apoplexy or spinal cord injury (as the further nerve injury of blocking-up and suppress the means of axon regeneration) for example; Reactive compound of the present invention and neurotrophic factor can also be used for the medicine of neurodegenerative disease as NGF, BDNF or other, as Exelon ^TMOr levodopa is co-administered.As used herein, when using two kinds of materials simultaneously or so that during two kinds of materials of mode individual application that described material worked in the identical time, claiming that two kinds of materials are co-administered.

Can take from the current edition of standard outline " Merck index " or from the data base by the structure of the active component of code, common name or trade name definition, for example international monopoly (for example IMS world publication).Introduce its content corresponding here as a reference.Any those skilled in the art can identify described active component fully and based on these with reference to preparing equally and in the standard testing model, testing its medicine indication and characteristic in the external and body.

For above-mentioned indication, suitable dosage will depend on natch, concrete molecule for example used in the present invention, method of application and sanatory character and seriousness and change.

Embodiment

Implement following method to carry out the embodiment of following discloses:

Transfection.The DMEM of the levoproline that contains 10% hyclone, 5% penicillin/streptomycin and 22mg (Sigma Chemical, St.Louis, coating CHO K1 cell in flat board MO) (ATCC, Manassas, VA).Flat board is placed 37 ℃ of hydraulic pressure CO ₂Overnight incubation in the cell culture incubator.Use Qiagen according to manufacturer's explanation ^SuperFect reagent carries out cotransfection with 1: 15 ratio (cDNA:APPwt (695)) with purpose cDNA and total length APP.With 5 * 10 ⁵Cell is layered on the DMEM that contains 10% hyclone, 5% penicillin/streptomycin, and (Sigma Chemical, St.Louis is in six orifice plates MO) and cultivated 24 hours.The SuperFect mixture is made up of the total DNA of 100 μ l serum-free mediums (DMEM), 3 μ g and the SuperFect of 20 μ l.Remove culture medium and add the 1mL fresh culture from cell.Whole SuperFect mixture are added in the culture medium and at 37 ℃ hatched 2 hours.Remove mixture then and use 3mL PBS washed cell once.In cell, add fresh culture and hatched 24 or 48 hours.

Immunocytochemistry--as described in manufacturer (Life Technologies), in OPTIMEM (not containing blood serum medium), use Lipofectamine with suitable construct transfection CHO cell.As people such as Dev 1999, Neuropharmacol., (1999) 38,635-644 are described, to the Chinese hamster ovary celI immunostaining and observe immunoreactivity.Use anti--Flag M2 monoclonal mouse antibody (Sigma) to detect the Flag-protein expression, two anti-ly are texas Red-X goat anti-rabbit igg (Molecular Probes).

The flow cytometry of Caspase-3.With CyD or CPZ with APPwt transient transfection HEK cell 24 hours.With cell fixation, change processing and using thoroughly from the dyeing of the Caspase-3 apoptosis cell apoptosis test kit (#550914) of BD Biosciences.From 〉=10 ¹The painted cell of gate FITC is also measured it as positive cell of Caspase-3.Scattering and sidescattering are with the health of check cell mass before checking simultaneously.

Compound treatment.With cyclosporin A, Sangliferhin A, FK506 (as people such as Sedrani, J.Am.Chem.Soc.125,3849-3859 page or leaf (2003) is described, and its integral body is incorporated herein by reference) and N-methyl-4-valine-cyclosporin concentrate handles the HEK293 cell 24 hours of stably express APPswe mutant.Use is measured cell survival from the CellTiter-Glo test kit (#G7573) of Promega.Use self-control double sandwich enzyme-linked immunosorbent assay (except that the antibody that uses the Novartis development replaces the Biosource antibody, the self-control enzyme-linked immunosorbent assay is identical with described form) to measure A β 40 and A β 42 levels in the cell conditioned medium.Use stably express 5 * Gal4 response element luciferase reporter gene and C99-Gal4-VP16 or carve the cell line measurement C99 that lacks albumen-Gal4-VP16 construct or carve and lack albumen cutting (disclosed) as people such as Maltese, Wilson 2001.Gal4-VP16 is released and is attached to reporter gene increase uciferase activity after by the gamma-secretase cutting.With the compound treatment stabilized cell and measure IC ₅₀Value.

A β enzyme-linked immunosorbent assay.With commercially available targeting A β peptide NH ₂Terminal mouse monoclonal antibody is as wrapping by the capture antibody of 96 orifice plates (for A β 40 is BiosourceCat#KBH3481/PPO81, is Cat#KBH3441/PPO81 for A β 42) in advance.Dilute with 1/220 from Biosource acquisition polyclone detection antibody (anti-hA β 40 antibody Cat#44-348 and anti-hA β 42 antibody Cat#44-344) and all 15mM Hydrazoic acid,sodium salt.Two anti-(A β 4 is BiosourceCat#KBH3481, and A β 42 is Cat#KBH3441) are the anti-rabbit igg of horseradish peroxidase-labeled.Anti-with the 3.3mM thymol by 1/100 dilution two.(Winooski VT) goes up washing four times for Bioteck Instruments, Inc in the microtest plate scrubber with PBS-TE (1mM EDTA and 0.05% polysorbas20, lavation buffer solution) with the antibody sandwich plate before using.Take out 100 μ l transfectional cells conditioned medium and contain 1mM AEBSF (Biosource, Camarillo, in diluents CA) with 1: 2 the dilution.100 these mixture of μ l are added in 96 orifice plates of the antibody sandwich of washing, with the adhesive tape covering and 4 ℃ of overnight incubation.Remove sample and wash flat board four times with lavation buffer solution.Add antibody test solution by 100 μ l/ holes, and with flat board room temperature wave and culture 2 hours.Dull and stereotyped four times and add two anti-and shake and hatch 2 hours by 100 μ l/ holes of reuse lavation buffer solution washing.Reuse lavation buffer solution washing flat board 5 times also pats dry on napkin.Every hole is added 100 μ l and is stablized chromogen (tetramethyl benzidine) and flat board was hatched 30 minutes in the dark.In flat board, add 100 μ l stop buffer (1NH ₂S) with cessation reaction.1 hour inherent microplate readers (microplate reader) (Molecular Devices) read flat board in 450nM.

Antibody and Western engram analysis.CDNA with APP wild type and APP swedish mutant body as discussed previously inserts pCI plasmid expression vector cytomegalovirus promoter downstream (Promega, Madison, WI) (Bodendorf, U., Fischer, F., Bodian, D., Multhaup, G., Paganetti, P.2001 J.Biol.Chem.276:12019 12023).The N-terminal (people 1996 such as Thinakaran, Borchelt) of PS1 NTF antibody recognition PS1.Cell transfecting contained protease inhibitor (Complete after 24 hours ^Roche Molecular Biochemicals) extract in the RIPA buffer (10mM Tris pH 7.5,150mM sodium chloride, 1mM EDTA, 1%Nonidet P-40,0.5% NaTDC, 1%SDS) the HEK293 cell cultivated and 4 ℃ with 10000 * g centrifugal 10 minutes.Collect supernatant and abandon precipitation.Use sds polyacrylamide gel electrophoresis isolated cell extract subsequently, be transferred to PVDF Immobilon-P ^Film (Millipore) is also used one of indication to resist and is surveyed.It is as discussed previously that (Manni, M. wait the people, and 1998.FEBS.427:367-370) (Amersham Pharmacia Biotech, Piscataway NJ) carry out immune detection with the ECL detection system.

Immunocytochemistry--as described in manufacturer (Life Technologies), in OPTIMEM (serum-free medium), use Lipofectamine with suitable construct transfection CHO cell.As people such as Dev 1999, Neuropharmacol., (1999) 38,635-644 are described, to the Chinese hamster ovary celI immunostaining and observe immunoreactivity.Use anti--Flag M2 monoclonal mouse antibody (Sigma) to detect the Flag-protein expression.Two anti-are texas Red-X goat anti-rabbit igg (Molecular Probes).

The CPZ construct--from available from Life Technologies, the normalized cDNA library (L0001) of the pure people DRG of Inc (LTI, article number: 11315-017, lot number: 81027-242) obtain wild type human carboxylpeptidase (hCPZ).CDNA is inserted fragment is cloned into the compatible carrier pCMVSPORT6 of Gateway-with 5 ' to 3 ' direction EcoR V and Not I site.Use primer to finish corresponding to disappearance, the point mutation (aminoacid E251A) of catalysis (Cat) domain of curl (FZ) domain and the amino acid/11 79-568 of aminoacid 42-161 and introduce N-end FLAG label (DYKDDDDK Seq ID.No 1) aminoacid 29 after by site-directed mutation, described primer is:

5 ' CCTCCAGGCCTCCCCGAAGCTTCTCGGCGCTGTCTGCAGCTGGTGGCCTGTGG-3 ' (Seq Id No.2) and

5 ' CCACAGGCCACCAGCTGCAGACAGCGCCGAGAAGCTTCGGGGAGGCCTGGAGG-3 ' (Seq Id No.3) is used for the disappearance in coiled structure territory,

5 ' CCACACGGCCAGCCCTCTTCATCCGGGCCAGCCCTGAGGGCAGTGCCTCGTCAGC-3 ' (Seq Id No.4) and

5 ' GCTGACGAGGCACTGCCCTCAGGGCTGGCCCGGATGAAGAGGGCTGGCCGTGTGG 3 ' (Seq Id No.5) is used for the disappearance of catalyst structure domain,

5 ' CATCTCCCGGCCCGCCACCGCGTTGCCATGAATGTTGC-3 ' (Seq Id No.6) and

5 ' GCAACATTCATGGCAACGCGGTGGCGGGCCGGGAGAIGC-3 ' (SeqId No.7) is used for point mutation E251A, and

5 ' GGTGGCCTGTGGCATTCACCCTTGTCATCGTCGTCCTTGTAGTCGGCGGGGTTCCG CTCAAACTCG-3 ' (Seq Id No.8) and

5 ' CGAGTTTGAGCGGAACCCCGCCGACTACAAGGACGACGATGACAAGGGTGAATGCC ACAGGCCACC-3 ' (Seq Id No.9) is used to introduce the FLAG label.Use the Qiagen plasmid kit from the escherichia coli that transform, to separate all DNA.Use ABI Prism 3700 DNA analysis instrument systems to verify the sequence of all open reading-frames by dna sequencing.

In situ hybridization--use the oligonucleotide primers of polymerase chain reaction (PCR), can produce the rna probe template from any known sequence without any need for clone's step ground with the self-priming of 5 ' side joint SP6 promoter and T7 Promoter Recognition sequence.It is as follows that PCR reaction is carried out: 95 ℃ of degeneration 45 seconds, 58 ℃ of annealing stages 30 seconds, 70 ℃ of 1 minute extension stages, totally 40 circulations.After separating on 4% agarose gel, according to manufacturer (Qiagen, Switzerland) purified pcr product are described with the QiAQuick purification kit.The dNTP that use contains digoxin-UTP with T7-RNA polymerase (antisense) and SP6-RNA polymerase (justice) in 37 ℃ of PCR products of transcribing purification in 2 hours.Remove not the nucleotide of integrating, be dissolved in the 50 μ l water with the ethanol precipitation probe and with it and be stored in-20 ℃ then.With the gradient dilution point of the contrast RNA (concentration known) of DIG-probe and labelling on nylon membrane.After hatching with the alkali phosphatase of puting together with anti-DIG antibody, then use NBT/BCIP (be dissolved in the 75mg/ml nitroblue tetrazolium in 70% dimethyl formamide and 30% water and be dissolved in 50mg/ml 5-bromo-4-chloro-3-indyl phosphate in 100% dimethyl formamide) to carry out development step as alkaline phosphatase substrate.Estimate the CPZ concentration and probe concentration by the strength ratio of ordering with the contrast RNA of labelling.Use in situ hybridization and immunochemical full-automatic instrument Discovery ^TM(Ventana MedicalSystems Strasbourg) carries out ISH.In the RNA assay laboratory, set up and use paraffin-embedded tissue slice to locate the method for this gene, and this method is as described below, under condition of no solvent, use EZprep solution (Ventana Medical Systems SA, Strasbourg) in 75 ℃ 8 minutes, that continues carried out deparaffinization and rehydration to tissue slice in 8 minutes in 42 ℃.Use RiboMap ^TM(Ventana Medical Systems SA, Strasbourg) finish all pre-treatment step according to manufacturer's explanation and use enzymic digestion as an additionalization step: the E.C. 3.4.21.64 with 12 μ g/ml obtained optimum in 16 minutes in 37 ℃ of digestion to test kit.(Ventana Medical Systems SA, an amount of DIG-riboprobe Strasbourg) (CPZ probe=5ng/ microscope slide) carried out the CPZ probe hybridization 6 hours at 45 ℃ with being diluted in RiboHybe solution.Hybridization back at high stringent condition (under 0.1 * SSC) in 50 ℃ of washings in 8 minutes 3 times.In order to detect the DIG-labelling, with the antibody dilution agent afterwards, apply described antibody 30 minutes, the use BlueMap that continues in 37 ℃ with the mouse anti DigiTAb (JacksonImmunoresearch Inc.) of 1/2000 dilution biotin-conjugated ^TM(Ventana Medical Systems SA Strasbourg) carries out the BCIP/NBT color developing detection according to manufacturer's explanation to test kit.Have the best and believe/make an uproar that equilibrated substrate incubation time is 4 hours.Use ISH nuclear red soon (nuclear fast red) to redye 10 minutes.Fixing section and use Permount mounting in quartzy cover plate (Crystalmount).

Embodiment 1

The processing of senilism albumen: observed the A β level that cDNA improves the C99 substrate of expressing, the APP cutting of prompting GACE mediation improves.Because CPZ and CyD significantly improve the processing of C99 and improve A β 42 secretions, select their further research.Detected with PS1 N-terminal fragment (NTF) level in the HEK293 cell of CPZ or CyD transfection.In cell, can easily detect total length PS1 and NTF with the PS1 stable transfection.On the contrary, in the HEK of untransfected cell, endogenous total length PS1 and NTF level are lower.Expressing in the cell of CPZ or CyD excessively, significantly improving of PS1 NTF level indicates these fragments stabilized during 48 hours transfection with higher rate generation or endogenous NTF.

CPZ analyzes: CPZ is the member of carboxypeptidase (CP) gene family, belongs to the CPE subfamily, known its processing biological activity neuropeptide (Song and Fricker 1997).The CPE relevant enzyme participates in selectivity processing reaction usually, and it is from the C-terminal selective removal alkaline residue of processing intermediate.For whether catalytic activity, expression or the Subcellular Localization of determining CPZ is to induce A β necessary, produces three kinds of CPZ constructs that suddenly change.In first kind of construct, remove the only remaining signal peptide of catalyst structure domain (Δ cat), coiled structure territory and C-terminal fully.Disappearance coiled structure territory (Δ fz) in second kind of construct, and in the third construct, in catalyst structure domain, introduce a single point sudden change (Glu251Ala) of damage CPZ catalytic activity.Every kind of CPZ variant and APPwt or C99 substrate cotransfection are entered the HEK293 cell.Compare with the CPZ of wild type CPZ or flag labelling, three sudden change constructs all do not have activity separately.It is similar using the result of APPwt and C99 substrate.

Possible being interpreted as to the active disappearance of CPZ mutant lacks expression or this proteinic location of mistake in cell.In order to understand fully this problem, cross expression with flag labelling CPZ mutant and in Chinese hamster ovary celI.In the cell of saturatingization processing, the CPZ wild type has clarifying cytoplasma membrane or slow excretion vesicles location.This result and the research consistent (people 2000 such as Novikova, Reznik) that detects the interior distribution of CPZ born of the same parents before.The Subcellular Localization of CPZ sudden change construct Δ cat, Δ fz and Glu251Ala remains unchanged.These find the catalytic activity of prompting CPZ but not interior location of its cell or expression are to induce A β level necessary in Chinese hamster ovary celI.

Destroying the CPZ catalytic activity stops A β generation and does not influence its expression or Subcellular Localization.Cross and express CPZ and can cause still less beta-catenin activation (people 1998 such as Tesco, Kim) with Wnt competition and combining of endogenous curling receptor.The active A β generation that reduces and improve of beta-catenin is relevant, thinks that the A β generation of described raising improves beta-catenin and the associating ability mediation of GSK3 β (people 1999 such as Kang, Soriano) by PS1.Possible CPZ can induce A β to produce by activate the PS1 cutting via Caspase-3, causes PS1/ beta-catenin complex (people 1998 such as Tesco, Kim) still less.If PS1 does not combine with beta-catenin, then it can more easily associate with the GACE complex.Because the coiled structure territory is characterized by the protein interaction domain, the coiled structure territory of possible CPZ can interact with the component (being PS1 or APP self) of gamma-secretase complex.Studying these interactions at present.

It should be noted that and think that the C-terminal of CPZ domain contains the furin cleavage site (people 2000 such as Novikova, Reznik) that CPZ is released into the supposition of born of the same parents' external environment from plasma membrane.Furin also participates in carving and lacks protein ligands δ from cell surface scarce proteic release (Ikeuchi and Sisodia 200) at quarter.Although the furin site on the CPZ still unconfirmed is the furin substrate, having proved can be from emiocytosis CPZ (people 2000 such as Novikova, Reznik).This points out CPZ may be positioned on the processing path identical with carving scarce albumen, but it be not immediately clear how the scarce albumen cutting at quarter of CPZ relates to A β generation (people 1999 such as Kim, Wang).Yet, observe, cross to CyD and express similarly, CPZ can make cell enter apoptosis, indicate the common mechanism that two kinds of protein exist influences A β generation.

Previous discovery (Novikova and Fricker 1999) prompting, CPZ expresses in rat brain pia-arachnoid cell, but does not show that as yet CPZ expresses (Novikova and Fricker 1999) in the known brain zone that attacked by Alzheimer.In order to determine that CPZ expresses wherein, analyze the mouse brain section by in situ hybridization.In wild type C7BL-6 mice (Jackson Laboratories), the CPZ antisense probe is presented at the combination that extensive distribution is arranged in the brain, has the top level signal in cerebellum and volume cortex.In further checking, also find CPZ in Hippocampus near CA1 and CA3 zone, most probable is expressed in pyramidal cell.In cerebellum, CPZ expresses in the molecular domains near Perkinje and granular cell layer.The distribution of CPZ in volume cortex do not show and is arranged in particular cell types, but express fifty-fifty in this zone.The location of CPZ in mice Hippocampus, cerebellum and volume cortex illustrates the zone expression of its known people's of being subjected to Alzheimer invasion and attack in brain.

The sign of CyD: but exempt from the protein binding chemical compound and whether can regulate APP processing in order to measure these, handle the HEK/APPswe stabilized cell with cyclosporin A, Sangliferhin A, FK506 or N-methyl-4-valine-cyclosporin concentrate of finite concentration scope.According to manufacturer's explanation (Promega Cat#G5421) each concentration is measured viability and measured excretory A β 40 and A β 42 with enzyme-linked immunosorbent assay described herein by the MTS algoscopy.The viability IC of all concentration ₅₀Equal＞40 μ M.Cyclosporin A, Sangliferhin A are for the inhibition IC of A β 40 and A β 42 ₅₀Be＜3 μ M, the excretory strong inhibition to A β is described.N-methyl-4-valine-cyclosporin is for A β 40 and A β 42 excretory IC ₅₀Be＜10 μ M, and FK506 is for not influence of A β secretion.On the contrary, FK506 handles and causes that A β 42 improves from the dose dependent of 3-20 μ M.

Proved that immunosuppressant such as cyclosporin A can suppress in some animal models neural degeneration and apoptosis and suppress the beta induced mitochondrial injury (Kim 2002) of A in the isolating mice mitochondrion.Because CyD is the target of cyclosporin A and is positioned in the mitochondrion that it may work in this site A β processing, prompting CyD may be important to the development of people AD.Although do not test other isoform as yet, CyD may be because it be that processing has unique effect to unique known locations to A β in mitochondrial cyclophilin isoform.Discovery is crossed expression CyD and can be improved Caspase-3 activity and stablize PS1 NTF in the HEK cell, point out it can directly modify the PS1 activity.In addition, can combine and suppress its active chemical compound strong inhibition A β secretion and C99 cutting with CyD.

Concrete, 3 μ M Sanglifehrin A strong inhibition A β and before being lower than 20 μ M pair cell do not have toxicity.Known Sanglifehrin A is in conjunction with CyD and suppress its PPIase activity, and does not influence the binding ability of CyD and adenylic acid translocator (ANT), and does not suppress the activity as the calcineurin of CsA (Samantha J.Clarke 2002).Can explain the steep dose response of Sanglifehrin A (steep dose response) in a number of ways.Have only as Sanglifehrin A to combine with quite a lot of partial C yD, be enough to destroy it when MPT active, Sanglifehrin A just can suppress duct opening (Clarke 2002).Because ANT exists with dimer, CyD must be to combine to induce conformation change with ANT more than a molecule probably.Because Sanglifehrin A must be in conjunction with a plurality of CyD molecules suppressing the MPT complex activity, when this will be explained in 3 μ M threshold values to the excretory quick inhibition of A β.In case reach this threshold value, the MPT complex can be suppressed and be stoped the duct open fully.The threshold value that this MPT suppresses is A β processing seemingly and/or excretory important instrumentality also.Perhaps, Sanglifehrin A can have different with CyD or also have a plurality of targets except that CyD.It is unclear that CyD is directly to regulate the MPT chemical compound by its PPIase activity or by the MPT conformation change.In any case given here data play a crucial role in A β processing with data consistent explanation CyD that is provided by other research and MPT duct.

Although these chemical compounds can significantly reduce A β secretion and suppress the C99 cutting, they also suppress to carve scarce albumen cutting.Consider that data show CyD and CPZ cross expression and influence the GACE activity, this result is not astonishing.These data illustrate that first but exempting from chemical compound can suppress the GACE activity and define can influence A β in the cell and carve the new path that lacks albumen processing.

It should be noted that the FK506 processing improves A β 42 secretions significantly with dose-dependent form.But known FK506 is in conjunction with exempting from albumen but not CyD.The specificity of A β 42 improves metabolism or the generation that prompting FK506 target protein may be regulated A β 42.FK506 and CsA can both suppress calcineurin but they have Different Effects to A β secretion, so calcineurin may not influence A β level.But the unique influence of this of FK506 is further supported to exempt from the albumen path and is regulated A β processing.

Embodiment 2

Lack the albumen cutting at C99 and quarter

As described in people such as Maltrese, Wilson 2001, use have signal peptide, TGN reservation queue and be inserted in Q56/Y57 place the GAL4-NLS-VP16 sequence modification C99 or carve and lack albumen and stride film sequence generation HEK 293 stable cell lines.Described cell is stably express 5 * GAL4RE-luciferase reporter gene construct (RD-2002-01437 and RD-2001-02419) also.When thereby cutting C99 transgenic activates the Gal4 luciferase reporter gene, measure the GACE activity of these cells.Handle these HEK stabilized cells and determine the IC that C99 cuts with cyclosporin A, Sangliferhin A, FK506 or N-methyl-4-valine-cyclosporin ₅₀GALVP can be separately as negative control.The GALVP IC of cyclosporin A, N-methyl-4-valine-cyclosporin, Sangliferhin A and FK506 ₅₀Be respectively 6.9,9.9,＞20 and＞20 μ M, because before＞40 μ M and avirulence, showing to exist between the inhibition of C99 cutting and cell survival, these chemical compounds obviously get in touch.Yet cyclosporin A, N-methyl-4-valine-cyclosporin, Sangliferhin A suppress the C99 cutting at 0.71,0.87 and 0.85 μ M respectively, and suppress to carve scarce albumen cutting at 1.7,2.7 and 3.2 μ M respectively.FK506 is to C99 or carve not influence of scarce albumen cutting.Therefore can and carve scarce albumen cutting with the bonded part strong inhibition of CyD C99 and show the GACE activity inhibited.

Embodiment 3

Caspase-3 activates

The recent research prompting activates Caspase-3 can cause that A β secretes the protein (people 2003 such as Tesco, Koh) in raising and the stable GACE complex.Because CyD and CPZ improve A β secretion and stablize PS1 NTF, analyze the activated level of Caspase-3 in CyD and CPZ overexpressing cell.With APPwt transient transfection HEK 293 cells 24 hours, saponin was changed processing thoroughly, monoclonal antibody fixing and that use the FITC at active Caspase-3 to put together is surveyed with CyD and CPZ.Negative control cell empty carrier and APPwt transfection, and positive control cell was handled 6 hours with 1 μ M D-82041 DEISENHOFEN before analysis.In the cell of expressing CyD, 52% cell that detects is that active Caspase-3 is male, and in the cell of expressing CPZ, 48% cell is that Caspase-3 is male.These results show these proteinic activation of crossing induced expression Caspase-3, and pointing out this is the excretory possibility mechanism of stabilisation PS1 NTF and raising A β.

Claims

1. non-immunosuppressant cyclophilin in conjunction with cyclosporin preparation be used for the treatment of or prevention and A β produce and/or the medicine of secretion related pathologies state in purposes, described pathological state is Alzheimer, parkinson disease, tau protein disease, prion disease, volume temporo type dementia, striatonigral degeneration, Lu Yi body dementia, Huntington Chorea, Pick disease, amyloidosis and other and the relevant neurodegenerative disorders of excessive A β generation for example.

2. be used for the treatment of or prevention and A β produce and/or the method for the pathological state that secretion is relevant, it comprises uses the non-immunosuppressant cyclophilin of effective dose in conjunction with cyclosporin to described patient.

3. according to the purposes of claim 1 or according to the method for claim 2, wherein non-immunosuppressant cyclophilin is chemical compound and the officinal salt thereof of formula A in conjunction with cyclosporin

Wherein B is the amino acid residue of formula B:

Wherein a represents the key of the α Abu residue of link position 2;

B represents the key of the C residue of link position 4;

The R representative:

Carboxyl or alkoxy carbonyl;

-NR ₁R ₂Base, wherein R ₁And R ₂Be identical or different and represent hydrogen, alkyl, C _2-4Alkenyl, C _3-6Cycloalkyl, phenyl (randomly replacing) or benzyl or contain 5 or 6 annular atomses and 1 to 3 heteroatomic saturated or unsaturated heterocycle base by halogen, alkoxyl, alkoxy carbonyl, amino, alkyl amino or dialkyl amido; Or R wherein ₁And R ₂Form saturated or undersaturated heterocycle with the nitrogen-atoms that they connected, described heterocycle contains 4 to 6 annular atomses, randomly contains other hetero atom that is selected from nitrogen, oxygen or sulfur, and is randomly replaced by alkyl, phenyl or benzyl;

The atomic group of following formula:

R wherein ₁And R ₂As above definition, R ₃Represent hydrogen or alkyl and n to be from 2 to 4 integer,

And wherein alkyl represents to contain the straight or branched alkyl of from 1 to 4 carbon atom;

C is MeLeu or 4-hydroxyl-MeLeu.

4. according to the purposes of claim 1 or according to the method for claim 2, wherein non-immunosuppressant cyclophilin is the chemical compound of formula I in conjunction with cyclosporin:

Wherein W be MeBmt, dihydro-MeBmt or 8 '-hydroxyl-MeBmt;

X is α Abu, Val, Thr, Nva or O-methylthreonine (MeOThr);

R is Sar or (D)-MeAla;

Y is MeLeu, γ-hydroxyl-MeLeu, MeIle, MeVal, MeThr, MeAla, Me Tyr, MeTyr (O-PO (OH) ₂), MeaIle or MeaThr or Pro;

Z is Val, Leu, N-Alk-Val or N-Alk-Leu,

Wherein Alk represents the Me of Me or following replacement:

By the Me of vinyl substituted, described vinyl is randomly by phenyl or contain N, the S of 6 yuan of rings or the assorted aromatic radical of O replaces, or

By the Me that phenyl replaces, described phenyl is randomly replaced by halogen;

Q is MeLeu, γ-hydroxyl-MeLeu or MeAla.

5. according to the purposes of claim 1 or according to the method for claim 2, wherein non-immunosuppressant cyclophilin is to be selected from following chemical compound in conjunction with cyclosporin:

A) [dihydro-MeBmt] ¹-[γ-hydroxyl-MeLeu] ⁴-ciclosporin;

B) [MeVal] ⁴-ciclosporin;

C) [MeIle] ⁴-ciclosporin;

D) [MeThr] ⁴-ciclosporin;

E) [γ-hydroxyl-MeLeu] ⁴-ciclosporin;

F) [Nva] ²-[γ-hydroxyl-MeLeu] ⁴-ciclosporin;

G) [γ-hydroxyl-MeLeu] ⁴-[γ-hydroxyl-MeLeu] ⁶-ciclosporin;

H) [MeVal] ⁵-ciclosporin;

I) [MeOThr] ²-[(D) MeAla] ³-[MeVal] ⁵-ciclosporin, or

J) [8 '-hydroxyl-MeBmt] ¹-ciclosporin.

M) [N-benzyl-Val] ⁵-ciclosporin,

N) [N-5-fluoro-benzyl-Val] ⁵-ciclosporin,

O) [N-pi-allyl-Val] ⁵-ciclosporin,

P) [N-3-phenyl-pi-allyl-Val] ⁵-ciclosporin,

Q) [Pro] ⁴-ciclosporin, or

R) [γ-hydroxyl-MeLeu] ⁹-ciclosporin.

6. according to the purposes of claim 1 or according to the method for claim 2, wherein non-immunosuppressant cyclophilin is [MeVal] in conjunction with ciclosporin ⁴-ciclosporin.