WO2009083612A2

WO2009083612A2 - Flavonoid as means for the prophylaxis and treatment of neurodegenerative and other protein folding-aberration diseases

Info

Publication number: WO2009083612A2
Application number: PCT/EP2009/050020
Authority: WO
Inventors: Martin H. Groschup; Martin Eiden
Original assignee: Groschup Martin H; Martin Eiden
Priority date: 2008-01-02
Filing date: 2009-01-02
Publication date: 2009-07-09
Also published as: DE102008003467A1; WO2009083612A3

Abstract

The invention relates to the use of synthetically produced or plant-derived flavonoids, the derivatives thereof, and/or degradation products thereof for the therapeutic and prophylactic treatment of aggregation- and folding-aberration diseases. This means particularly preparations of skullcap (scutellaria) and plants with similar agents (e.g. parsley, celery, chamomile, and chrysanthemum), as well as the agents contained therein in their pure form or in combinations (including the already know materials baicalein, baicalin, etc) as therapeutically and/or prophylactically active substances for prion infections and other neurodegenerative protein folding-aberration diseases (Alzheimer's disease, Parkinson's disease, Huntington's disease, senile amyloidosis, tauopathies, etc.) for humans and animals.

Description

Flavonoids as agents for the prophylaxis and treatment of neurodegenerative and other protein deficiency diseases

description

[0001] The present invention presents a new possibility for the treatment of protein deficiency and protein aggregation diseases.

[0002] The invention relates to the use of synthetically produced (including chemical modifications, for example acetylation, hydroxylation, halogenation, methylation, carbonylation, alkylation, glycosylation, esterification, oxidation, hydrolysis, condensation, polymerization) or plant-derived flavonoids, the Derivatives or degradation products for the therapeutic and prophylactic treatment of these aggregation and misfolding diseases. These include, in particular, preparations of scullcap (Scutellaria) and plants with similar ingredients (eg parsley, celery and chamomile and chrysanthemums) and the constituents contained therein in pure form or in combinations (including the already known substances baicalein, baicalin, etc.). as therapeutically and / or prophylactically active substances in prion infections and other neurodegenerative protein deficiency diseases (Alzheimer's disease, Parkinson's disease, Huntington's disease, senile amyloidosis, tauopathies, etc.) in humans and animals. To date, there is no sufficiently effective causative therapy for these protein misfolding and aggregation diseases.

State of the art

1. Protein Misfolding and Protein Aggregation Diseases

1.1. Forms of the disease

[0003] The proteins involved in protein aggregation and misfolding diseases are listed in Table 1.

Table 1: Proteins involved in protein aggregation and misfolding diseases

The molecular basis of these diseases is the misfolding and aggregation of each specific protein (see Table 1). This misfolding can occur either spontaneously or by a mutation in this protein, be triggered by a cofactor, aging processes or infections. During the aggregation process, the misfolded protein forms oligomers that assemble into multimers and / or fibrils. The misfolding is the cause of the disease either by loss of the physiological activity of the protein or by direct or indirect toxic effects on the surrounding cells or the whole organism. The aggregates can be deposited as miniscule particles (which can not be visualized by light or electron microscopy) or as amyloids (s.u.) in body cells. Neurodegenerative protein deficiency diseases primarily affect neurons, astrocytes, micro- and / or macroglial cells and affect the surrounding extracellular spaces or body fluids. After staining with Congo red they can be visualized. The aggregation is usually associated with a refolding of the protein into β-sheet-rich structures. It is still unclear which component is responsible for the cytotoxic and neurotoxic effects during the aggregation process.

1.2 Mechanisms of inhibition

The following different therapeutic strategies are currently being followed closely to treat these diseases: a) Direct interaction with the aggregating protein b) Influencing post-translational processes that promote protein aggregation c) Dissolution of the fibrils and aggregates a) Direct interaction with the aggregating protein

One way to inhibit aggregation is the use of small peptides that are incorporated into the resulting fibrils and terminate the aggregation process. These peptides were synthesized by "structure based designs" and are effective in vitro inhibitors of the aggregation of the amyloid β-protein (Aβ) in Alzheimer's disease and the amylin in diabetes (type II), but the therapeutic applications are limited because of the Peptides in the body can easily be degraded and are immunogenic, and there is also a danger that toxic intermediates may form during the inhibition process. [0007] Another possibility for finding inhibitors is the testing of substance libraries by means of high-throughput methods (high-throughput methods). These screenings are performed with cell-based and cell-free assays and have already identified numerous molecules that inhibit the aggregation of amyloid β-protein, prions, tau protein and huntingtin in vitro, a therapeutic effect of these proteins in vivo however, it has not been described so far. [0008] A third possibility, an aggregation To prevent n is the stabilization of the native protein by the direct binding of a molecule. This has already been demonstrated with tansthy- drin and superoxide dismutase.

[0009] Antiaggregatory agents include: N-methyl peptides (Sciarretta et al 2006) β-sheet intercalators (Sato-T et al., 2006)

4,5-dianilinophthalimide (Blanchard-BJ et al., 2004)

N744 (Chirita et al., 2004)

Juglone, Celastrol (Wang et al 2005) C2-8 (Zhang, et al., 2005)

Fosfosal, levonordefrin, (Desai et al., 2006)

nadolol

N'-benzylidenes-benzohydrazides (Bertsch et al., 2005)

Substances that stabilize the native conformation include: dicloflenac, diflunisal (Johnson et al., 2005).

flufenamic

b) Influencing Post-translational Processes Promoting Protein Aggregation Another approach is to inhibit secondary processes that affect protein aggregation. An example is proteases that proteolytically cleave the amyloid precursor protein (APP) and thereby the one responsible for Alzheimer's disease Forming amyloid β-protein. By inhibition of these proteases (ß- and γ-secretase), therefore, the accumulation of Aß protein can be prevented. Various substances have already been found in vitro that can specifically inhibit these proteases. However, with few exceptions, these substances can not overcome the blood-brain barrier. Another problem is that the inhibition, especially of ß-secretase toxic side effects occur, which can negatively influence other processes such as synaptic plasticity and myelin formation of the nerves.

The phosphorylation of proteins is a target in the search for active substances. Examples are the (hyper) phosphorylation of the tau protein in tauopathies and the α-synuclein in Parkinson's disease. Starting point here is the inhibition of the corresponding kinases, which are responsible for the phosphorylation. Also, various substances have been identified in vitro that inhibit the kinases. A serious problem, however, is that these kinases have multiple substrates and therefore toxic side effects occur again by inhibiting other metabolic pathways. Finally, oxidation processes in fibril formation play an important role. During the aggregation of Aβ, α-synuclein and huntingtin, so-called "reactive oxygen species (ROS)" occur which are neurotoxic and, moreover, accelerate the aggregation itself The use of antioxidants such as polyphenols (eg curcumin) and metal chelators is therefore a possibility to halt or stop these processes, and in vitro inhibitory effects have already been demonstrated.

Substances which inhibit proteolysis of the amyloid precursor protein include:

NSAID (inhibits γ-secretase) (Beher et al., 2004).

Lithium (inhibits γ-secretase) (Phiel et al., 2003).

Gleevec (inhibits γ-secretase / presilin) (Netzer et al., 2003).

MRK 560 (inhibits γ-secretase) (Best et al., 2007)

R-flurbiprofen (flurizan) (inhibits γ-secretase) (Eriksen et al 2003) Acylguanidines (inhibit β-secretase) (CoI et al., 2006).

Carboyclic and heterocyclic (Hanessian et al., 2005)

Piptidomimetics (inhibit ß-secretase)

[0014] Substances that inhibit phosphorylation include: AR-A014418 (Bhat et al., 2003)

Lithium (Noble et al., 2005) The substances that inhibit oxidation include: curcumin (Yang et al., 2005)

Melatonin (Matsubara et al., 2003)

c) Resolution of the aggregates

Another therapeutic option is an attempt to enhance the protein degradation mechanisms present in the cell. This can either induce the formation of specific chaperones or promote autophagic processes ("macroautophagy"): chaperones serve for the correct folding of a protein or can be used to induce misfolded proteins to undergo cellular degeneration.A chaperone - hsp70- has been shown to that it is able to induce both Aβ oligomers and mutant huntingtin proteins to undergo proteolytic degradation, and to identify several compounds that induce the formation of hsp70 and inhibit the formation of aggregates in vitro, but these substances usually also contain other chaperones Serious side effects in vivo can not be ruled out, since aggregation of α-synuclein and mimicking huntingtin has been shown to be transported by the cell to certain regions, the so-called aggresomes, in which the aggregates are secreted either by macroautophagy mined or in us harmful fibrils are stored. Therefore, a therapeutic target is the increase in autophagic activity. There are a number of substances that have this effect, but one problem is that they also degrade proteins with anti-tumor properties.

[0017] Chaperone-enhancing substances include: geldanamycin (Auluck et al., 2005)

Celastrole (Westerheide et al., 2004)

Activation of the resolution of aggregates rapamycin (Ravikumar et al., 2004) trehalose (Sarkar et al., 2007)

1.3. Inhibitors of prion diseases

[0019] Prion infections are transmissible protein deficiency diseases in humans and animals for which in vitro as well as in vivo (animal) models exist in which a possible prophylactic and / or therapeutic effect of chemical substances can be detected. Prion diseases (Creutzfeldt-Jakob disease, BSE, scrapie) are triggered by the misfolding of a body protein, the prion (PrP ^c ). These errors Convolution, also called conversion, can be triggered by infection, mutation, or sporadic (random) events. The misfolded protein (PrP ^Sc ) has the property of transferring this misfolding to other PrP ^c molecules. This causes aggregation and accumulation of misfolded PrP ^Sc molecules, especially in the CNS. Along with the formation of the aggregates, neurodegenerative events occur in the CNS that ultimately lead to death.

The following substances or substance classes have been described with which the prion infections and pathogenesis can be influenced:

• multicyclic anionic molecules (Congo Red): Gervasoni et al. 2004

• polyanionic sulph. Polysaccharides (pentosan polysulphate): Ouidja et al. 2007

• Polyene antibiotics (amphotericin B): McKenzie et al. 1994

Anthracycline (Iodoxorubicin): Tagliavini et al. 1997

Cyclic tetrapyrroles (porphyrins, phthalocyanines): Priola et al. 2000 • Peptides (β-sheet breaker): Soto et al. 2000

• * acridine derivatives (quinacrine): Benito-Leόn et al. 2004

• * Phenothiazine derivatives (chlorpromazine): Korth et al. 2001

Polyamines (SuperFect, DOSPA): Supattapone et al. 2001

• Inducers of aggregation (suramin): Gilch et al. 2001 • mAbs against PrP; recombinant anti-PrP Fab fragments: White et al. 2003

• RNAi: Pfeifer et al. 2006

Curcumin: Caughey et al., 2003

• Cyclodextrins: Prior et al. 2007

However, all of these substances are characterized by a high toxicity and significant side effects, so that a prophylactic or therapeutic effective dose in vivo is only marginal or absent. Furthermore, they often do not have sufficient penetrance through the blood-brain barrier, so that only an insufficient drug concentration in the CNS comes about.

1.4 Conclusion

It should be noted that there are several strategies for prophylactic and / or therapeutic treatment of protein misfolding diseases. However, so far no chemical substance is available for which a sufficient prophylactic or therapeutic effect could be demonstrated on the living animal or on the human body. An important aspect of this is, above all, that in a large part of the chosen therapy pathways severe side effects occurred by the administered agent. 2. Specialist and patent literature on flavonoids

Document EP 1 808 169 A2 describes polyhydroxylated aromatics for the treatment of amyloidoses and alphasynuclein fibril disorders. A treatment for Alzheimer's and Parkinson's is proposed. Mechanisms of action are the inhibition of aggregate formation and dissolution of the aggregates. The compounds 1,2,4-benzenetriol, ellagic acid, ethyl gallate, exifon. Gallamide, bile acid, 5-hydroxydopamine, phloroglucide, propyl gallate, quercetin, quinic acid, tannic acid. The mode of action is demonstrated by the dissolution of Aβ 1-42 aggregates (thioflavine assay), concentration-dependent dissolution of Aβ 1-40 fibrils by gallic acid and tannic acid (thioflavine fluorescence), resolution of Ab 1-40 fibrils by polyhydroxylated aromatics (Congorot spectrophotometry). , Concentration-dependent dissolution of Aβ 1-40 fibrils by gallic acid and tannic acid (Congo red spectrophotometry), dissolution of amylin by myricetin, exifon, tannic acid. The reduction of the neurotoxic action of the peptides is effected by prior or simultaneous addition of polyhydroxylated aromatics. As a cell culture, primary rat neurons were used. In women with early Alzheimer's disease, one group was treated with placebo, the other with substance from the description of the invention (without specification) (oral therapy over 6-36 months). As evidence of the activity of the substance, delayed onset of typical cognitive disorders and behavior has been identified.

In the patent application WO 02/087567 A2 polymethoxylated flavones are proposed for the treatment of insulin resistance.

Porat et al. (2006 Chem. Biol. Drug Des, Review) report inhibiting the formation of amyloid fibrils by polyphenols. The effect of the polyphenols is therefore based on the reduction of the amyloigene protein forms, the enhancement of the degradation of aggregated proteins, the direct inhibition of the self-assembly process, a specific incorporation of the aromatic ring structures into aggregated or aggregated proteins or the abolition of the cytotoxic effects of the fibrils. especially by the antioxidant properties. The in vitro inhibition of α-synuclein and β-amyloid by apomorphine, the inhibition of β-amyloid by resveratrol and catechol or polyphenols of wine or curcumin and rosmarinic acid, the inhibition of β-amyloid and PrP ^Sc by tannic acid or epigallocatechin gallate. It is further reported that purpurrogalin, exifon, hypericin, myricetin, gossypetine, pentahydroxybenzophenone, Epicatechingallate and trihydroxybenzophenone polyphenols inhibit tau protein and ß-amyloid, inhibit phensulfonphthalein insulin, amyloid deposits in islets of Langerhans and ß-amyloid, and inhibit baicalin α-synuclein.

Rong-Hua et al (Chin, J. Biotech, 2006) investigated the effect of baicalein on the proliferation and differentiation of porcine predipocytes. Inhibition of proliferation between 140-640 μM, inhibition of differentiation between 40-320 μM, concentration-dependent inhibition of PPARγ2 (peroxysome proliferator actovant receptor) expression between 40 μM and 160 μm, and concentration-dependent inhibition of FAS activity were found.

The prior art also includes the publication by Bomhoff et al. (2006, Arch. Biochem. Biophys.). It reports that Mg ions accelerate the aggregation of 1 SS-CC-lactalbumin fibrils (thioflavin assay), sucrose and TMAO (trimethylamine oxide) accelerate the aggregation of lSS-α-lactalbumin fibrils and baicalin accelerate the concentration-dependent inhibition of aggregation causes.

Xin et al (2007, Zhongguo Zhong Xi Yi Jie He Za Zhi.) Describe protective effects of baicalin on Parkinson's mice induced by MPTP. The mice show typical Parkinsonsymptome after injection of MPTP. MPTP therefore destroys neurons in the black substance or causes a reduction of dopamine in the striatum. Treatment with Baicalin (Gastric Perfusion [100 mg / kg] for 15 days prevents the loss of neurons in the black substance and inhibits the reduction of dopamine, but there is no improvement in motor dysfunction However, the results of Xin et al note that: To investigate the pathogenesis of Parkinson's disease in animal experiments, MPTP was used to copy the major symptoms of idiopathic disease, but the α-synuclein is not involved in the pathogenesis of MPTP-induced Parkinsonism Syndrome involved in mice.

Jung et al. (2007 Se Pu) studied a Xiao-Xu-Ming Sud, a traditional anti-Alzheimer's drug also used for stroke. The analysis was carried out by HPLC: the brew contains peniflorin (99.1%), primary oglucosylcimifugin (98.4%), baicalin (98.4%), 4'-O-beta-D-glucosyl-5-O-methyl-4-visinol (99.9%). ), Quinoline (99.6%), tetrandrine (102%). Healing (curative) effects were found in aging rats. Baicalein reduces 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y cells. This is reported by Jung et al. (2005, Eur. J. Cell Biol). Thus, baicalein protects SH-SY5Y cells from their 6-hydroxydopamine (6-OHDA) induced destruction by reducing reactive oxygen radicals. In addition, Baicalin reduces mitochondrial dysfunction triggered by 6-OHDA.

Veestergaard et al. (2005 Anal. Chim. Acta) describe an electrochemical approach to detect copper-binding properties of flavonoids using graphite electrodes and discuss possible effects on copper-dependent diseases. The oxidation potential of 6 different flavonoids (myricetin, catechin gallate, quercetin, delphinidin, baicalein, catechin, cyanidin, luteolin) was determined. The antioxidant potency of each flavonoid was determined as follows: myricetin> catechin gallate> quercetin> delphinidin> baicalein> catechin> cyanidin> luteolin. The analysis of the copper-binding properties by determining the reactivity of the corresponding flavonoid towards Cu (II) revealed: myricetin = catechin gallate> quercetin> delphinidin = baicalein> cyanidin> catechin.

The efficacy of some flavonoids on cultured cerebral neurons after the addition of β-amyloid is the subject of the publication by Kim et al., (2004, Europ Journal of Neurology suppl., 2, poster presentation). A murine neuronal cell culture (cortex) was investigated. Addition of Aβ fragment 25-35 results in concentration-dependent neuronal cell death. Ten different flavonoids (apigenin, baicalein, catechin, epicatechin, epigallocatechin gallate, kaempferol, luteolin, myricetin, quercetin, rutin) were tested. The ability to scavenge free radicals was accomplished with the use of diphenylpycrylhydrazole radicals. Except for apigenin, all flavonoids possess this property. In addition, these flavonoids (with the exception of apigenin) can also prevent the cell death caused by Aβ fragment 25-35.

The anti-aggregating properties of flavonoids given in EP 1 808 169 A2 are based on investigations on fibril formation or dissolution of pure amyloid fibrils. This also applies to the publications by Porat et al. (2006 Chem. Biol. Drug Des, Review) and Bomhoff et al. (2006, Arch. Biochem. Biophys.). The reported cell-based effects of flavonoids, particularly baicalein and baicalin, described in Jung et al., (2005, Eur. J. Cell Biol.), Xin et al., (2007, Zhongguo Zhong Xi Yi Jie He Za Zhi.) and Kim et al. (2004, Europ Journal of Neurology suppl.2, poster presentation), however, show only general effects with regard to cytotoxic / neuroprotective and antioxidant Properties. The effect of flavonoids in vivo has hitherto only been described by an unspecific improvement in behavior or motor skills (see EP 1 808 169 A2, section [0083]). The beneficial effect after substance administration found there can not be assigned to a specific mechanism of action and may possibly be attributed to general cytoprotective and antioxidant effects of the flavonoids, which are described in the other documents mentioned above. The effect of flavones shown in WO 02/087567 A2 is based on the influence of different flavones on the metabolism or on metabolic products in insulin resistance in vitro and in vivo. The described Xiao-Xu-Ming Sud as a traditional remedy for Alzheimer's disease (and also stroke) contains Baicalein, but the causal relationship between Baicalein and (nonspecific) curative effects was not shown. Reference has only been made to a curative effect on aging rats Although the anti-aggregatory properties of baicalein and other flavonoids are described in EP 1 808 169 A2, the causative relationship between these properties and that in vivo has not been described The effects of the baicalins and baicalins described in Porat et al (2006, Chem., Biol., Drug Des, Review) and Bomhoff et al., (2006, Arch. Biochem., Biophys.), is based on inhibition of the aggregation of pure fibrils or the dissolution of purified aggregates.

In the o.g. Documents showed neuro- and cytoprotective properties of bacellein / baicalin, as well as additional antioxidant effects of baicalein. Baicalin has been used to treat the pathogenesis of Parkinson's disease in animal experiments induced by MPTP. However, the effect was not caused by the anti-aggregatory effect of baicalin, since α-synuclein is not involved in the pathogenesis of MPTP-induced Parkinson's disease in mice.

In summary, in all of these documents, effects of the flavonoids have been described which have shown general positive therapeutic effect, in particular with regard to motor function and behavior in vivo. Particularly in EP 1 808 169 A2, no causal relationship between the anti-aggregatory properties of flavonoids in vitro and the unspecific therapeutic effects in vivo could be demonstrated.

3. Skullcap (scutellaria) and its ingredients

In the context of the present invention, two phytochemicals (Baicalein, Baicalin) from the group of flavonoids were found, which specifically in vitro the misfolding of the Inhibit prion protein (conversion) and dissolve existing aggregates. These substances come from the scullcap Scutellaria lateriflora. Even the tea made from this herb has this in vitro activity. It was finally shown that the tea can significantly delay the onset of prion disease in animal experiments. Pathological examinations of the treated mice showed that the application is harmless. Since the molecular mechanisms of prion diseases are similar to those of other protein aggregation diseases, the effect of the ingredients from the skullcap can also be transferred to other diseases such as Alzheimer's, Parkinson's, etc. Helmetrope tea thus represents, among other things, an effective therapeutic agent against these aggregative diseases. In the following, therefore, we will discuss the scleropod, its ingredients and their previously known therapeutic effectiveness.

3.1. Analysis of scutellaria (scutellaria) and its ingredients

The helmet herbs (Scutellaria) belong to the family Lamiaceae (Lamiaceae). There are about 360 species known to be used in traditional medicine in Asia, North America and Europe (Joshee et al., 2002). Tea preparations or extracts should have nonspecific anti-inflammatory, anti-viral, sedidative and neuroprotective effects. They are also said to have anti-cancerogenic effects and are used to treat epilepsy, insomnia and neuralgia. The effect is based on the ingredients of the helmet herb, the flavonoids.

3.2. flavonoids

The flavonoids are a group of water-soluble plant dyes and play an important role in the metabolism of many plants. They belong to the polyphenols together with the phenolic acids. So far, more than 8,000 polyphenols are known. Flavonoids can be divided into the following groups:

Table 2:

Antho cyanidine cyanidin, delphinidin, malvidin, pelargonidin, peonidin, chalcone (anthocyanins)

Isoflavones Biochanin A, Equol

3.3. Previously known effects of flavonoids

Antibacterial: Cushnie TP, & Lamb AJ. (2005) Antiviral: Serkedjieva J et al. (2007) Anti-Inflammatory: Talhouk et al. (2007) Anti-Cancerogen: Li al (2007) Anti-amyloid: Porat et al. (2006), Riviere et al. (2006), Shoval et al. , (2007)

In these three publications, however, in vitro data on the anti-amyloid effect of the flavonoids were invariably shown. So far, there are no data on the efficacy of flavonoids in vivo (for example in animal experiments).

3.4. Baicalein and Baicalin

Baicalein and Baicalin belong to the group of flavones. Baicalein is a 5,6,7-trihydroxy flavone, Baicalin has an additional sugar ring compared to Baicalein and is called Biacalein-7-Glucuronide (Figure 8). Other flavones are luteolin (ingredient of parsley and celery), apigenin (ingredient of parsley, chamomile and celery), di- orcin, acetoacetin and tangeretin (ingredients of chrysanthemums).

3.5. Scutellaria species with Baicalein and Baicalin:

Both flavonoids can be isolated from several species of Scutellaria, with Scutellaria baicalensis being the most intensively studied species (Kim et al., 2007). In addition, Baicalein and Baicalin were isolated from Scutellaria lateriflora (Awadv et al., 2003). Other scutellaria species are S. rivularis, S. discolour, S. indica, S. scadens (Joshee et al., 2002).

3.6. Inhibitory effects of Baicalein and Baicalin

3.6.1. Anti-aggregatory properties of Baicalein in vitro:

a) Inhibition of lSS-alpha-lactalbumin aggregation (Bomhoff et al., 2006)

b) Inhibition of aggregation of alpha-synuclein (Zhu et al., 2004) c) indirect inhibition of platelet aggregation by inhibition of oxygenases (Kälvegren et al., 2007)

Anti-aggregatory properties of Baicalein in vivo have not been described so far.

3.6.2. Antioxidative properties of Baicalein in vitro and in vivo:

Ciesielska et al. 2002

3.6.3. Anti-cancerogenic effects of Baicalein in vitro

Chao et al. (2007).

Object of the invention

The invention has for its object to find substances that are suitable for the therapeutic treatment of protein deficiency and aggregation diseases.

Solution of the task

The object has been solved according to the features of the claims. As a starting point and exemplary example prion diseases were chosen.

The present invention presents a new possibility for the treatment of protein misfolding and protein aggregation diseases. According to the invention, agents are provided for the prophylaxis and treatment of neurodegenerative protein deficiency and aggregation diseases which contain flavonoids as active components in vivo. The agents are further characterized in that they are in identically or modified (eg acetylation, hydroxylation, halogenation, methylation, carbonylation, alkylation, glycosylation, esterification, oxidation, hydrolysis, condensation, polymerization) form synthetically produced flavonoids or derivatives thereof be used. According to a preferred embodiment of the invention, different flavonoids are combined. This creates - compared to the individual components - a surprising synergistic effect.

The compositions of the invention may be derived from plants containing flavonoids or from parts of plants containing flavonoids, preferably from the herb or root of plants containing flavonoids. The most suitable is the Plant scutellaria (skullcap). But also plants with structure-like flavonoids such as parsley, paprika, chamomile, celery or chrysanthemums can be used. A preferred embodiment of the invention is the combination of the flavonoids Baicalein and Baicalin. It can also be structurally similar flavonoids from the group of flavones, such as acetoacetin, diurinary tangeretin, luteolin, apigenin or their derivatives or degradation products can be combined.

The invention relates not only to individual substances but also to mixtures, solutions or suspensions of flavonoids or extracts of plants containing flavonoids. Preferably, extracts are obtained from plants containing flavonoids. The extractants are hot or cold water, alcohols or organic solvents, e.g. Ether. Extraction may be by maceration, dimacification, digestion, re / percolation, soxhlet, turbo (vortex), ultra-turrax, ultrasound and countercurrent extraction. A particularly simple but effective remedy is a tea available from scutellaria. The concentration of flavonoids in the extract is in the range from 0.1 nM to 10 mM.

Protein deficiency diseases are: a) Parkinson's disease, Lewy body dementia, multisystem atrophy b) Alzheimer's disease c) familial amyloidosis d) diabetes (type II) e) Huntington's disease f) spinocerebellar ataxia g) amyotrophic sclerosis h ) Tauopathies (Alzheimer's disease, frontotemporal dementia) i) senile amyloidosis j) Creutzfeldt-Jakob disease, vCJD, GSS, FFI, Kuru bovine spongiform encephalopathy, scrapie k) senile amyloidosis (ATTR amyloidosis)

The inventive method for preparing the means is to pour over the plants or parts of plants with the extractant, allowed to draw for a few minutes and strain. In a preferred embodiment, 29 g of herb are infused with 1 liter of boiling water, then allowed to draw for 10 minutes and strain. The use of the agent according to the invention is preferably the oral treatment of animals suffering from protein misfolding and aggregation diseases, which is carried out once, several times or regularly. Also intravenous, intramuscular, intraperitoneal, intraventricular and / or intracerebral administration, which are performed once, several times or regularly, are possible. Likewise, applicators that form an inhalable aerosol are possible.

With the present invention, the anti-aggregatory properties of baicalein / baicalin could be demonstrated for the first time in a cell-based assay. In addition, the specific effect of the invention could be demonstrated for the first time in an animal model for prion diseases. Scrapie-infected mice treated with Scolutolia lateriflora tea showed a significant increase in incubation time. In addition, another study with a mouse model of Alzheimer's disease was performed. Here, for the first time, a specific relationship between substance application and aggregate formation or dissolution could be demonstrated: After administration of the substance, the proportion of amyloid plaques in different brain regions of the mice was significantly reduced. That the properties of the substances shown in vitro (inhibition of aggregate formation, dissolution of existing aggregates) also show these effects in vivo (reduction of the plaque count in the Alzheimer mouse model). In this invention, it is demonstrated that flavonoids in vitro specifically inhibit fibril formation or dissolve aggregates and that this mechanism of action is also successful in vivo. In summary, the specific action of the S. scutellaria tea is based on the anti-aggregating action of its components baicalein and baicalin. This effect has been demonstrated in vitro as well as in vivo in 2 different disease models. For the first time a causal relationship between application of a substance (group) and its specific therapeutic effect could be shown.

The inhibition of fibril formation or dissolution of aggregates by flavonoids described according to the invention shows a specific action against protein-folding diseases. This effect relates to the general disease mechanism of these neurodegenerative diseases (Skovronsky et al., 2006): A cellular, endogenous protein sporadically misfolded by mutations or by other events associated with other also misfolded proteins. These form oligomers, which then form superordinate structures such as protofibrils and ultimately amyloid plaques. For this reason, the observed effect, which the applicants have so far proven in prion diseases and Alzheimer's disease, can also be transferred to other protein misfolding diseases such as Parkinson's disease. For the treatment of these diseases, tea from S. lateriflora was used which contains as the (main) components, the flavonoids analyzed according to the invention, baicalein and baicalin (Awad et al., 2003, see bibliography). The effect is based on the anti-aggregatory properties of these substances and can be demonstrated specifically in vivo (see below).

All flavonoids have a common basic structure of two aromatic and one heterocyclic ring. The large number of flavonoids can be explained by the combination of different hydroxyl and methoxyl groups on this backbone (Moon et al., 2006). Since the anti-aggregatory effect is structurally conditioned, the patent application covers all major groups of flavonoids, which include flavanones, flavones, flavonols, flavanols, anthocyanidins and isoflavones.

In the present invention, in contrast to the prior art, a specific biochemical mechanism of action of a substance (group) could be shown for the first time, which occurred both in vitro and in vivo in two disease models and thus used prophylactically and therapeutically using a defined application can be. The in vivo effect was shown in the significant prolongation of the incubation period in scrapie-infected mice or in the significant reduction of amyloid plaques in a transgenic mouse model for Alzheimer's disease. The effect of flavonoids on the reduction of amyloid plaques in the animal model was further demonstrated in two other publications published after the priority date of the patent application (Rezai-Zadeh, K et al and Onozuka, H et al.).

The invention will be described in more detail by means of exemplary embodiments, without reducing them to these examples.

embodiments

Demonstration of the efficacy of flavonoids as inhibitors of protein deficiency and aggregation disease

The effectiveness of flavonoids against these diseases could be demonstrated by the example of the helmet and its ingredients in the context of in vivo studies on living organism. In addition, the antiaggregatory activity could also be detected in vitro. The substances not only inhibit the formation of new blood, but also lead to the dissolution of already existing aggregates. Example 1 Effect in vitro

The efficacy was tested in vitro in 2 test systems. In a cell-free conversion assay, recombinantly produced prion protein is incubated together with infectious PrP ^Sc . By means of specific antibodies, the subsequent misfolding (conversion) of the prion protein as well as the susceptibility of the PrP ^Sc aggregates to the proteolytic

Degradation are detected. This assay was developed by the authors / inventors and has already been published (Eiden et al., 2006). The second, cell-based assay uses cells that are permanently infected with scrapie pathogens. Here the PrP ^Sc formation can be detected by immunochemical methods.

Example 2 Cell-free Assay In the cell-free assay it was shown that the flavonoids baicalein as well as bacellin can inhibit the conversion of the prion protein in a concentration-dependent manner (FIG. 1). Here Bacalein has a much higher _(IC50 <20 microns) activity as baicalin. Other structurally similar flavonoids (epicatechin, morin, naringenin, quercitin, kaempferol) had no inhibitory activity in the studies so far performed. In this assay it could also be shown that both flavonoids also make the already existing PrP ^Sc aggregates (amyloid) susceptible to proteolytic degradation (FIG. 2). This degradation can be done by cellular or exogenously supplied proteases. Again, Baicalein has a much higher activity than Baicalin. The other flavoids studied were again ineffective. The tea of the helmet herb itself can both inhibit the conversion concentration-dependent and make the aggregates susceptible to proteolytic degradation (Figure 3). The results show that Baicalem / Baicalin interact specifically with PrP ^c molecules, preventing their conversion. In addition, because of their planar structure, you may also be able to store in PrP ^Sc aggregates and then make them proteolytically accessible.

Example 3 Cell Based Assay

In the second, cell-based assay, the anti-prion effect could also be detected. Here, we worked with cells that are permanently infected with a scrapie pathogen. Again, both Baicalein, Baicalin (Figure 4) and the tea extract (Figure 5) show inhibitory effect, which was concentration-dependent. Baicalein in turn has a much higher activity than Baicalin (Figure 6). In a cell cytotoxic Test (MTT test) showed both substances and the tea no cytotoxic effects. However, as the other flavonoids are also inhibitory, it can be assumed that in addition to the specific effects of baicalein / baicalin, the group of polyphenolic flavonoids has a general inhibitory effect on PrP ^Sc formation. This may be due to the antioxidant effects of this group of substances.

Example 4 Effect in vivo

Finally, the therapeutic effect of the helmetcap tea in the mouse model was examined. To this end, six wild-type mice were used, which were infected (inoculated) intracerebrally (i.e.) with a scrapie pathogen (Figure 7). Two weeks prior to inoculation, the infusion of the mice was switched from water to freshly brewed tea and maintained until death. As a control group, 6 mice were used, which were also were infected, but were further soaked with water. In addition, 3 mice were used, which were soaked with the Helmkraut tea at the same time, but were not infected. The untreated infected mice died within a narrow time window of 147.3 days, while the tea-treated animals lived significantly longer with one exception (148 days). Three mice lived 60 days longer than the animals in the untreated control group. The uninfected but tea-treated animals showed no pathological changes indicative of a side effect from the flavonoids.

It has been shown that tea (hot water extract) of the scull (scutellaria) is effective in the incubation period of a prion infection, i. for up to 60 days, which equates to a 100,000-fold pathogen titer reduction. The effect is probably based on 2 ingredients, the flavonoids Baicalein and Baicalin. The results show that the treatment of scrapie-infected animals with helmet scarf tea has a significant therapeutic effect. This effect can be attributed on the one hand to a specific interaction of Baicalein / Baicalin with the misfolded or aggregated prion protein on the other hand to general neuroprotective effects of flavonoids.

Example 5

A study with a mouse model for Alzheimer's disease was performed. For this purpose, transgenic mice B6.Cg-Tg (APPswe, PSEN ldE9) 85Dbo / J (reference: The Jackson Laboratory) were used, which have a mutation in the human amyloid precursor protein and in the presenilin-1 protein and amyloid deposits early after Form 6-7 months. 8 weeks after birth drinking water was in 4 animals by tea from S. replaced lateriflora. As control, 3 animals were used, which were further supplied with pure drinking water. After an average incubation period of 213 days, the animals were sacrificed and immunohistochemistry of the cortex and hippocampus was performed with three different antibodies (monoclonal antibodies 6E10, β1-40, and β-42) specifically detecting the amyloid deposits. The number of small amyloid deposits was evaluated (see Table 3).

Table 3. Immunohistochemistry of 4 brain regions (frontal cortex, parietal cortex, occipital cortex, hippocampus) from treated (therapy) and untreated (control) mice, respectively. The number of small plaques was determined in each case from the mean of 4 (therapy) and 3 (control) mice, respectively.

The study, with one exception (#), showed a significant reduction in amyloid plaques in all areas of the cortex and hippocampus after S. lateriflora tea therapy.

Legend to the figures:

Figure 1: Inhibition of PrP ^res formation in the cell-free conversion assay.

The detection in Western blot was carried out with the monoclonal antibody P4 by means of chemiluminescence.

A: Baicalein efficiently inhibits the formation of PrP ^res . In lanes 1-2 are proteinase K (PK) to see -resistant PrP ^res fragments in samples without baicalein, in the product lanes 3-4 after incubation with 10.0 mM Baicalein, in lanes 5-6 with 1.0 mM Baicalein, and in lanes 7-8 after incubation with 0.1 mM Baicalein.

B: inhibition of PrP ^res formation by Baicalin. PK-resistant fragments without baicalin (lanes 1-2), with 10 mM Baicalin (lanes 3-4), 1.0 mM Baicalin (lanes 5-6) and 0.1 mM Baiquinin (lanes 7-8).

C: no inhibition of PrP ^res formation by the flavonoid epicatechin: samples without epicatechin (lanes 1-2) after incubation with 10 mM epicatechin (lanes 3-4), with 1.0 mM epicatechin (lanes 5-6) and 0.1mM epicatechin (lanes 7-8).

FIG. 2: Dissolution of PrP ^Sc aggregates in a cell-free conversion assay.

These are the same Western blots of Figure 1, which after removal of the antibody by means of "stripping" with a new antibody (Ra 10) were re-incubated.

A: Baicalein dissolves PrP ^Sc aggregates efficiently after incubation with PK: lanes 1-2 show PK-resistant PrP ^Sc aggregates without baicalein, lanes 3-4 samples after incubation with 10.0 mM Baicalein, lanes 5-6 with 1.0 mM Baicalein, and Lanes 7-8 samples after incubation of 0.1 mM Baicalein.

B: dissolution of PrP ^Sc aggregates by Baicalin. PK-resistant aggregates without baicalin (lanes 1-2), after incubation with 10 mM baicalin (lanes 3-4), 1.0 mM baicalin (lanes 5-6) and 0.1 mM baicalin (lanes 7-8).

C: No resolution of the PrP ^Sc aggregates by epicatechin: samples without epicatechin (lanes 1-2) after incubation with 10 mM epicatechin (lanes 3-4), with 1.0 mM and 0.1 mM epicatechin epicatechin.

FIG. 3: Inhibition of PrP ^res formation and dissolution of PrPSc aggregates in a cell-free conversion assay by Helmkraut tea.

A: Helmkrauttee inhibits the formation of PrP ^res . Lanes 1-2 show PK-resistant PrP ^res fragments in samples without tea, lanes 3-4 samples after incubation with tea, lanes 5-6 with tea (1:10 dilution), and lanes 7-8 after incubation with tea ( 1: 100 dilution). B: Helmkrauttee dissolves PrP ^Sc aggregates after incubation with PK.

These are the same Western blots of Figure that after removal of the antibody by "stripping" with a new antibody (Ra 10) were re-incubated. Lanes 1-2 show PK-resistant PrP ^Sc aggregates without tea, lanes 3-4 samples after incubation with tea, lanes 5-6 with tea (1:10 dilution), and lanes 7-8 after incubation with tea (1: 100 Dilution).

FIG. 4. Inhibition of PrP ^Sc formation in ScN-A and SMB cells in a cell-based assay

PK-resistant PrP signals on sections of a 96-well dot blot after detection with the p Ab RaIO.

A: Cells were treated with decreasing concentrations of Baicalein: Incubation of ScN ₂ A cells with 10 mM Baicalein (lane 1), 0.1 mM Baicalein (lane 2), 0.01 mM Baicalein (lane 3) and untreated ScN ₂ A cells. (Lane 4). Incubation of SMB cells with 1.0 mM Baicalein (lane 5), 0.1 mM Baicalein (lane 6), 0.01 mM Baicalein (lane 7) and untreated SMB cells (lane 8).

B: Cells were treated with decreasing concentrations of Baicalin: Incubation of ScN2A cells with 10 mM Baicalin (lane 1), 0.1 mM Baicalin (lane 2), 0.01 mM Baicalin (lane 3) and untreated ScN2A cells. (Lane 4). Incubation of SMB cells with 1.0 mM (lane 5), 0.1 mM Baicalin (lane 6), 0.01 mM Baicalin (lane 7) and untreated SMB cells (lane 8).

C: Cells were treated with decreasing concentrations of epicatechin: Incubation of ScN ₂ A cells with 10 mM epicatechin (lane 1), 0.1 mM epicatechin (lane 2), 0.01 mM epicatechin (lane 3), and untreated ScN ₂ A cells. (Lane 4). Incubation of SMB cells with 1.0 mM (lane 5), 0.1 mM epicatechin (lane 6), 0.01 mM epicatechin (lane 7) and untreated SMB cells (lane 8).

Figure 5. Inhibition of the PrP / PrP ^res in scrapie-formation infϊzierten cells after incubation with Helmkrauttee

A: Helmkrauttee inhibits the PrP / PrP ^res formation in ScN ₂ A cells. Lane 1 shows the PK-resistant fragment of the untreated control, the lanes 2-5 samples incubated with increasing concentrations of helmet-scab tea: 1: 500 (lane 2), 1: (lane 3), 1:20 (lane 4) and 1:10 (Lane 5). B: Helmkrauttee inhibits PrP / PrP ^res formation in SMB cells. Lane 1 shows the PK-resistant fragment of the untreated control, the lanes 2-5 samples incubated with increasing concentrations of helmet-scab tea: 1: 500 (lane 2), 1:50 (lane 3), 1:20 (lane 4) ) and 1:10 (lane 5). The detection in Western blot was carried out with the polyclonal antibody RaIO by means of chemiluminescence.

FIG. 6: EC50 values of the flavonoids investigated

FIG. 7: Survival times of the mice after infection with the mouse scrapie strain RML in comparison to control mice.

[0071] C57 / B16 mice were inoculated intracerebrally with 30 μl of a 1% RML brain homogenate.

Blue line: Treatment with helmet herb tea. The survival times were up to 207 days. Red line: control mice. All control mice died between 145-153 days.

Figure 8: Structure of Baicalein and Baicalin

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Claims

claims

1. An agent for the prophylaxis and treatment of neurodegenerative protein misfolding and aggregation diseases, characterized in that they contain as in vivo active components combinations of different flavonoids.

2. Composition according to claim 1, characterized in that they in identical or modified (eg acetylation, hydroxylation, halogenation, methylation, carbonylation, alkylation, glycosylation, esterification, oxidation, hydrolysis, condensation, polymerization) form synthetically produced flavonoids or their Become derivatives.

3. Composition according to claim 1, characterized in that the flavonoids from a) plants containing flavonoids or b) parts of plants containing flavonoids are obtained.

4. Composition according to claim 3, characterized in that the flavonoids from the herb or the root of plants containing flavonoids are obtained.

5. Composition according to claim 3 or 4, characterized in that it is a) the plant Scutellaria (skullcap) and / or b) plants with structure-like flavonoids such as parsley, paprika, chamomile, celery or chrysanthemums.

6. A composition according to any one of claims 1 to 5, characterized in that it a) the flavonoids Baicalein and Baicalin or b) structurally similar flavonoids from the group of flavones, such as acetaminophen, diurinary tangeretin, luteolin, apigenin or c) derivatives or degradation products contain.

7. Composition according to one of claims 1, 3, 4, 5 or 6, characterized in that they are individual substances, mixtures, solutions or suspensions of flavonoids or extracts of plants containing flavonoids is.

8. Composition according to claim 7, characterized in that the extract by means of a) hot or cold water or b) alcohols or c) organic solvents, such as ethers and / or d) maceration, dimacification, digestion, re-/ percolation, Soxhletverfahren, Turbo-

(Vortex), ultra-turrax, ultrasound and countercurrent extraction from plants containing flavonoids.

9. Composition according to one of claims 1 to 8, characterized in that it is tea, available from scutellaria.

10. Composition according to one of claims 7 to 9, characterized in that the concentration of flavonoids in the extract in the range of 0.1 nM to 100 mM.

11. Composition according to one of claims 1 to 10, characterized in that it is in the

Protein Deficiency Diseases around:

1) Parkinson's disease, Lewy body dementia, multisystem atrophy m) Alzheimer's disease n) familial amyloidosis o) diabetes (type II) p) Huntington's disease q) spinocerebellar ataxia r) amyotrophic sclerosis s) tauopathies (Alzheimer's, frontotemporal dementia) t) senile Amyloidosis u) Creutzfeldt-Jakob disease, vCJD, GSS, FFI, Kuru bovine spongiform encephalopathy, scrapie v) senile amyloidosis (ATTR amyloidosis).

12. A process for the preparation of the agent according to one of claims 3 to 9, characterized by the following steps:

• pour over the plant or parts of the plant with solvent,

• let it rest for a few minutes and strain.

13. Use of the agents according to any one of claims 1 to 11 for the oral treatment of animals suffering from protein misfolding and -aggregationskrankheiten that is performed once, several times or regularly.

14. Use of the agents according to any one of claims 1 to 11 for the treatment of animals suffering from protein misfolding and -Aggregationskrankheiten, by intravenous, intramuscular, intraperitoneal, intraventricular and / or intracerebral administration, which is carried out once, repeatedly or regularly.

15. Use of the agents according to any one of claims 1 to 11 for the treatment of animals suffering from protein misfolding and aggregation diseases, via applicators which form an inhalable aerosol which is carried out once, repeatedly or regularly.