JP2008512351A - Cyclosporine for treating Alzheimer's disease - Google Patents

Abstract

  Non-immunosuppressive, cyclophilin-binding cyclosporine is useful as a neuroprotective agent, for example in the prevention or treatment of pathologies associated with Aβ secretion and / or production.

Description

  The present invention relates to a novel use of cyclosporine and in particular to a new pharmaceutical use of non-immunosuppressive, cyclophilin-binding cyclosporine. (In this specification, cyclosporin is transliterated as cyclosporin, and Cicrosporin is transliterated as cyclosporin.)

Cyclosporin A (CsA) binds to immunophilin proteins such as cyclophilin (CyP), while FK506 and rapamycin are both immunophilin binding compounds but bind to FK506 binding protein (FKBP). . Immunophilin binding is necessary but not sufficient for the immunosuppressive activity of these drugs. Biological effects are observed by the interaction of the drug / immunophilin complex and a third effector protein. For example, the CyP-CsA and FKBP-FK506 complex inhibits the serine / threonine phosphatase activity of calcineurin, thereby blocking the production of cytokines such as interleukin-2 ⁴ . On the other hand, the FKBP-rapamycin complex inhibits a kinase called FRAP (also known as RAFT or mTOR) ⁵ , which is involved in interleukin-2 receptor-mediated T cell proliferation.

  A new class of compounds called sanglifehrins has been isolated from Streptomyces genus A92-308110. Of the 20 different sanglifehrins isolated to date, sanglifehrin A (SFA) is the most abundant compound and exhibits strong immunosuppressive activity. SFA represents a new type of immunosuppressive agent whose mechanism of action differs from all other known immunophilin binding compounds, namely CsA, FK506, and rapamycin. SfA has been shown to bind directly to the mitochondrial transition pore (MTP) complex at the site different from other immunophilins and cyclophilin A (CpA) to the immunophilin protein, cyclophilin D (CyD), and inhibit MTP opening. ing.

  Non-immunosuppressive, cyclophilin-binding cyclosporine and their use in the treatment and prevention of AIDS and AIDS-related disorders are described in EP 484281, which is a cyclosporine class of compounds, their nomenclature and mechanism of action. Including the general disclosure of The disclosure of EP 0,484,281B, in particular the general disclosure referred to above and the disclosure of other parts referred to below, is incorporated by reference in the teaching of the present invention.

Surprisingly, cyclosporine that binds to cyclophilin but is not immunosuppressive treats pathological conditions associated with Aβ production and / or secretion, including but not limited to Alzheimer's disease (“AD”). It was found useful as a neuroprotective agent. AD is characterized by the extracellular accumulation of amyloid plaques in the brain consisting mainly of Aβ40 or 42 amino acid peptides. Extracellular accumulation of these peptides is a characteristic pathology of this disease (Selkoe 1999). Aβ peptide is produced by intracellular proteolysis of amyloid precursor protein (APP), a ubiquitously expressed type I transmembrane protein (Selkoe 1999; Sisodia 2000). The two enzymes that cleave APP in the amylogenic pathway are called β- and γ-secretases, which cleave APP from the N- and C-termini, respectively. In this pathway, β-secretase (BACE1) is the first enzyme to cleave APP and produce secreted sAPPβ fragments and membrane-bound C-terminal fragments (CTF, C99) (Vassar, Bennett et al. 1999). . C99 fragment is the substrate for γ- secretase complex (GACE), which produce Aβ and AICD cleaves C99 (APP intracellular domain (A PP I ntra C ellular D omain)). AICD binds to the complex of Tip60 and Fe65 and derepresses KAI1 (tetraspanin cell surface molecule), a gene in the NFκ-B pathway (Baek, Ohgi et al. 2002). The GACE complex consists of four major components, presenilin 1 (PS1), nicastrin (NCSTN), Aph1, and Pen2 (Edbauer, Winkler et al. 2003; Kimberly, LaVoie et al. 2003). The PS1 functional homologue, presenilin 2 (PS2) is responsible for ~ 20% of Aβ produced in cells (Kimberly, Xia et al. 2000). While these four components are necessary and sufficient for reconstitution of GACE activity, there is evidence that non-GACE-mediated cleavage of APP results in Aβ production (Tesco, Koh et al. 2003; Nunan, Shearman et al. 2001).

DETAILED DESCRIPTION OF THE INVENTION Elucidation of novel genes involved in the APP pathway is an important step in determining the complete etiology of the disease as well as developing a good understanding of the complex mechanisms that drive Aβ production. The determination of new genes and pathways that control Aβ will aid in the development of new therapeutic strategies to treat disease progression. Genetic linkage and chromosomal region of several species, associated in particular tardive Alzheimer's disease (L ate O nset A lzheimer's D isease) (LOAD) ( onset from beyond 65 years of age) and especially (Ertekin-Taner Graff-Radford et al. 2000; Bertram, Blacker et al. 2000; Scott, Hauser et al. 2003). A subset of LOAD-related genes, as well as novel genes involved in APP processing are described in co-pending application USSN ____________________________________________________________, large-scale functional screen to test cDNA clones for the ability to modulate Aβ production in CHO K1 cells. Including the disclosure. The disclosure of USSN_, particularly the identification of genes that regulate Aβ secretion and the disclosure of other parts referred to below, is incorporated by reference in the teachings of the present invention.

  The discovery of genes discovered in the immunophilin pathway that serves as an important regulator of intracellular Aβ production provides a drug target in Alzheimer's disease. Several cDNAs found in functional screens involved in the immunophilin pathway include Map kinase 4, calmodulin, acid ceramidase, and TOB3, AAA-ATPase.

  Mitogen-active protein kinase (MAPK), also known as extracellular signal-repressed kinase (ERK), is a family of serine / threonine protein kinases that phosphorylate and positively or negatively regulate target substrates that initiate signal transduction cascade events It is. ERK transmits signals from cell membranes to the nucleus in response to a wide range of stimuli and plays an important role in the control of gene expression, mitosis, proliferation, motility, metabolism, and apoptosis (Wada and Penninger 2004 ). Inhibitors of MEK and ERK activity have been shown to inhibit APP catabolism. In addition, MAPK is a known pathway that can activate JNK, trigger apoptosis and increase Aβ production (Tesco, Koh et al. 2003). Without wishing to be bound by theory, MAPK4 cDNA could work through the activation of APP catabolism or apoptosis.

  Similarly, APP intracellular domain (AICD) fragments interact with cJun N-terminal kinase (JNK), linking the MAP kinase pathway to APP processing (Scheinfeld, Roncarati et al. 2002). MAPK4 is an ERK that activates JNK, so overexpression of MAPK4 can lead to overactivation of JNK, which can increase JNK-AICD interactions leading to increased Aβ production. It is also possible that JNK activation induces apoptosis. Alternatively, MAPK4 has been shown to alter the phosphorylation state of APP, which affects its preferential processing by β- and α-secretases by acting on APP transport and blocking PS1 / β-catenin interactions. (Hung and Selkoe 1994; Walter, Capell et al. 1997). Furthermore, cellular phosphorylation status has also been shown to be important for PS1 activity (Seeger, Nordstedt et al. 1997). JNK activation and protein phosphorylation are also some other pathways by which MAPK4 can work to increase Aβ production.

Another gene identified in this screen is calmodulin. Calmodulin transmits a Ca ²⁺ signal by interacting with specific target proteins including CaMKII, CaMKIV, calcineurin, spectrin A2, p21, and neuronal nitric oxide synthase, loop-helix-loop Ca ²⁺ binding protein (Means 1981). When Ca ²⁺ binds to calmodulin, it undergoes a conformational change that allows it to bind to the target protein and stimulate or inhibit their activity. Calmodulin has been shown to control Aβ production in cell-free preparations and intact cells using the calmodulin antagonists W-7 and trifloperazine. These known inhibitors of calmodulin activity also inhibit Aβ production (Desdouits, Buxbaum et al. 1996). Thus, intracellular Ca ²⁺ concentration and / or calmodulin target protein can affect APP processing. This compound data confirms the fact that an increase in Aβ production is observed when calmodulin is overexpressed.

Ca ²⁺ dysregulation, particularly a decrease in cytoplasmic Ca ²⁺ levels, coincides with an increase in Aβ production associated with PS1 FAD mutations (Yoo, Cheng et al. 2000). PS1 FAD mutations can significantly attenuate capacitive calcium entry (CCE) and store depletion activation currents, suggesting that reduced CCE can increase Aβ production (Yoo, Cheng et al. 2000) . The overexpression of the calmodulin gene seen in this screen is also responsible for the reduced intracellular calcium level and can block proper CCE. A decrease in intracellular Ca ²⁺ may directly or indirectly increase PS1 activity, leading to more Aβ secretion from the cell. The data detailed below suggests that SfA effectively inhibits Aβ40 and Aβ42 production and C99 and Notch cleavage, suggesting that gamma secretase activity is linked to Ca ²⁺ homeostasis via the immunophilin protein CyD. .

  Another gene, human acid ceramidase, catalyzes the hydrolysis of ceramide to sphingosine and fatty acids (Ferlinz, Kopal et al. 2001). Ceramide is a signaling molecule that acts as a precursor to most sphingolipids and induces apoptosis of many different cell types, typically through caspase-3 activation. Increased ceramide levels are associated with Alzheimer's disease and are considered part of the oxidative neurotoxic pathway in the elderly AD brain. Overexpression of ceramide increases Aβ production, linking ceramidase cleavage and Aβ. Since ceramide is thought to work through apoptosis, acid ceramidase may increase apoptosis as well.

  The ceramide state of cells has been shown to regulate both BACE stability and Aβ biosynthesis (Puglielli, Ellis et al. 2003). Previous studies have demonstrated that high levels of ceramide can increase Aβ secretion in an apoptosis-independent manner (Puglielli, Ellis et al. 2003). Ceramidase overexpression reduces ceramide levels, a pathway known to alter tau phosphorylation and Aβ polymerization, increases sphingosine and free fatty acid levels (FFA), and possibly alters membrane fluidity (Wilson and Binder 1997) . Although the mechanism is not clear, it is likely that ceramidase affects FFA levels leading to alterations in APP processing and more Aβ production and / or secretion.

  TOB3 is an AAA-ATPase that performs chaperone-like functions that assist in the association, activation, or disassembly of protein complexes, including protein secretion (Strausberg, Feingold et al. 2002). This class of proteins helps maintain the integrity of the endoplasmic reticulum (ER) as the protein is processed continuously. Without AAA-ATPase activity, excessive accumulation of misfolded proteins occurs, causing ER elongation and cell death (Kobayashi, Tanaka et al. 2002). The most likely explanation for TOB3 overexpression increasing Aβ secretion is related to its ability to perform protein folding and transport in the ER. When this process is stimulated with TOB3 overexpression, APP processing may increase.

  Our screening data show that TOB3 overexpression alters APP processing leading to more Aβ production and fewer C99 and C83 C-terminal fragments. TOB3 expression also reduces APP and sAPPα levels in N2A cells more than in HEK 293 cells (data not shown). This suggests that TOB3 affects one or more components of the APP processing mechanism leading to increased APP and C99 cleavage. Since TOB3 is involved in protein transport and processing, the exact mechanism and properties of TOB3 for APP processing are currently being measured.

  Carboxypeptidase Z (CPZ), another cDNA identified from functional screening, is a member of the metallocarboxypeptidase gene family along with CPE and CPD, which is the intracellular processing of bioactive peptides and proteins prior to their secretion It is believed to function in CPZ is a unique carboxypeptidase because it contains a functional N-terminal cysteine-rich frizzled domain that binds Wnt and wingless proteins, which are important components for Wnt signaling (Moeller, Swindell et al. 2003). ).

  Another interesting cDNA is cyclophilin D (CyD, also known as CyF. A nomenclature quote can be found in Current Medicinal Chemistry, 2003, 10, 1485-1506 1485 Cyclophilin D as a Drug Target, Waldmeier, et al). . CyD has been found to be involved in Aβ production. Cyclophilin proteins such as CyD are peptidyl prolyl isomerases involved in protein transport and maturation. Cyclophilin is mainly localized in the cytoplasm, except for CyD, which is exclusively localized in the mitochondrial matrix (Waldmeier, Zimmermann et al. 2003). When CyD is overexpressed, endogenous PS1 N-terminal fragment (NTF) can be stabilized. A common lesion in AD brain is the presence of intracellular neurofibrillary tangles composed of abnormally phosphorylated tau, a microtubule-associated protein (Johnson and Bailey 2002). When overexpressed with APP, CyD may activate caspase-3 activity, suggesting a possible mechanism by which Aβ secretion is regulated. These results suggest that CyD, when overexpressed, is an important factor required for the cleavage of APP and C99 via the γ-secretase pathway.

  In one aspect, CyD is an essential component of the mitochondrial permeability transition pore that is thought to bind the adenine nucleotide transporter and regulate open pores (Waldmeier, Zimmermann et al. 2003). Mitochondrial membrane permeabilization is the result of cellular stress, leading to loss of mitochondrial membrane potential, release of apoptotic proteins, and ultimately apoptotic cell death (Waldmeier 2002).

  Mitochondrial failure has been suggested to have an important role in AD neuropathology in Down syndrome patients by promoting abnormal β-APP processing and intracellular accumulation of Aβ (Busciglio 2002). Increased intracellular calcium levels have also been shown to reveal intracellular Aβ accumulation (LaFerla 2002). Surprisingly, in the brain of a transgenic mouse model of AD, full-length APP is not only transported along the secretory pathway, but is also targeted to the mitochondria of cultured cortical neurons. Incomplete translocation and progressive accumulation of β-APP in the mitochondrial membrane can lead to mitochondrial dysfunction and may have an important role in the pathogenesis of AD (Anandatheerthavarada, Biswas et al. 2003). It is known that overexpression of mitochondrial proteins can lead to the formation of insoluble aggregates within the matrix (eg uncoupling protein-3, UCP3). These aggregates can both disrupt the normal function of mitochondria and increase the passive permeability of the inner membrane. CyD may have an important role in mitochondria that processes excess APP or C-terminal fragment processing.

  CyD is a known target for immunosuppressive drugs such as cyclosporine. These compounds block mitochondrial permeability transition (MPT) and prevent apoptosis (Samantha J. Clarke 2002; Waldmeier 2002). The FK506 compound is also known to bind to immunophilin proteins but not to CyD (Uchino 2003).

Cyclosporine binds at least 1/5 of cyclosporine (also known as cyclosporin A) to human recombinant cyclophilin in a competitive ELISA test described by Quesniaux in Eur. J. Immunol. 1987, 17 , 1359-1365. When considered, it is considered to be cyclophilin. In this test, the cyclosporine to be tested is added during the incubation of cyclophilin and coated BSA-cyclosporine and the concentration required to produce 50% inhibition of the control reaction without competitor (IC ₅₀ ) is calculated. To do. The results are shown as binding ratio (BR), which is the logarithm of the base 10 of the ratio of the IC ₅₀ of the test compound to the IC ₅₀ in a similar test using cyclosporine instead of the test cyclosporine. Thus, a BR of 1.0 indicates that the test compound binds to cyclophilin less than one-tenth of cyclosporine, and a negative value indicates that it binds more strongly than cyclosporine.

Cyclosporine active as a neuroprotective agent has a BR, preferably less than or equal to 0.7 (since log _1O 5 = about 0.7), preferably 0 or less.

Cyclosporine is considered non-immunosuppressive when it has no more than 5%, preferably no more than 2%, activity of cyclosporine in a mixed lymphocyte reaction (MLR). The mixed lymphocyte reaction is described by T. Meo in “Immunological Methods”, L. Lefkovits and B. Peris, Eds., Academic Press, NY pp. 227-239 (1979). Spleen cells (0.5 × 10 ⁶ ) from Balb / c mice (female, 8-10 weeks) were irradiated (2000 rads) or mitomycin C from CBA mice (female, 8-10 weeks) for 5 days. Co-incubate with treated 0.5 × 10 ⁶ spleen cells. Irradiated allogeneic cells elicit a proliferative response in Balb c spleen cells, which can be measured by incorporation of labeled precursor into DNA. Because the stimulator cells were irradiated (or treated with mitomycin C), they do not proliferate to Balb / c cells but maintain their antigenicity. The IC ₅₀ seen with the test compound in the MLR is compared to that seen with cyclosporine in parallel experiments.

  Compounds that are determined to be non-immunosuppressive in the MLR are often found to be inactive in the IL-2 reporter gene assay, and therefore the IL-2 reporter gene assay is for use in the present invention. It can be used as a primary screen for selection of non-immunosuppressive, cyclophilin-binding cyclosporine compounds, for example.

  Non-immunosuppressive, cyclophilin-binding cyclosporin compounds that are active as inhibitors for the extracellular accumulation of amyloid plaques in AD, for example, as agents for treating pathologies associated with Aβ secretion are hereinafter referred to as active compounds.

  The active compounds are therefore useful for the treatment of any clinical condition involving Aβ peptide secretion, endogenous PS1 N-terminal fragment (NTF) expression or increased gamma-secretase activity. Furthermore, the active compounds are useful in the treatment of conditions involving apoptosis that increase Aβ secretion. Aβ peptide formation and subsequent aggregation is not only a feature of AD, but is also an essential component of other neurological diseases such as Parkinson, Huntington, and other systemic amyloidosis (Selkoe 1989; Price, Borchelt et al. 1993; Citron, Vigo-Pelfrey et al. 1994). From these results it is clear that apoptosis and Aβ production are completely linked.

  The active compounds also have utility for modulating “peripheral Aβ modifiers” that are not expressed in the brain. Peripheral amyloidosis can result in a phenotype like heart and dermatological amyloidosis (Yamaguchi, Yamazaki et al. 1992), (Selkoe 1989). If a key regulator of peripheral Aβ is discovered, new therapeutic agents can be delivered to such targets, eliminating the need to cross the blood brain barrier. Decreased peripheral Aβ levels have been shown to reduce Aβ levels and reduce plaque formation in transgenic mouse brain (Bohrmann, Tjernberg et al. 1999; DeMattos, Bales et al. 2002).

  It has been found that many of the active compounds have a structure that differs from cyclosporine, especially at the 4 and / or 5 positions. Other positions where the structure of the active compound may differ from cyclosporine are the 6 and 7 positions.

One group of active compounds is an N-methylated amino acid with a different MeLeu group at position 4, such as γ-hydroxy-MeLeu, MeIle, MeVal, MeThr, MeAla, MeTyr or MeTyr (O—PO (OH) ₂ ), or Cyclosporine substituted with Pro. In addition to MeIle and MeThr, allo-form MealIle and MeaThr can also be used. In the variant, the stereochemistry at the β-position has the opposite configuration as that of the natural amino acid, so that the normal and variant forms diastereoisomeric pairs.

A further group of active compounds are those in which the Val at position 5 is substituted with an N-alkyl-amino acid, preferably N-methyl-amino acid. Preferably, the N-alkylated amino acid is Val or Leu. Preferably, the hydrogen of the imino group of [Val] ⁵ is substituted with an unbranched C _1-6 alkyl group, preferably methyl, ethyl or n-propyl, especially methyl. The latter preferred group of active compounds is all novel.

  Additionally or alternatively, certain active compounds may differ from cyclosporine at the 1, 2, 3, and / or 6 position.

〔式中、Ｂは式Ｂ

[式中、ａは２位のαＡｂｕ残基への結合を意味し；
ｂは、４位の残基Ｃへの結合を意味し；
Ａｌｋは２〜６個の炭素原子を含む直鎖または分枝鎖アルキレンまたは３〜６個の炭素原子を含むシクロアルキレンを意味し、そして
Ｒは
カルボキシまたはアルキルオキシカルボニルラジカル；
ラジカル−ＮＲ_１Ｒ_２(ここで、Ｒ_１およびＲ_２は同一または異なって、水素、アルキル、Ｃ_２−４アルケニル、Ｃ_３−６シクロアルキル、フェニル(所望によりハロゲン、アルコキシ、アルコキシカルボニル、アミノ、アルキルアミノまたはジアルキルアミノで置換されていてよい)またはベンジル、または５または６環原子および１〜３個のヘテロ原子を含む飽和もしくは不飽和ヘテロシクリルラジカルであるか、またはＲ_１およびＲ_２は、それらが結合している窒素原子と一体となって、４〜６環原子を含み、そして窒素、酸素または硫黄から選択されるヘテロ原子をさらに含んでいてよく、かつアルキル、フェニルまたはベンジルで置換されていてよい飽和または不飽和ヘテロ環を形成する)；
式

(式中、Ｒ_１およびＲ_２は上記で定義の通りであり、Ｒ_３は水素またはアルキルラジカルであり、そしてｎは２〜４の整数であり、
そして、アルキルは１〜４個の炭素原子を含む直鎖または分枝鎖アルキルである。)
のラジカルである。]
のアミノ酸残基であり；
ＣはＭｅＬｅｕまたは４−ヒドロキシ−ＭｅＬｅｕである。〕
のシクロスポリン誘導体およびその薬学的に許容される塩である。 A particular class of active compounds for use in the present invention is of formula A

[Where B is the formula B

[Wherein a represents a bond to the αAbu residue at position 2;
b means a bond to residue C at position 4;
Alk means a straight or branched alkylene containing 2 to 6 carbon atoms or a cycloalkylene containing 3 to 6 carbon atoms, and R is a carboxy or alkyloxycarbonyl radical;
Radical —NR ₁ R ₂ (wherein R ₁ and R ₂ are the same or different and are hydrogen, alkyl, C _2-4 alkenyl, C _3-6 cycloalkyl, phenyl (optionally halogen, alkoxy, alkoxycarbonyl, amino Is optionally substituted with alkylamino or dialkylamino) or benzyl, or a saturated or unsaturated heterocyclyl radical containing 5 or 6 ring atoms and 1 to 3 heteroatoms, or R ₁ and R ₂ are Together with the nitrogen atom to which they are attached, it contains 4-6 ring atoms and may further contain a heteroatom selected from nitrogen, oxygen or sulfur and is substituted with alkyl, phenyl or benzyl. A saturated or unsaturated heterocycle which may be
formula

Wherein R ₁ and R ₂ are as defined above, R ₃ is hydrogen or an alkyl radical, and n is an integer from 2 to 4,
Alkyl is a straight or branched alkyl containing 1 to 4 carbon atoms. )
The radical of ]
Amino acid residues;
C is MeLeu or 4-hydroxy-MeLeu. ]
Cyclosporine derivatives and pharmaceutically acceptable salts thereof.

であり、そしてＣがアミノ酸残基４−ヒドロキシ−ＭｅＬｅｕである、式Ａの化合物である。 This class of cyclosporine derivatives is further described in published international patents WO 98/28328, WO 98/28329 and WO 98/28330. Particularly preferred compounds of this class are those in which B is the amino acid residue B ′

And C is the amino acid residue 4-hydroxy-MeLeu.

〔式中、
ＷはＭｅＢｍｔ、ジヒドロ−ＭｅＢｍｔまたは８'−ヒドロキシ−ＭｅＢｍｔであり；
ＸはαＡｂｕ、Ｖａｌ、Ｔｈｒ、ＮｖａまたはＯ−メチルスレオニン(ＭｅＯＴｈｒ)であり；
ＲはＳａｒまたは(Ｄ)−ＭｅＡｌａであり；
ＹはＭｅＬｅｕ、γ−ヒドロキシ−ＭｅＬｅｕ、ＭｅＩｌｅ、ＭｅＶａｌ、ＭｅＴｈｒ、ＭｅＡｌａ、ＭｅＴｙｒ、ＭｅＴｙｒ(Ｏ−ＰＯ(ＯＨ)_２)、ＭｅａＩｌｅまたはＭｅａＴｈｒ、またはＰｒｏであり；
ＺはＶａｌ、Ｌｅｕ、Ｎ−Ａｌｋ−ＶａｌまたはＮ−Ａｌｋ−Ｌｅｕであり、
ここで、Ａｌｋは、Ｍｅまたビニル(これは所望によりフェニル、または６環員を含むＮ、ＳもしくはＯヘテロアリールで置換されていてよい)もしくはフェニル(これは所望によりハロゲンで置換されていてよい)で置換されたＭｅを意味し、そして
ＱはＭｅＬｅｕ、γ−ヒドロキシ−ＭｅＬｅｕまたはＭｅＡｌａである。〕
の化合物およびその薬学的に許容される塩から成る。 A particularly preferred group of active compounds is of formula I:

[Where,
W is MeBmt, dihydro-MeBmt or 8′-hydroxy-MeBmt;
X is αAbu, Val, Thr, Nva or O-methylthreonine (MeOTTh);
R is Sar or (D) -MeAla;
Y is MeLeu, γ-hydroxy-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;
Where Alk is Me or vinyl (which is optionally substituted with phenyl or N, S or O heteroaryl containing 6 ring members) or phenyl (which is optionally substituted with halogen) ) And Me is MeLeu, γ-hydroxy-MeLeu or MeAla. ]
And a pharmaceutically acceptable salt thereof.

The groups W, X, Y, Z and Q independently have the following preferred meanings:
W is preferably W ′, where W ′ is MeBmt or dihydro-MeBmt;
X is preferably X ′, where X ′ is αAbu or Nva, more preferably X ″, where X ″ is αAbu;
Y is preferably Y ′, where Y ′ is γ-hydroxy-MeLeu, MeVal, MeThr, MeAla or MeTyr (O—PO (OH) ₂ );
Z is preferably Z ′, where Z ′ is Val or MeVal; and Q is preferably Q ′, where Q ′ is MeLeu.

  One particularly preferred group of active compounds are those compounds of formula I wherein W is W ′, X is X ′, Y is Y ′, Z is Z ′ and Q is Q ′. is there.

Particularly preferred active compounds of the formula I are:
a) [dihydro-MeBmt] ^1- [γ-hydroxy-MeLeu] ⁴ -cyclosporine,
b) [MeVal] ⁴ -cyclosporine,
c) [MeIle] ⁴ -cyclosporine,
d) [MeThr] ⁴ -cyclosporine,
e) [γ-hydroxy-MeLeu] ^4- cyclosporine,
^{f) [Nva] 2 - [} γ- hydroxy-MeLeu] ⁴ - cyclosporine,
g) [.gamma.-hydroxy -MeLeu] ⁴ - [γ- hydroxy-MeLeu] ⁶ - cyclosporin,
h) [MeVal] ⁵ -cyclosporine,
i) [MeOThr] ² -[(D) MeAla] ^3- [MeVal] ⁵ -cyclosporine,
j) [8′-hydroxy-MeBmt] ¹ -cyclosporine,
k) [MeAla] ⁶ -cyclosporine,
l) [DMeAla] ^3- [MeTyr (OPO (OH) ₂ )] ⁴ -cyclosporine,
m) [N-benzyl-Val] ⁵ -cyclosporine,
n) [N-5-fluoro-benzyl-Val] ⁵ -cyclosporine,
o) [N-allyl-Val] ⁵ -cyclosporine,
p) [N-3-phenyl-allyl-Val] ⁵ -cyclosporine,
q) [Pro] ⁴ -cyclosporine.

Particularly preferred active compounds are [MeIle] ⁴ -cyclosporine and [γ-hydroxy-MeLeu] ⁴ -cyclosporine, most particularly [MeIle] ⁴ -cyclosporine.

In addition to compounds of formula I, preferred active compounds include, for example, r) [γ-hydroxy-MeLeu] ⁹ -cyclosporine.

The active compound is:
1) Fermentation 2) Biotransformation 3) Derivatization 4) Partial synthesis 5) It can be obtained by a method including total synthesis.

  These methods are described generally and more specifically in Examples 0 to 10 of EP 0484281B and US Pat. No. 5,767,069. This general description and the teachings of these examples are incorporated herein by reference. Example 11 of EP 0484281B describes the measurement of the immunosuppressive and cyclophilin binding activity of each active compound against cyclosporine and the teaching of this example is also included within the scope of the disclosure herein.

  Thus, the present invention relates to Alzheimer's disease, Parkinson's disease, tauopathy, prion disease, frontotemporal dementia, striatal nigra degeneration, Lewy body dementia, Huntington's disease, Pick's disease, amyloidosis, and excessive Aβ Provided is the use of a non-immunosuppressive, cyclophilin-binding cyclosporine in the manufacture of a medicament for the treatment or prevention of conditions associated with Aβ secretion, such as other neurodegenerative disorders associated with production.

  The invention further includes Alzheimer's disease, Parkinson's disease, tauopathy, prion disease, frontotemporal dementia, striatal nigra degeneration, Lewy body dementia, Huntington's disease, Pick's disease, amyloidosis, and other A method of treating a condition associated with Aβ secretion, such as a neurodegenerative disorder, comprising administering to the patient an effective amount of an active compound of the invention.

  The active compounds may be administered by any conventional route, in particular enterally, for example orally, for example in the form of a drinking liquid, tablet or capsule, or parenterally, for example in the form of an injectable solution or suspension. Can be administered. The daily dose indicated may be 1-20 mg / kg, preferably 3-10 mg / kg by intravenous route and 1-50 mg / kg, preferably 10-30 mg / kg by oral route.

  It is believed that the active compound is less toxic than cyclosporine. Because the active compound is not immunosuppressive, certain side effects of cyclosporine associated with immunosuppression are avoided. Side effects associated with other cyclosporine, particularly nephrotoxicity and central nervous system toxicity in long-term use, are conveniently less than that of cyclosporine.

  Preferred galenical formulations for the active compounds are based on microemulsions as described in UK patent application 2222770A including topical as well as oral forms; also fatty acid saccharide monoesters such as saccharose as described in UK patent application 2209671A Includes solid solutions containing monolaurate. Suitable unit dosage forms for oral administration contain, for example, 25-200 mg of active compound per dose.

Formulation examples A, B, C and D of EP 0484281B are incorporated herein by reference:
The ingredients here of these formulations, as well as their method of manufacture, are fully described in British Patent Application 22222770, the contents of which are hereby incorporated by reference.

The usefulness of the active compounds as neuroprotective agents can be demonstrated in in vivo or in vitro tests, for example:
The active compounds of the invention can be provided alone, in combination with other agents, or in continuous combination. For example, an active compound of the invention may be combined with an anti-inflammatory agent, such as but not limited to a corticosteroid, to block further neuropathy and inhibit axonal regeneration after a stroke or spinal cord injury, and NGF Can be administered in combination with neurotrophic factors such as BDNF or other agents for neurodegenerative diseases such as Exelon ^™ or levodopa. As used herein, two agents are said to be administered in combination when the two agents are administered simultaneously or when administered independently in a form in which the agents act simultaneously.

  The structure of the active ingredient identified by code number, generic name or trade name can be taken from the current edition of the standard introduction “The Merck Index” or from databases such as Patents International (eg IMS World Publications). Their corresponding contents are hereby incorporated by reference. Those skilled in the art are well capable of identifying active ingredients and can similarly produce and test pharmaceutical indications and properties both in vitro and in vivo in standard test models.

  In the above indications, the appropriate dosage will, of course, vary depending on, for example, the particular molecule of the invention used, the mode of administration and the nature and severity of the condition being treated.

The following method is performed to carry out the examples described below:
Transfection. CHO K1 cells (ATCC, Manassas, VA) are plated with DMEM, 10% fetal calf serum, 5% penicillin / streptomycin (Penn / Strep), and 22 mg L-proline (Sigma Chemical, St. Louis, MO). . Plates are incubated overnight at 37 ° C. in a water jacketed CO ₂ cell culture chamber. The cDNA of interest is cotransfected with full length APP at a 1:15 ratio (cDNA: APPwt (695)) using Qiagen® SuperFect reagent as per manufacturer's instructions. In a 6-well dish, 5 × 10 ⁵ cells are plated with DMEM, 10% fetal calf serum, 5% penicillin / streptomycin (Sigma Chemical, St. Louis, MO) and grown for 24 hours. A SuperFect mix is made with 100 μl serum free medium (DMEM), 3 μg total DNA, and 20 μl SuperFect. Remove the medium from the cells and add 1 mL of fresh medium. Add the entire SuperFect mixture to the medium and incubate at 37 ° C. for 2 hours. The mixture is then removed and the cells are washed once with 3 mL PBS. Fresh media is added back to the cells and they are incubated for 24 or 48 hours.

Immunocytochemistry-CHO cells are transfected with the appropriate construct using Lipofectamine as described by the manufacturer (Life Technologies) in OPTIMEM (serum-free medium). CHO cells are immunostained and the immunoreactivity is observed as described by Dev et al 1999, Neuropharmacol., (1999) 38, 635-644. Flag-protein expression is detected using anti-Flag M2 monoclonal mouse antibody (Sigma), secondary antibody is Texas Red-X goat anti-rabbit IgG (Molecular Probes).

Flow cytometric analysis of caspase-3. HEK cells are transiently transfected with APPwt with CyD or CPZ for 24 hours. Cells are fixed, permeabilized and stained using the caspase-3 apoptosis kit from BD Biosciences (# 550914). FITC stained cells were> allowed pass through 10 ^first gate, measured as cells positive for active caspase-3. Forward and side light scatter is also tested for confirmation of the health of the cell population.

Compound treatment. HEK 293 cells stably expressing APPswe mutants were treated with cyclosporin A, sanglifehrin A, FK506 (Sedrani, et al., J. Am. Chem. Soc. 125, which is incorporated by reference in its entirety) for 24 hours. , pp 3849-3859 (2003)) and N-methyl-4-valine-cyclosporin concentrate. Cell viability is measured using Promega's CellTiter-Glo Kit (# G7573). Aβ40 and 42 levels are measured in the cell supernatant using an in-house 2 sandwich ELISA (the in-house ELISA is the same form as described except that the antibody developed at Novartis is used in place of the Biosource antibody). Cell lines that stably express the 5xGal4 response element-luciferase reporter and C99-Gal4-VP16 or Notch-Gal4-VP16 constructs are used to measure C99 or Notch cleavage (as described in Maltese, Wilson et al. 2001). ). When cleaved with γ-secretase, Gal4-VP16 is released and binds to the reporter, increasing luciferase activity. Stable cells are treated with the compound and IC ₅₀ values are determined.

Aβ ELISA. A commercially available mouse monoclonal antibody against the NH ₂ terminus of the Aβ peptide is used as a capture antibody in a pre-coated 96 well plate (Biosource Cat # KBH3481 / PPO81 for Aβ40 and Cat # KBH3441 / PPO81 for Aβ42). Polyclonal detection antibodies are obtained from Biosource (anti-hAβ40 Cat # 44-348 and anti-hAβ42 Cat # 44-344) and diluted 1/220 in 15 mM sodium azide. Secondary antibodies (Biosource Cat # KBH3481 for Aβ40 and Cat # KBH3441 for Aβ42) are horseradish peroxidase labeled anti-rabbit IgG. The secondary antibody is diluted 1/100 in 3.3 mM thymol. Antibody-coated plates are washed 4 times with PBS-TE (1 mM EDTA and 0.05% Tween 20, wash buffer) with a microplate washer (Bioteck Instruments, Inc, Winooski, VT) prior to use. Take 100 μl of conditioned medium of transfected cells and dilute 1: 2 in sample diluent containing 1 mM AEBSF (Biosource, Camarillo, Calif.). 100 μl of this mixture is added to a washed, antibody-coated 96 well plate, covered with tape and incubated at 4 ° C. overnight. Remove sample and wash plate 4 times with wash buffer. Detection antibody solution is added at 100 μl / well and incubated for 2 hours at room temperature with shaking. The plate is washed again 4 times with wash buffer and the secondary antibody solution is added at 100 μl / well and incubated for 2 hours with shaking. Wash plate 5 times with wash buffer and dab onto paper towel to dry. 100 μl of stabilized chromogen (tetramethylbenzidine) is added to each well and the plate is incubated for 30 minutes in the dark. 100 μl of stop solution (1N H ₂ S) is added to the plate to stop the reaction. The plate is read with a microplate reader at 450 nM (Molecular Devices) for 1 hour.

Antibody and Western blot analysis. APP wild type and APP Swedish mutant cDNAs are inserted downstream of the cytomegalovirus promoter of the pCl plasmid expression vector as previously described (Promega, Madison, Wis.) (Bodendorf, U., Fischer, F., Bodian , D., Multhaup, G., Paganetti, P. 2001 J. Biol. Chem. 276: 12019 12023).

PS1 NTF antibody recognizes the N-terminus of PS1 (Thinakaran, Borchelt et al. 1996). Cultured HEK 293 cells were treated with RIPA buffer (10 mM Tris, pH 7.5, 150 mM sodium chloride, 1 mM EDTA, 1% Nonidet P-) containing protease inhibitor (Complete® Roche Molecular Biochemicals) 24 hours after transfection. 40, 0.5% sodium deoxycholate, 1% SDS) and centrifuged at 10,000 × g for 10 minutes at 4 ° C. Collect the supernatant and discard the pellet. Subsequently, cell extracts are separated by SDS polyacrylamide gel electrophoresis, transferred to PVDF Immobilon-P® membrane (Millipore) and probed with primary antibody as indicated. Immunological detection is performed with the ECL detection system (Amersham Pharmacia Biotech, Piscataway, NJ) as previously described (Manni, M., et al., 1998. FEBS. 427: 367-370).

Immunocytochemistry-CHO cells are transfected with the appropriate construct using Lipofectamine as described by the manufacturer (Life Technologies) in OPTIMEM (serum-free medium). CHO cells are immunostained and the immune activity is observed as previously described (Dev et al 1999, Neuropharmacol., (1999) 38, 635-644). Flag-protein expression is detected using anti-Flag M2 monoclonal mouse antibody (Sigma), secondary antibody is Texas Red-X goat anti-rabbit IgG (Molecular Probes).

CPZ construct-Wild type human carboxypeptidase (hCPZ) from a standardized cDNA library of pure human DRG (L0001) purchased from Life Technologies, Inc (LTI, Catalog Number: 11315-017, Lot Number: 81027-242) obtain. The cDNA insert is cloned in the 5 'to 3' direction into the Gateway compatible vector, EcoR V and Not I sites in pCMV • SPORT6. Frizzled (FZ) domain corresponding to amino acids 42-161, deletion of the catalytic (Cat) domain of amino acids 179-568, point mutation (amino acid E251A), and N-terminal FLAG label after amino acid 29 (DYKDDDDK SEQ ID NO: 1) Is performed by site-directed mutagenesis using the following primers:
5 'CCTCCAGGCCCTCCCCGAAGCTTCTCCGCGGCTGTCTGCAGCTGGTGGCCTGTGGG-3' (SEQ ID NO: 2) and 5 'CCCAGGGCACCAGCTGCAGACAGCGCCGAGAAGCTTCGGGGGAGCCCTGGAGG-3' for frizzled domain removal
5 'CCACACGGCCAGCCCTCTTCCATCCGGGGCCAGCCCTGAGGGCAGTGCCCTCGTCAGC-3' (SEQ ID NO: 4) and 5 'GCTGACGGAGCACTGCCCCTCAGGGCTGGCCCGGGATGAGGGTGGGCCGTGGTG
5 ′ GCATCTCCCGGCCCGCCCACCCGCGTGCCATGAATGTTGC-3 ′ (SEQ ID NO: 6) and 5 ′ GCAACATTCATGGCAACGCGGGTGGCGGGCCGGGAGATCC-3 ′ (SEQ ID NO: 7) for point mutation E251A
As well as 5'GGTGGCCCTGTGGGATTCACCCTTGTCATCGTCCGTCCTTGTAGTCCGGGCGGGTCTCCGTCCAAACTCG-3 '(SEQ ID NO: 8) and 5'CGAGTTGAGCCGGAACCCGCGCCGATACCAGAGACGCG Isolation of total DNA from transformed E. coli is performed using the Qiagen plasmid kit. All open reading frames are sequenced by DNA sequencing using the ABI Prism 3700 DNA Analyzer system.

In situ hybridization-Using polymerase chain reaction (PCR) with self-priming oligonucleotide primers flanked by 5 'SP6- and T7-promoter recognition sequences, the riboprobe template can be obtained without any cloning steps. It can be produced from any known gene sequence. The PCR reaction is performed in 40 cycles of a denaturation step at 95 ° C. for 45 seconds, an annealing phase of 58 ° C. for 30 seconds and an extraction phase of 1 minute at 70 ° C. After separation on a 4% agarose gel, the PCR product is clarified with the QiAQuick purification kit according to the manufacturer's instructions (Qiagen, Switzerland). The clarified PCR product is transcribed using digoxigenin-UTP-containing dNTPs at 37 ° C. for 2 hours using T7-RNA polymerase (antisense) and SP6-RNA polymerase (sense). Exclude unincorporated nucleotides and probe the ethanol, dissolve in 50 μl water, and then store at −20 ° C. Serial dilutions of DIG probe and labeled control RNA (known concentration) are spotted onto a nylon membrane. Incubation with alkaline phosphatase-conjugated anti-DIG antibody was performed using NBT / BCIP (50 mg / ml 5-bromo-4-chloro-3- in 75% / ml nitroblue tetrazolium and 100% dimethylformamide in 70% dimethylformamide and 30% water. Indoyl phosphate) is followed by a chromogenic phase which is used as a substrate for alkaline phosphatase. CPZ probe concentration is estimated by comparison with the spot intensity of the labeled control RNA. ISH is performed using the fully automated device Discovery ^™ (Ventana Medical Systems, Strasbourg) for in situ hybridization and immunochemistry. The protocol performed to localize this gene using paraffin-embedded tissue sections was developed within the RNA Analytics Laboratory and is described below, and deparaffinization and rehydration of tissue sections is done in solvent-free conditions. EZprep solution (Ventana Medical Systems SA, Strasbourg) for 8 minutes at 75 ° C., followed by an additional 8 minutes at 42 ° C. All pretreatment steps were performed with the RiboMap ^™ kit (Ventana Medical Systems SA, Strasbourg), with one additional permeabilization step using enzymatic digestion according to the manufacturer's instructions: the best results were obtained at 12 μg / ml Obtained with proteinase K at 37 ° C. in 16 minutes. CPZ probe hybridization is performed at 45 ° C. for 6 hours with matched amounts of DIG riboprobe (CPZ probe = 5 ng / slide) diluted in RiboHybe solution (Ventana Medical Systems SA, Strasbourg). Post-hybridization washing is performed 3 times at 50 ° C. for 8 minutes under high stringency conditions (0.1 × SSC). For DIG-labeled detection, after 1/2000 dilution in antibody diluent, biotin-conjugated mouse anti-digoxin antibody (Jackson Immunoresearch Inc.) was applied for 30 minutes at 37 ° C., and BCIP / NBT chromogenic detection was performed using BlueMap ^™. Use kit (Ventana Medical Systems SA, Strasbourg) according to manufacturer's instructions. The substrate incubation time for optimal signal / noise ratio is 4 hours. Perform ISH Nuclea Fast Red for 10 minutes. Sections are mounted in Crystal mounts and post-mounted using Permount.

Example 1
Presenilin processing: The observation that overexpression of cDNA increased Aβ levels and C99 substrate suggests an increase in GACE-mediated cleavage of APP. Because CPZ and CyD significantly increased C99 processing and increased Aβ42 secretion, they are selected for further testing. PS1 N-terminal fragment (NTF) levels are tested in HEK 293 cells transfected with CPZ or CyD. In cells stably transfected with PS1, both full-length PS1 and NTF are readily detectable. In contrast, endogenous full-length PS1 and NTF levels are low in untransfected HEK cells. There is a clear increase in PS1 NTF levels in cells overexpressing CPZ or CyD, indicating that these fragments are produced at a high rate during the 48 hour transfection period, or that endogenous NTF is stabilized. Suggest.

Analysis of CPZ: CPZ is a member of the carboxypeptidase (CP) gene family within the CPE subfamily known to process bioactive neuropeptides (Song and Fricker 1997). CPE-related enzymes are generally involved in selective processing reactions by selective removal of basic residues from the C-terminus of the processing intermediate. To determine whether CPZ catalytic activity, expression, or subcellular localization is required for Aβ secretion induction, three mutant constructs of CPZ are produced. In the first construct, the catalytic domain is completely removed (Δcat), leaving the signal peptide, the frizzled domain, and the C-terminus. In the second construct, the frizzled domain (Δfz) is removed, and in the third construct a single point mutation (Glu251Ala) that would impair the catalytic activity of CPZ is inserted into the catalytic domain. Each CPZ variant is co-transfected into HEK 293 cells with either APPwt or C99 substrate. None of the three mutant constructs is active compared to wild-type CPZ or Flag-tagged CPZ. The results are similar for both APPwt and C99 substrates.

  A possible explanation for the lack of activity of the CPZ variant is a loss or mislocalization of protein expression in the cell. To solve this question, CPZ mutants are Flag labeled and overexpressed in CHO cells. In permeabilized cells, CPZ wild type shows clear plasma membrane or late secretory vesicle localization. This result is consistent with previous studies that examined the intracellular distribution of CPZ (Novikova, Reznik et al. 2000). Intracellular distribution remains unchanged with CPZ mutant constructs; Δcat, Δfz, and Glu251Ala. These findings suggest that CPZ is required for the induction of Aβ levels in CHO cells, rather than the level of its intracellular localization or expression.

  Disruption of CPZ catalytic activity eliminates Aβ production without affecting expression or subcellular distribution. Overexpression of CPZ can compete with Wnt binding to the endogenous frizzled receptor, resulting in low β-catenin activation (Tesco, Kim et al. 1998). The decrease in β-catenin activity is associated with increased Aβ production and is thought to be mediated by the ability of PS1 to increase the association of β-catenin with GSK3β (Kang, Soriano et al. 1999). . It is possible that CPZ can induce Aβ production through activation of caspase-3 PS1 cleavage, leading to fewer PS1 / β-catenin complexes (Tesco, Kim et al. 1998). If PS1 is not bound by β-catenin, it can more easily associate with the GACE complex. Since the frizzled domain is characterized as a protein interaction domain, it is possible for the frizzled domain in CPZ to interact with components of the γ-secretase complex, ie, -PS1, or APP itself. These interactions are currently under investigation.

  Interestingly, the C-terminus of CPZ contains a putative furin cleavage site and is thought to release CPZ from the plasma membrane to the extracellular environment (Novikova, Reznik et al. 2000). Furin is also involved in the release of Notch ligand Delta at cell surface Notch (Ikeuchi and Sisodia 2003). Although the furin site in CPZ has not been identified as a furin substrate, it has been demonstrated that CPZ can be secreted from cells (Novikova, Reznik et al. 2000). This is because CPZ is in the same processing pathway as Notch, but it is currently unclear how CPZ furin cleavage is associated with Aβ production (Kim, Wang et al. 1999). However, the observation that CPZ can induce cells to undergo apoptosis, as well as CyD overexpression, suggests a common mechanism for these two proteins that affect Aβ production.

  Previous findings (Novikova and Fricker 1999) suggest that CPZ is expressed in rat brain leptomeningeal cells, but CPZ is not expressed in areas of the brain known to be affected by Alzheimer's disease Shown (Novikova and Fricker 1999). To determine where CPZ is expressed, mouse brain sections are analyzed by in situ hybridization. In wild type C7BL-6 mice (Jackson Laboratories), CPZ antisense probes show binding as a broad distribution in the brain, with the cerebellum and frontal lobe having the highest level of signal. More detailed experiments have shown that CPZ is also most likely expressed in pyramidal cells in the hippocampus close to the CA1 and CA3 regions. In the cerebellum, CPZ is expressed in close proximity to molecular regions in the Perkinje and granule cell layers. The CPZ distribution in the frontal lobe did not appear to be localized to specific cell types, but instead was expressed uniformly throughout the entire region. CPZ localization in the mouse hippocampus, cerebellum, and frontal lobe demonstrates its expression in regions of the brain known to be affected by Alzheimer's disease in humans.

CyD characterization: To test whether these immunophilin-binding compounds can modulate APP processing, HEK / APPswe stable cells were treated with a range of concentrations of cyclosporin A, sanglifehrin A, FK506 or N Treat with methyl-4-valine-cyclosporin concentrate. For each concentration, viability is measured with the MTS assay according to the manufacturer's instructions (Promega Cat # G5421) and secreted Aβ40 and Aβ42 are measured using the ELISA assay described herein. The viability IC _{50 for} all compounds is> 40 μM. Inhibition IC ₅₀ for cyclosporine A, sanglifehrin A for Aβ40 and Aβ42 is <3 μM, indicating strong inhibition of Aβ secretion. The IC ₅₀ for Aβ40 and Aβ42 secretion of N-methyl-4-valine-cyclosporine is <10 μM, while FK506 has no effect on Aβ40 secretion. In contrast, FK506 treatment results in a concentration-dependent increase in Aβ42 at 3-20 μM.

  It has been demonstrated that immunosuppressive agents such as cyclosporin A can inhibit neurodegeneration and apoptosis in several animal models and inhibit Aβ-induced mitochondrial damage in isolated mouse mitochondria (Kim 2002). Since CyD is the target of cyclosporin A and is localized to mitochondria, it may have an important role in Aβ processing at this site, suggesting that CyD may be important for AD development in humans . Although no other isoforms have been tested, CyD should have a unique effect on Aβ processing, probably because it is the only cyclophilin isoform known to localize to mitochondria . It has been found that CyD overexpression in HEK cells can increase caspase-3 activity and stabilize PS1 NTF, suggesting that it may directly modify PS1 activity. Furthermore, compounds that can bind and inhibit CyD activity strongly inhibit both Aβ secretion and C99 cleavage.

  In particular, sanglifehrin A strongly inhibits Aβ at 3 μM and is nontoxic to cells up to 20 μM. Sanglifehrin A can bind to and inhibit the PPIase activity of CyD without affecting the ability of CyD to bind to the adenine nucleotide transporter (ANT) and without inhibiting calcineurin activity such as CsA. Known (Samantha J. Clarke 2002). The steep dose response of sangliferin A could be explained in several ways. Sanglifehrin A can only inhibit opening when a significant portion of CyD is bound, and its activity is sufficient to be interrupted by MPT (Clarke 2002). Since ANT exists as a dimer, it is likely that CyD should bind more than one molecule to ANT in order to induce a conformational change. This explains the rapid inhibition of Aβ secretion at the 3 μM threshold, since sanglifehrin A must bind to multiple CyD molecules to inhibit its activity on the MPT complex. When this threshold is reached, the MPT complex can be completely inhibited and open pores can be prevented. This threshold of MPT inhibition is considered to be an important regulator of Aβ processing and / or secretion. Alternatively, sanglifehrin A may have multiple targets other than or in addition to CyD. It is also unclear whether CyD controls the MPT complex directly through PPIase activity or by conformational changes in MPT. In any case, the data presented here and by others is consistent with the CyD and MPT pores having an important role in Aβ processing.

  While these compounds can dramatically reduce Aβ secretion and inhibit C99 cleavage, they can also inhibit Notch cleavage. This was not surprising because the data suggested that CyD and CPZ overexpression affected GACE activity. This data is the first suggestion that immunophilin compounds inhibit GACE activity and define a novel pathway that can affect Aβ and Notch processing in cells.

  Interestingly, FK506 treatment dramatically increases Aβ42 secretion in a dose response manner. FK506 is known to bind to immunophilin proteins but not to CyD. A specific increase in Aβ42 suggests that the FK506 target protein controls Aβ42 metabolism or production. Both FK506 and CsA can inhibit calcineurin, but have different effects on Aβ secretion, so calcineurin probably does not affect Aβ levels. This unique effect of FK506 further directs that the immunophilin pathway controls Aβ processing.

Example 2
C99 and Notch cleavage The HEK 293 stable cell line was inserted into the modified C99 or Notch transmembrane sequence, signal peptide, TGN retention sequence, and Q56 / Y57 as described by Maltrese, Wilson, et al. 2001. Produced using the GAL4-NLS-VP16 sequence. The cells also stably express 5xGAL4RE-luciferase reporter constructs (RD-2002-01437 and RD-2001-02419). These cells are indicative of GACE activity when the C99 transgene is cleaved, which activates the Gal4 luciferase reporter. These HEK stable cells are treated with cyclosporin A, sanglifehrin A, FK506 or N-methyl-4-valine-cyclosporin and the IC ₅₀ for C99 cleavage is determined. GALVP alone may possibly be a negative control. The IC ₅₀ of GALVP for cyclosporin A, N-methyl-4-valine-cyclosporin sangiferin A, and FK506 are 6.9, 9.9,> 20, and> 20 μM, respectively, and these compounds are Since it is not toxic up to> 40 μM, it shows a clear range between C99 cleavage and cell viability. However, cyclosporin A, N-methyl-4-valine-cyclosporine sanglifehrin A all inhibited C99 cleavage at 0.71, 0.87, and 0.85 μM, respectively, and Notch cleavage was 1. Inhibit at 7, 2.7 and 3.2 μM. FK506 has no effect on C99 or Notch cleavage. Therefore, a ligand capable of binding CyD strongly inhibits C99 and Notch cleavage, suggesting that GACE activity is inhibited.

Example 3
Caspase-3 activation Recent studies suggest that caspase-3 activation can lead to increased Aβ secretion and stabilization in the GACE complex of this protein (Tesco, Koh et al. 2003). Since both CyD and CPZ increase Aβ secretion and stabilize PS1 NTF, caspase-3 activation levels are analyzed in CyD and CPZ overexpressing cells. HEK 293 cells are transiently transfected with APPwt for 24 hours with CyD and CPZ, saponin permeabilized, immobilized, and probed with a FITC-conjugated monoclonal antibody against active caspase-3. Negative control cells are transfected with empty vector and APPwt, and positive control cells are treated with 1 μM staurosporine for 6 hours prior to analysis. In CyD expressing cells, 52% of the tested cells are positive for active caspase-3, while in CPZ expressing cells 48% of the cells are positive for caspase-3. These results suggest that overexpression of these proteins induces caspase-3 activation, which may be a mechanism by which PS1 NTF is stabilized and Aβ secretion is secreted.

Claims (6)

  Alzheimer's disease, Parkinson's disease, tauopathy, prion disease, frontotemporal dementia, striatal nigra degeneration, Lewy body dementia, Huntington's disease, Pick's disease, amyloidosis, and others associated with excessive Aβ production Use of non-immunosuppressive, cyclophilin-binding cyclosporine in the manufacture of a medicament for the treatment of pathological conditions associated with Aβ production and / or secretion, such as neurodegenerative disorders.   A method of treating or preventing a pathological condition associated with Aβ production and / or secretion, comprising administering to the patient an effective amount of a non-immunosuppressive, cyclophilin-binding cyclosporine. A non-immunosuppressive, cyclophilin-binding cyclosporine has the formula A

[Where B is the formula B

[Wherein a represents a bond to the αAbu residue at position 2;
b means a bond to residue C at position 4;
Alk means a straight or branched alkylene containing 2 to 6 carbon atoms or a cycloalkylene containing 3 to 6 carbon atoms, and R is a carboxy or alkyloxycarbonyl radical;
Radical —NR ₁ R ₂ (wherein R ₁ and R ₂ are the same or different and are hydrogen, alkyl, C _2-4 alkenyl, C _3-6 cycloalkyl, phenyl (optionally halogen, alkoxy, alkoxycarbonyl, amino Is optionally substituted with alkylamino or dialkylamino) or benzyl, or a saturated or unsaturated heterocyclyl radical containing 5 or 6 ring atoms and 1 to 3 heteroatoms, or R ₁ and R ₂ are Together with the nitrogen atom to which they are attached, it contains 4-6 ring atoms and may further contain a heteroatom selected from nitrogen, oxygen or sulfur and is substituted with alkyl, phenyl or benzyl. A saturated or unsaturated heterocycle which may be
formula

Wherein R ₁ and R ₂ are as defined above, R ₃ is hydrogen or an alkyl radical, and n is an integer from 2 to 4,
Alkyl is a straight or branched alkyl containing 1 to 4 carbon atoms. )
The radical of ]
Amino acid residues;
C is MeLeu or 4-hydroxy-MeLeu. ]
The use according to claim 1 or the method according to claim 2, which is a compound of claim 1 and pharmaceutically acceptable salts thereof. Non-immunosuppressive, cyclophilin-binding cyclosporine is represented by the formula I:

[Where,
W is MeBmt, dihydro-MeBmt or 8′-hydroxy-MeBmt;
X is αAbu, Val, Thr, Nva or O-methylthreonine (MeOTTh);
R is Sar or (D) -MeAla;
Y is MeLeu, γ-hydroxy-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;
Where Alk is Me or vinyl (which is optionally substituted with phenyl or N, S or O heteroaryl containing 6 ring members) or phenyl (which is optionally substituted with halogen) ) Means Me substituted with
Q is MeLeu, γ-hydroxy-MeLeu or MeAla. ]
The use according to claim 1 or the method according to claim 2, which is a compound of Non-immunosuppressive, cyclophilin-binding cyclosporine:
a) [dihydro-MeBmt] ^1- [γ-hydroxy-MeLeu] ⁴ -cyclosporine;
b) [MeVal] ⁴ -cyclosporine;
c) [MeIle] ⁴ -cyclosporine;
d) [MeThr] ⁴ -cyclosporine;
e) [γ-hydroxy-MeLeu] ^4- cyclosporine;
^{f) [Nva] 2 - [} γ- hydroxy-MeLeu] ⁴ - cyclosporin;
g) [.gamma.-hydroxy -MeLeu] ⁴ - [γ- hydroxy-MeLeu] ⁶ - cyclosporin;
h) [MeVal] ⁵ -cyclosporine;
i) [MeOThr] ² -[(D) MeAla] ^3- [MeVal] ⁵ -cyclosporine, or j) [8'-hydroxy-MeBmt] ¹ -cyclosporin.
m) [N-benzyl-Val] ⁵ -cyclosporine,
n) [N-5-fluoro-benzyl-Val] ⁵ -cyclosporine,
o) [N-allyl-Val] ⁵ -cyclosporine,
p) [N-3-phenyl-allyl-Val] ⁵ -cyclosporine,
3. Use according to claim 1 or method according to claim 2 selected from q) [Pro] ⁴ -cyclosporine, or r) [[gamma] -hydroxy-MeLeu] ⁹ -cyclosporine. The use according to claim 1 or the method according to claim 2, wherein the non-immunosuppressive, cyclophilin-binding cyclosporine is [MeVal] ⁴ -cyclosporine.