US20130184268A1 - 2-(r2-thio)-10-[3-(4-r1-piperazin-1-yl) propyl]-10h-phenothiazine for treating a beta-amyloidopathy or an alpha-synucleopathy, and method for the diagnosis or prediagnosis thereof - Google Patents

2-(r2-thio)-10-[3-(4-r1-piperazin-1-yl) propyl]-10h-phenothiazine for treating a beta-amyloidopathy or an alpha-synucleopathy, and method for the diagnosis or prediagnosis thereof Download PDF

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US20130184268A1
US20130184268A1 US13/821,515 US201113821515A US2013184268A1 US 20130184268 A1 US20130184268 A1 US 20130184268A1 US 201113821515 A US201113821515 A US 201113821515A US 2013184268 A1 US2013184268 A1 US 2013184268A1
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amyloidopathy
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Jens Pahnke
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    • A—HUMAN NECESSITIES
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/5415—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with carbocyclic ring systems, e.g. phenothiazine, chlorpromazine, piroxicam
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    • C07D279/10—1,4-Thiazines; Hydrogenated 1,4-thiazines
    • C07D279/14—1,4-Thiazines; Hydrogenated 1,4-thiazines condensed with carbocyclic rings or ring systems
    • C07D279/18—[b, e]-condensed with two six-membered rings
    • C07D279/22—[b, e]-condensed with two six-membered rings with carbon atoms directly attached to the ring nitrogen atom
    • C07D279/24—[b, e]-condensed with two six-membered rings with carbon atoms directly attached to the ring nitrogen atom with hydrocarbon radicals, substituted by amino radicals, attached to the ring nitrogen atom
    • C07D279/28—[b, e]-condensed with two six-membered rings with carbon atoms directly attached to the ring nitrogen atom with hydrocarbon radicals, substituted by amino radicals, attached to the ring nitrogen atom with other substituents attached to the ring system
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
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    • A—HUMAN NECESSITIES
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    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D279/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one sulfur atom as the only ring hetero atoms
    • C07D279/10—1,4-Thiazines; Hydrogenated 1,4-thiazines
    • C07D279/14—1,4-Thiazines; Hydrogenated 1,4-thiazines condensed with carbocyclic rings or ring systems
    • C07D279/18—[b, e]-condensed with two six-membered rings
    • C07D279/22—[b, e]-condensed with two six-membered rings with carbon atoms directly attached to the ring nitrogen atom
    • C07D279/24—[b, e]-condensed with two six-membered rings with carbon atoms directly attached to the ring nitrogen atom with hydrocarbon radicals, substituted by amino radicals, attached to the ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4709—Amyloid plaque core protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette

Definitions

  • AD Alzheimer's dementia
  • CAA cerebral ⁇ -amyloidopathy
  • a ⁇ proteostasis i.e. the equilibrium of production and degradation/removal by means of receptors or proteases is disturbed in AD and CAA.
  • ABSC transporters cellular transporters
  • Parkinson's disease the protein ⁇ -synuclein accumulates, which inter alia regulates the dopamine release in the substantia nigra.
  • ⁇ -synucleinopathy it is known that ABC transporters play a crucial role for transport (Kortekaas et al., Ann Neural 2005, 57, 176-179).
  • A-G which can alternatingly transport various substrates (metabolites, medicaments, peptides, proteins, ions) and are even able to replace each other in the transport function (e.g. ABCB1 and ABCC1, Tao et al. Cancer Chemotherapy and Pharmacology, 64, 5, 961-969).
  • ABC transporter a common structural element of the ABC transporter is an ATP-binding cassette and a transport pore
  • ABCC1 is an important protein/peptide transporter, in particular A ⁇ transporter, which has extraordinary functional effects on the cerebral protein accumulation.
  • ABCC1 is also an important ⁇ -synuclein transporter.
  • R 1 and R 2 are the same or different and each independently of one another are C 1 -C 6 alkyl groups, which independently of one another optionally comprise another substituent selected from alkyl, aryl, acyl (preferably acetyl), amino, nitro, sulfonyl, hydroxyl, alkoxy, aryloxy, arylthio, alkylthio groups and halogen atoms, wherein the respective alkyl groups optionally comprise at least one further halogen atom and the residue
  • R 3 is located at one of the positions 6-9 of the phenothiazine ring system and is a hydrogen atom or an alkyl, aryl, acyl (preferably acetyl), amino, nitro, sulfonyl, hydroxyl, alkoxy, aryloxy, arylthio or alkylthio group or a halogen atom, wherein the respective alkyl groups optionally comprise at least one further halogen atom or an NR 4 R 5 or OR 6 group, wherein R 4 , R 5 and R 6 are the same or different and each independently of one another are selected from hydrogen and C 1 -C 3 alkyl groups and the residue
  • R 7 is located at one of the positions 1, 2 or 4 of the phenothiazine ring system and is a hydrogen atom or an alkyl, aryl, acyl (preferably acetyl), amino, nitro, sulfonyl, hydroxyl, alkoxy, aryloxy, arylthio or alkylthio group or a halogen atom, wherein the respective alkyl groups optionally comprise at least one further halogen atom or an NR 8 R 9 or OR 10 group, wherein R 8 , R 9 and R 10 are the same or different and each independently of one another are selected from hydrogen and C 1 -C 3 alkyl group, for treating a ⁇ -amyloidopathy or an ⁇ -synucleinopathy accompanied by a cerebral protein deposit.
  • the object of the invention to provide a method with which ⁇ -synucleinopathies and also ⁇ -amyloidopathies can be diagnosed or prediagnosed.
  • This object is solved by a method according to claim 11 .
  • Preferred embodiments are obtained from the dependent claims.
  • the object is solved by a method for the diagnosis or prediagnosis of a ⁇ -amyloidopathy or ⁇ -synucleopathy or for determining the risk of a proband to develop such an illness, wherein the proband already takes substances transported by the cerebral ABCC1 transporter, consisting of the following steps:
  • step b) repeating the determination of step a) at at least one further later time point;
  • step c) comparing the quantities determined in step a) and b) with quantities which had been defined as characteristic at the same time points for probands who at the time of the sampling showed no clinical symptoms of a ⁇ -amyloidopathy or an ⁇ -synucleopathy.
  • the proband already takes at least one substance which is transported via the cerebral ABCC1 transporter means that this substance needs not to be administered. On the contrary it is already present in the body of the proband, for example, as a result of a drug treatment of another disease.
  • the body fluid samples of the proband which are studied are preferably samples from blood plasma, blood serum and/or cerebral spinal fluid.
  • the ⁇ -amyloidopathy is preferably an Alzheimer's dementia, the ⁇ -synucleinopathy is preferably Parkinson's disease.
  • the ⁇ -synucleinopathy can also be a dementia with Lewy bodies (DLB).
  • Substances which are transported via the cerebral ABCC1 transporter are preferably selected from antibiotics (e.g. difloxacin, grepafloxacin), virostatics/antiviral medicaments (e.g. saquinavir, ritonavir), anti-allergics/antihistamines (e.g. cimetidine), cardio-vascular medicaments (e.g. verapamil), antidepressants (e.g.
  • citalopram antihyperuricemics
  • probenecid e.g. probenecid
  • cytostatics e.g. methotrexate, etoposit, edatrexate, ZD1694
  • vitamins/vitaminanalogues e.g. methotrexate, folic acid, L-leucovorin
  • antiphlogistics e.g. indomethacin
  • anti-epileptics e.g. valproic acid
  • hormones/hormone derivatives e.g. 17 ⁇ -estradiol
  • leukotrienes e.g. LTC4
  • fluorescent samples e.g.
  • This indirect analysis of the transport activity of ABCC1 transporters can be used for the diagnosis/prediagnosis of a corresponding disease.
  • the profile of the active substance concentration in body fluids preferably blood plasma, serum and/or cerebrospinal fluid can be investigated.
  • a time-dependent measurement for probands in whom there is a reduced ABCC1 transport activity compared with healthy probands shows a delayed or shifted substance concentration curve (concentration c plotted over time t), i.e. the maximum of the curve varies with time.
  • mice The relative significance of members of the ABC transporter family was then investigated in vivo using newly generated ABC transporter-deficient Alzheimer mouse models.
  • the genetically modified mice each exhibit a deficiency (knock out) at specific ABC transporters ABCG2, ABCB1 or ABCC1.
  • the A ⁇ immunohistochemistry of brain sections showed:
  • ABCB1-deficient mice showed a smaller increase in the number and size of A ⁇ -plaques than ABCC1-deficient mice.
  • enzyme-coupled immune adsorption tests were used for A ⁇ .
  • the ABCC1-deficient mice showed a significant increase in aggregated A ⁇ compared to the control mice at all measurement time points.
  • the cerebral loading with A ⁇ was greatest at an age of 25 weeks.
  • the A ⁇ values (A ⁇ 42) were 12 times higher than in the control mice.
  • Buffer-soluble A ⁇ also increased with age but after 25 weeks, at the time of the highest plaque loading, the values of the soluble A ⁇ in the ABCC1-deficient group decreased substantially.
  • ABC transporters depend inter alia on specific protein/peptide characteristics such as the specific charge.
  • the Dutch-type variant of the amyloid precursor protein Dutch mutant, APP dt ) which introduces an additional negative charge near the interface of the ⁇ -secretase of the APP and thus results in a severe cerebral amyloidangiopathy (CAA) influence the elimination of A ⁇ dt via the blood-brain barrier.
  • the Western blot analyses of brain capillaries and plexuschoroideus (CP) from control mice showed a strong expression of ABCB1 in cerebral capillary endothelial cells (BC) and of ABCC1 in CP ( FIG. 3 d ).
  • Thiethylperazine proved to be a particularly efficient activator of the ABCC1 transporter.
  • Other derivatives starting from the same scaffold also showed good results in the activation of the ABCC1 transporter.
  • the corresponding derivatives are represented in the general formula I
  • R 1 and R 2 are the same or different and each independently of one another are C 1 -C 6 alkyl groups, which independently of one another optionally comprise another substituent selected from alkyl, aryl, acyl (preferably acetyl), amino, nitro, sulfonyl, hydroxyl, alkoxy, aryloxy, arylthio, alkylthio groups and halogen atoms, wherein the respective alkyl groups optionally comprise at least one further halogen atom and the residue
  • R 3 is located at one of the positions 6-9 of the phenothiazine ring system and is a hydrogen atom or an alkyl, aryl, acyl (preferably acetyl), amino, nitro, sulfonyl, hydroxyl, alkoxy, aryloxy, arylthio, alkylthio group or a halogen atom, wherein the respective alkyl groups optionally comprise at least one further halogen atom or an NR 4 R 5 or OR 6 group, wherein R 4 , R 5 and R 6 are the same or different and each independently of one another are selected from hydrogen and C 1 -C 3 alkyl groups and the residue
  • R 7 is located at one of the positions 1, 2 or 4 of the phenothiazine ring system and is a hydrogen atom or an alkyl, aryl, acyl (preferably acetyl), amino, nitro, sulfonyl, hydroxyl, alkoxy, aryloxy, arylthio, alkylthio group or a halogen atom, wherein the respective alkyl groups optionally comprise at least one further halogen atom or an NR 8 R 9 or OR 10 group, wherein R 8 , R 9 and R 10 are the same or different and each independently of one another are selected from hydrogen and C 1 -C 3 alkyl group
  • the halogen atom/the halogen atoms are preferably selected from fluorine and chlorine.
  • the residues R 1 and R 2 are the same or different and each independently of one another are a C 1 -C 6 alkyl group or a C 1 -C 6 alkyl group (preferably C 1 alkyl) substituted with an acetyl group and the residues R 3 and R 7 are hydrogen or an acetyl group.
  • the residues R 1 and R 2 are the same or different and each independently of one another are a C 1 -C 3 alkyl group. It is further preferred that the residues R 3 and R 7 are hydrogen.
  • the residue R 1 is a methyl group
  • the residue R 2 is an ethyl group
  • the residues R 3 and R 7 are hydrogen (thiethylperazine, Torecan®).
  • further active substances preferably 1-benzohydrylpiperazines, most preferably 1-benzohydryl-4-cinnamyl piperazine (cinnarizine).
  • Various neurodegenerative diseases can be treated with the 2-(R 2 -thio)-10-[3-(4-R 1 -piperazin-1-yl)propyl]-10H-phenothiazine derivatives according to the invention or can be diagnosed by means of the indirect analysis described above.
  • the neurodegenerative disease is a ⁇ -amyloidopathy, in particular Alzheimer's dementia (AD).
  • AD Alzheimer's dementia
  • Another embodiment relates to the case that the neurodegenerative disease is an ⁇ -synucleinopathy, in particular Parkinson's disease (PD).
  • Both diseases, i.e. ⁇ -amyloidopathy and ⁇ -synucleinopathies are characterized by cerebral protein deposits which can be treated by means of an activation of the ABCC1 transporter or can be diagnosed by means of its activity.
  • LBD Lewy body dementia
  • Another embodiment relates to the case that the neurodegenerative disease is Huntington's disease (HD).
  • Another embodiment relates to the case that the neurodegenerative disease is a prion disease, in particular Creutzfeld-Jacob disease (CJD) orfatalfamilialinsomnia (FFI).
  • Another embodiment relates to the case that the neurodegenerative disease is a tauopathy, in particular cortico-basal degeneration (CBD), Steel-Richardson-Olszewski syndrome (PSP, progressivesupranuclearpalsy) or Pick's disease (PiD).
  • CBD cortico-basal degeneration
  • PSP Steel-Richardson-Olszewski syndrome
  • PiD Pick's disease
  • FTLD neurodegenerative disease frontotemporaldegeneration
  • FTLD neurodegenerative disease frontotemporaldegeneration
  • ALS amyotrophiclateralsclerosis
  • SCA spinocerebellarataxia
  • SPG spasticparaparesis
  • MS multiple sclerosis
  • ADEM Devic's syndrome
  • FIG. 1 a shows that the cortical density of neuritic plaques in ABCC1-deficient mice (ABCCI ko) is increased by ⁇ 75%;
  • FIG. 1 b,c shows that the mean plaque size is increased (+34%) as a result of the larger number of plaques (+63%) having a size of more than 700 ⁇ m 2 and a lower frequency of smaller plaques ( ⁇ 24%).
  • Error bars standard error (n ⁇ 3);
  • FIG. 1 d shows that the IHC staining in ABCG2-deficient (ABCG2ko), ABCB1-deficient (ABCBIko), ABCC1-deficient (ABCCIko) mice and in control mice shows a higher surface density of A ⁇ in ABCC1-deficient animals.
  • Typical plaques of the same size are shown in section, scaling bars represent 500 ⁇ m (overview) and 50 ⁇ m (section) (*p ⁇ 0.05);
  • FIG. 2 a shows that the plaque density in the cortex (coverage) in specific ABC-transporter knockout mice is increased.
  • FIG. 2 c shows that the total increase in the plaque size is associated with fewer smaller plaques and more larger plaques (>700 ⁇ m 2 ) whereas the number of medium-size plaques remains at the same value, error bars, standard error (n 5), *p ⁇ 0.05;
  • FIG. 3 shows that the deficiency of ABCC1 promotes the accumulation of A ⁇ and A ⁇ dt and that the activation of ABCC1 (by administration of Torecan) reduces the A ⁇ values;
  • FIG. 3 a shows that at an age of 25 weeks ABCC1 deficiency leads to a marked increase ( ⁇ 12 times) in insoluble A ⁇ ;
  • FIG. 3 b shows that the quantity of buffer-soluble A ⁇ 42 at an age of 25 weeks is noticeably reduced compared with 22 weeks ( ⁇ 56%). This is probably due to the deposition in insoluble deposits. At the same age the area covered by A ⁇ deposits which is measured in the immunohistochemistry is increased by 83% (error bars, standard error n 5, p ⁇ 0.05);
  • FIG. 3 d shows that the expression of ABCC1 can be seen predominantly in the plexus choroideus (CP) whereas ABCD1 is principally expressed in the capillaries of the brain (BP);
  • APP-transgenic mice (APP, APP dt ) were obtained from The Jackson Laboratory (Bar Harbor, USA) and the University of Tübingen (Tübingen, Germany).
  • the NEP-deficient mice were obtained from the Riken Brain Research Institute (Saitama, Japan).
  • ABCG2-, ABCB1-, and ABCC1-deficient mice were obtained from Taconic-Farms (Denmark). All transgenic and knockout mouse lines were hybridised for at least 9 generations in the genetic FVB-background. The mice were held in a 12 h/12 h light/dark cycle at 23° C. with free access to food and water.
  • mice were killed by cervical dislocation and perfused transcardially with PBS (phosphate-buffered, physiological saline solution).
  • PBS phosphate-buffered, physiological saline solution
  • the brain was removed and one hemisphere was stored in buffered 4% paraformaldehyde for paraffin embedding and immunohistochemistry.
  • the other hemisphere was shock-frozen in liquid nitrogen and stored at ⁇ 80° C. for biochemical analyses.
  • ELISA kits (TH40HS, TK42HS) from The Genetics Company (TGC, Schlieren, Switzerland) were used for the quantification of A ⁇ . Brain hemispheres were homogenised using PreCellys24 (12 s, 6,500 rpm). After adding carbonate buffer (pH 8.0) the homogenisates were mixed using PreCellys (5 s, 5,000 rpm) and centrifuged for 90 min at 4° C. and 24,000 g, in order to separate insoluble from soluble A ⁇ species. The remaining supernatant (buffer-soluble fraction) was mixed with 8M guanidine hydrochloride in a ratio of 1:1.6.
  • the pellet was dissolved in 8 volumes of 5M guanidine hydrochloride, agitated at room temperature for 3 h and centrifuged at 24,000 g for 20 min at 4° C. The remaining supernatant formed the guanidine-soluble fraction (GuaHCl). Protein contents of all the samples were measured three times, using a Nanodrop1000 spectrophotometer (ThermoFisher Scientific, Wilmington, USA). The ELISAs were carried out according to the manufacturer's instructions using suitable dilutions.
  • Tissue homogenisates were prepared for the Western Blots.
  • the total protein concentrations of the extracts were determined using a BCA assay (Pierce, part of Thermo Fisher Scientific, Rockford, USA). After electrophoresis of 10 ⁇ g total protein per trace, the proteins were blotted onto PVDF membranes. After blocking in 5% dry milk in TBST-buffer (50 mMTris pH 7.4, 150 mMNaCl, 0.1% Tween20) for 1 h at room temperature, the blots were studied either on ABCB1 (1:500, D-11, Santa Cruz), ABCC1 (1:200, Alexis Bio) or ⁇ -actin (1:20.000, Sigma) overnight at 4° C. Anti-mouse-HRP, anti-rat-HRP oranti-hare-HRP were used as detection antibodies. An Amersham ECL Plus Detectionkit and a RoperCoolSnap HQ 2 camera were used for visualisation.
  • Formalin-fixed brains were embedded in paraffin and cut into 4 ⁇ m thick sections. After removing the paraffin, the sections were further treated with a BondMaxTM Autostainer (Menarini/Leica, Germany). Immunostaining was initiated after blocking of endogenic peroxidase (5 min) and epitoperetrieval for 5 min using 95% formic acid (for antibody 6F3D, Dako, Germany) and 70% formic acid (for antibody 4G8, Millipore, Germany). Primary antibodies were routinely incubated at room temperature for 30 min with the following dilutions: 6F3D (1:100), 4G8 (1:500). Primary antibodies were detected with the BondMaxTM Bond Polymer Refinedetectionkit and according to the DAB R30 standard protocol. The sections were completely digitised with a resolution of 230 nm using a MiraxDesk/MiraxMidi scanner and then analysed automatically using the AxioVision software package (Zeiss, Germany).
  • Brain sections of APP dt were stained with 4G8-antibody. At least two non-consecutive sections were studied for CAA of the meningeal vessels in a masked manner. All meningeal vessels were counted manually and the severity of the CAA was categorised as follows:
  • the average number of vessels for each category was calculated relative to the total number of identified vessels.
  • Endothelial cells of mouse brain capillaries were prepared as described in Coisne et al. (Coisne, C. et al. Mouse syngenic in vitro blood-brain barrier model: a new tool to examine inflammatory events in cerebral endothelium. Laboratory Investigation; 85, 734-746 (2005)). At least 3-4 week old mice were beheaded and the brains removed. Following dissection of the brain stem, the white matter and the meninges, the tissue was homogenised in two volumes of wash buffer B (WBB) (Hanks bufferedsaltsolution (HBBS), 10 mM HEPES, 0.1% BSA) using a 15 ml glassdouncer (Wheaton Industries, Millville, N.J.; USA).
  • WBB wash buffer B
  • HBBS Hops bufferedsaltsolution
  • 10 mM HEPES 0.1% BSA

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WO2024083822A1 (fr) * 2022-10-18 2024-04-25 Immungenetics Ag Identification d'un sujet souffrant de la maladie d'alzheimer ou présentant un risque de développer la maladie d'alzheimer

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