EP1532170A2 - Beta-sheet breaking peptides - Google Patents

Beta-sheet breaking peptides

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Publication number
EP1532170A2
EP1532170A2 EP03753576A EP03753576A EP1532170A2 EP 1532170 A2 EP1532170 A2 EP 1532170A2 EP 03753576 A EP03753576 A EP 03753576A EP 03753576 A EP03753576 A EP 03753576A EP 1532170 A2 EP1532170 A2 EP 1532170A2
Authority
EP
European Patent Office
Prior art keywords
phe
compound
compound according
amyloid
disease
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03753576A
Other languages
German (de)
English (en)
French (fr)
Inventor
Claudio University of Texas SOTO-JARA
Céline Adessi
Serge Halazy
Thomas Rueckle
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merck Serono SA
Original Assignee
Applied Research Systems ARS Holding NV
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Priority to EP03753576A priority Critical patent/EP1532170A2/en
Publication of EP1532170A2 publication Critical patent/EP1532170A2/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • 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
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the field of ⁇ -sheet breaking peptides, particularly their use in the treatment of diseases such as Alzheimer's disease, Dementia pugilistica (including head trauma), Hereditary Cerebral Haemorrhage with amyloidosis of the Dutch type (HCHWA- D) and vascular dementia with amyloid angiopathy.
  • diseases such as Alzheimer's disease, Dementia pugilistica (including head trauma), Hereditary Cerebral Haemorrhage with amyloidosis of the Dutch type (HCHWA- D) and vascular dementia with amyloid angiopathy.
  • AD Alzheimer's disease
  • Alois Alzheimer in 1907 is a progressive neurological disorder that begins with short-term memory loss and is characterized by a progressive decline in cognitive function and behaviour. Progression of the disease leads to disorientation, impairment of judgment, reasoning, attention and speech and, ultimately, dementia. The course of the disease usually leads to death in a severely debilitated, immobile state between four and 12 years after onset. AD has been estimated to afflict 5 to 11 percent of the population over age 65 and as much as 47 percent of the population over age 85. The societal cost for managing AD is veiy high, primarily due to the extensive custodial care required for AD patients. Despite continuous efforts aimed at understanding the physiopathology of AD, there is currently no treatment that significantly retards the progression of the disease.
  • AD Alzheimer's disease
  • brain lesions include abnormal intracellular filaments called neurofibrillary tangles (NTFs) and extracellular deposits of amyloidogenic proteins in senile, or amyloid, plaques.
  • NTFs neurofibrillary tangles
  • Amyloid deposits are also present in the walls of cerebral blood vessels of AD patients.
  • the major protein constituent of amyloid plaques has been identified as a 4.3 kiloDalton peptide called ⁇ -amyloid peptide (A ⁇ ) 1 . Diffuse deposits of A ⁇ are frequently observed in normal adult brains, whereas AD brain tissue is characterized by more compacted, dense-core ⁇ -amyloid plaques.
  • HSHWA-D hereditary cerebral haemorrhage with amyloidosis-Dutch-type
  • a ⁇ has also been implicated in vascular dementia with amyloid angiopathy 6 and dementia pugilistica.
  • Natural A ⁇ is derived by proteolysis from a much larger protein called the amyloid precursor protein (APP) .
  • APP amyloid precursor protein
  • the APP gene maps to chromosome 21, thereby providing an explanation for the ⁇ -amyloid deposition seen at an early age in individuals with Down's syndrome, which is caused by trisomy of chromosome 21 9 .
  • Naturally-occurring A ⁇ derived from proteolysis of APP, is 39 to 43 amino acid residues in length, depending on the carboxyl-terminal end point, which exhibits heterogeneity.
  • the predominant circulating form of A ⁇ in the blood and cerebrospinal fluid of both AD patients and normal adults is A ⁇ l-40 10 .
  • a ⁇ 1- 2 and A ⁇ -43 are also found in ⁇ -amyloid plaques 11 .
  • Amyloid is a generic term that is applied to fibrillar aggregates that have a common structural motif: a ⁇ -pleated sheet conformation J . These aggregates exhibit special tinctorial properties, including the ability to emit a green birefringent glow after staining with Congo red, and the capacity to bind the fluorochrome Thioflavin S 14 . These tinctorial properties form the basis of assays used to detect ⁇ -amyloid deposits.
  • Alzheimer's disease has been to develop short peptides having some sequence homology to the natural protein sequence believed to be involved in amyloid formation, but also having one or more amino acids that disfavour or destabilise the formation of ⁇ -pleated sheet conformations 15 .
  • the peptides prevent the aggregation of ⁇ -amyloid, and thereby prevent its cytotoxic effects.
  • This approach has been suggested in Alzheimer's disease and in prion-related and has lead to the ⁇ -sheet breaking peptides shown below, amongst others:
  • peptides comprising a ⁇ -strand- forming section of peptide which forms a ⁇ -strand and associate as such with a target ⁇ -strand, and wherein the ⁇ -strand forming section of peptide comprises a sequence of at least four consecutive -L-amino acid, all of which sterically permit the ⁇ -stand forming section of peptide to form a ⁇ -stand, and at least one of which is a N ⁇ -substituted ⁇ -L- amino-acid residue, and any two successive N ⁇ -substituted ⁇ -L-amino-acid residues are
  • ⁇ -sheet breaking peptides While the known ⁇ -sheet breaking peptides have provided valuable information and may have utility in treating Alzheimer's disease, the development of peptide drags is severely limited by the fact that natural peptides are subject to degradation and rapid metabolism in the gut, the liver and in circulation. Furthermore, the desired site of action for treatment of many amyloid-related disorders is in the brain, and peptides, like many other molecules, may have difficulty penetrating the blood brain barrier. The brain itself is also replete with peptidases, which degrade peptide molecules.
  • the invention provides a compound of the general Formula I:
  • R 1 is selected from H and optionally substituted C 2 -C 6 -acyl, preferably acetyl;
  • R 2 , R 3 , R 4 and R 5 are independently selected from H and optionally substituted C ⁇ -C 6 -alkyl and wherein at least one among R 2 , R J , R and R 5 is optionally substituted C ⁇ -C 6 -alkyl, preferably methyl;
  • R 6 is selected from OH and NR 7 R 8 , wherein R 7 and R 8 are independently H and optionally substituted C ⁇ -C 6 -alkyl, preferably NH 2 ; and salts and tritiated derivatives thereof.
  • the invention provides a compound of Formula I for use as a medicament.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of Formula I, together with a pharmaceutically acceptable excipient or carrier.
  • the invention provides a use of a compound of Formula I for the preparation of a medicament for the treatment or prevention of a disease or condition selected from Alzheimer's disease, Dementia pugilistica (including head trauma), Hereditary Cerebral Haemorrhage with amyloidosis of the Dutch type (HCHWA-D) and vascular dementia with amyloid angiopathy.
  • a disease or condition selected from Alzheimer's disease, Dementia pugilistica (including head trauma), Hereditary Cerebral Haemorrhage with amyloidosis of the Dutch type (HCHWA-D) and vascular dementia with amyloid angiopathy.
  • the invention provides a use of a compound of Formula I for the treatment or prevention of a disease or condition selected from Alzheimer's disease, Dementia pugilistica (including head trauma), Hereditary Cerebral Haemorrhage with amyloidosis of the Dutch type (HCHWA-D) and vascular dementia with amyloid angiopathy.
  • a disease or condition selected from Alzheimer's disease, Dementia pugilistica (including head trauma), Hereditary Cerebral Haemorrhage with amyloidosis of the Dutch type (HCHWA-D) and vascular dementia with amyloid angiopathy.
  • the invention provides a method of treating Alzheimer's disease, Dementia pugilistica (including head trauma), Hereditary Cerebral Haemorrhage with amyloidosis of the Dutch type (HCHWA-D) and vascular dementia with amyloid angiopathy, comprising administering to a patient in need thereof an effective amount of a compound of Formula I.
  • the invention provides a use of a compound of Formula I for the preparation of a medicament for the treatment of a disease associated with abnormal protein folding into amyloid and amyloid-like deposits.
  • the invention provides a use of a compound of Formula I for the treatment of a disease associated with abnormal protein folding into amyloid and amyloid- like deposits.
  • the invention provides a method of treating a disease associated with abnormal protein folding into amyloid and amyloid- like deposits, comprising administering to a patient in need thereof an effective amount of a compound of Formula I.
  • Figure 1 shows the in vitro activity of compounds of the invention using a fibrillogenesis assay.
  • the compounds were screened for activity using an assay based on quantitating amyloid fibril formation of synthetic A ⁇ -42 . (110 ⁇ M) in 0.1 M Tris, pH 7.4 was incubated alone or in the presence of a ten- fold molar excess of each of the compounds during 5 days at 37°C. Thereafter, the amount of amyloid fibrils was measured using the Thioflavin T fluorescent method, as described in the Examples. Values correspond to the percentage of inhibition of amyloid formation and are expressed relative to the activity of the comparative compound of Example 8 (100%).
  • Figure 2 shows a graph representing the calculated % of injected dose of compound of Example 7 taken up per g of brain vs. time. Mice are injected with 0.2 ml of lactate Ringer's solution containing 1% BSA and tritium labelled peptide ("hot") of Example 7 (100 000 cpm/ml of tritium radioactive peptide).
  • Figure 3A shows the in vivo activity of the compound of Example 1 and the comparative compound of Example 8 in a rat model of cerebral amyloidosis.
  • a representative image shows the difference in amyloid size between the groups of animals treated with vehicle, the comparative compound of Example 8 and the compound of Example 1.
  • Figure 3B shows a graph depicting the amyloid area in the different animals as estimated by image analysis after immunohistochemistry.
  • the compounds of the invention are ⁇ -sheet breaking peptides with improved pharmacological profile over known ⁇ -sheet breaking peptides.
  • ⁇ -sheet breaking activity can be detected using, for example, an in vitro assay, such as that described by Soto et al. 14 , which measures the ability of test compounds to prevent amyloid fibril formation.
  • Amyloid fibrils are cytotoxic, inducing cell death by apoptosis 20 .
  • Compounds of the invention can be tested for their ability to prevent cell death induced by amyloid fibrils. Results are reported in the Examples.
  • a compound having an improved pharmacological profile is considered to be a compound having an increased in vitro activity, as measured by either or both of the in viti'o assays described herein, an increased stability in plasma and/or in brain homogenate, or an increased ability to prevent amyloid deposition in vivo in rat brain, as compared with known compounds.
  • "Improved” encompasses those compounds showing an increase in any one of these parameters, or in more than one.
  • the improvement will be a statistically significant one, preferably with a probability value of ⁇ 0.05.
  • R 1 is selected from formyl, acetyl, propionoyl and butyroyl. Particularly preferably R 1 is acetyl.
  • R is NHMe or NH 2 , particularly preferably NH 2 .
  • a particular embodiment of the invention includes compounds of Formula I wherein R 2 , IV, R and R 5 are selected from H, methyl and ethyl, particularly preferably H and methyl and at least one among R 2 , R J , R 4 and R 5 is selected from methyl and ethyl, preferably methyl.
  • W is methyl
  • R ⁇ is H
  • R and R 5 are selected from H, methyl.
  • R is methyl and R", R and R are H.
  • the stereochemistry at all the ⁇ -carbon atoms is L;
  • the compounds of the invention may be isolated and purified as salts. Such salts fall within the scope of the invention. For the purposes of administration to a patient, it is desirable that the salts be pharmaceutically acceptable.
  • C ⁇ -C 6 -alkyl refers to monovalent branched or unbranched alkyl groups having 1 to 6 carbon atoms. This term is exemplified by groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl and the like.
  • Ci-Cs-alkyl refers to monovalent branched or unbranched alkyl groups having 1 to 5 carbon atoms. This term is exemplified by groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl and the like.
  • C 2 -C 6 Acyl refers to a group -C(0)R where R includes “CrCs-alkyl” groups. This term is exemplified by groups such as formyl, acetyl, propionoyl and butyroyl.
  • “Pharmaceutically acceptable salts” refers to salts of the compounds of Formula I that retain the desired biological activity.
  • examples of such salts include, but are not restricted to, acid addition salts formed with inorganic acids (e.g. hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, fumaric acid, maleic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene sulfonic acid, naphthalene disulfonic acid, and polygalacturonic acid.
  • inorganic acids e.g. hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like
  • organic acids such as acetic acid, oxalic
  • Said compounds can also be administered as pharmaceutically acceptable quaternary salts known by a person skilled in the art, which specifically include the quaternary ammonium salts of the formula -NR, R', R" + Z " , wherein R, R', R" is independently hydrogen, alkyl, or benzyl, and Z is a counter ion, including chloride, bromide, iodide, alkoxide, toluenesulfonate, methylsulfonate, sulfonate, phosphate, or carboxylate (such as benzoate, succinate, acetate, glycolate, maleate, malate, ftimarate, citrate, tartrate, ascorbate, cinnamate, mandelate, and diphenylacetate).
  • the compounds of the invention are typically administered in the form of a pharmaceutical composition.
  • Such compositions can be prepared in a manner well known in the pharmaceutical art and comprise at least one active compound.
  • the compounds of the invention are administered in a pharmaceutically effective amount.
  • the amount of the compound actually administered will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.
  • the pharmaceutical compositions of the invention can be administered by a variety of routes including oral, rectal, transdermal, intrathecal, subcutaneous, intravenous, intramuscular, and intranasal.
  • the compounds of the invention are administered by subcutaneous, intramuscular or intravenous injection or infusion.
  • a compound of the invention is fused to a carrier molecule, a peptide or a protein that promotes the crossing of the blood brain barrier ("BBB").
  • BBB blood brain barrier
  • Modalities for drug delivery through the BBB entail disruption of the BBB, either by osmotic means or biochemically by the use of vasoactive substances such as bradykinin.
  • Other strategies to go through the BBB may entail the use of passive diffusion and the use of endogenous transport systems, including carrier-mediated transporters such as glucose and amino acid carriers; receptor-mediated transcytosis for insulin or transferrin; adsorptive-mediated transcytosis.
  • Strategies for drug delivery behind the BBB further include intracerebral implantation.
  • the compounds may be formulated as injectable or oral compositions.
  • the compositions for oral administration can take the form of bulk liquid solutions or suspensions, or bulk powders. More commonly, however, the compositions are presented in unit dosage forms to facilitate accurate dosing.
  • unit dosage forms refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a pre-determined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
  • Typical unit dosage forms include pre-filled, pre-measured ampoules or syringes of the liquid compositions or pills, tablets, capsules or the like in the case of solid compositions.
  • the compound of the invention is usually a minor component (from about 0.1 to about 50% by weight or preferably from about 1 to about 40%) by weight) with the remainder being various vehicles or carriers and processing aids helpful for forming the desired dosing form.
  • Liquid forms suitable for oral administration may include a suitable aqueous or non-aqueous vehicle with buffers, suspending and dispensing agents, colorants, flavours and the like.
  • Solid forms may include, for example, any of the following ingredients, or compounds of a similar nature: a binder such as macrocrystalline cellulose, gum tragacanth or gelatine; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavouring agent such as peppermint, methyl salicylate, or orange flavouring.
  • a binder such as macrocrystalline cellulose, gum tragacanth or gelatine
  • an excipient such as starch or lactose
  • a disintegrating agent such as alginic acid, Primogel, or corn starch
  • Injectable compositions are typically based upon injectable sterile saline or phosphate- buffered saline or other injectable carriers known in the art.
  • the compounds of this invention can also be administered in sustained release forms or from sustained release drug delivery systems.
  • sustained release forms or from sustained release drug delivery systems A description of representative sustained release
  • the compounds of the invention prevent the aggregation of A ⁇ associated with the onset and progression of Alzheimer's disease, Dementia pugilistica (including head trauma), Hereditary Cerebral Haemorrhage with amyloidosis of the Dutch type (HCHWA-D) and vascular dementia with amyloid angiopathy.
  • administration of the compounds is by injection or infusion, at periodic intervals.
  • the administration of a compound of the invention should preferably begin before any symptoms are detected in the patient, and should continue thereafter.
  • HCVWA-D Hereditary Cerebral Haemorrhage with amyloidosis of the Dutch type
  • vascular dementia with amyloid angiopathy include those with a familial history of these diseases.
  • Still a further aspect of the present invention is a process for preparing the compounds of Formula I.
  • the compounds of the invention may be prepared from readily available starting materials using the following general methods and procedures. It will be appreciated that where typical or preferred experimental conditions (i.e., reaction temperatures, time, moles of reagents, solvents, etc.) are given, other experimental conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimisation procedures.
  • the compounds of the invention may be prepared using methods of peptide synthesis known
  • the compounds of the invention are synthesised using solid-phase methods.
  • BOP benzotriazol-l-yl-oxcy-tris-(dimethylamino)- phosphonium hexafluorophosphate
  • BOP-Cl bis(2-oxo-3-oxazoldinyl) phosphinic chloride), Boc (butoxycarbonyl), BSA (Bovine Serum Albumin), Cbz (carboxybenzyl), DCM (dichloromethane), DIC (diisopropyl carbodiimide), DCC
  • ⁇ (Me), in the peptide sequence stands for a methyl group branched on the N atom of the amino acid.
  • the compounds according to formula I may be prepared from readily available starting materials. Examples of synthetic pathways will be described below.
  • the peptides of Formula (I) may be synthesized on a solid support using, for example, preferred Rink- Amide resin.
  • a solution of piperidine in DMF (20-50%>) is applied to remove the Fmoc-protecting group from the resin.
  • the resin is gently shacked for 30 minutes to lhour in such a solution. After draining, the resin is washed with DCM, DMF, THF and again DCM each three times.
  • a solution of Fmoc-aspartic acid ( ⁇ -O-t-butyl) and the resin are shaken with 1.5 equivalents of coupling reagent, such as PyBOP, TBTU, HATU, BOP-Cl, PyBrop, BOP, EDC, DIG, DCC, preferably HATU, and 3 equivalents of a tertiary base, such as NMP, triethylamine, diisopropyl-ethylamine or any tertiary base with similar pKa, preferably diisopropylethylamine.
  • coupling reagent such as PyBOP, TBTU, HATU, BOP-Cl, PyBrop, BOP, EDC, DIG, DCC, preferably HATU
  • a tertiary base such as NMP, triethylamine, diisopropyl-ethylamine or any tertiary base with similar pKa, preferably diisopropyle
  • the group R 1 is introduced as follows.
  • the Fmoc moiety is removed from compound of Formula (III) as described above in the deprotection step and the group R is introduced.
  • Tritiated derivatives of compound of Formula I can be prepared following the general protocol coupling a 3,4-dehydro-proline residue in step 4.
  • the Dehydro-Pro peptide (2 mg) is mixed with 10% palladium on calcium carbonate (10 mg) and DMF (1 ml) in a tritiation vessel. The mixture is stirred under tritium gas (5 Ci) for 3 hours. The solution is filtered and labile tritium removed by repeated evaporations to dryness with ethanol.
  • Crude reaction mixture was purified by high performance liquid chromatography in the following system: On a Vydac C18 Protein and Peptide column (250 x 9.6 mm) using a water: acetonitrile: TFA gradient system. The detection is performed by a radioactive detector and a UV detector (220 nm). Product collected, evaporated to dryness, re-dissolved in dispensing eluent.
  • the following building blocks are commercially available from Bachem, Switzerland, Fmoc-L-phenylalanine, Fmoc-( ⁇ -OtBu)-L-aspartic acid, Fmoc-L-leucine, Fmoc-L-proline, Fmoc-N-Me-L-phenylalanine, Fmoc-N-Me-(OtBu)-L-aspartic acid, Fmoc-N-Me-L-leucine.
  • Coupling reagents are commercially available from Novabiochem, Switzerland.
  • the peptide derivatives according to the general Formula (I) can be synthesized using standard peptide synthesis techniques either in solution or on solid phase. In both approaches typical coupling reagents are used, which are known to the person skilled in the art. It will be also appreciated that where typical or preferred experimental conditions (i.e. reaction temperatures, time, moles of reagents, solvents etc.) are given, other experimental conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions will be appreciated by the person skilled in the art.
  • Examples 1 to 7 are preferred embodiments of the invention. Structures of compounds of Examples 1 to 6 are presented in Table 1 below.
  • Compounds of Examples 1, 2, 3, 4, 5 and 6 may be synthesized according to protocol presented in scheme 1.
  • Compound of Example 7 stands for tritiated compound of Example 1 which may be synthesized according to protocol presented in scheme 1 using a 3,4-dehydro-proline amino acid instead of Proline in step 4. The peptide is then tritiated according to the tritiation protocol above.
  • Compound of Example 7 had the specific activity of 42 Ci/mmol.
  • the stability of the compounds of the invention can be assayed in comparison with the reference compound (Example 8).
  • Peptides were prepared as a l ⁇ g/ ⁇ l solution in water. 20 ⁇ l of the peptide solution was diluted in 80 ⁇ l of fresh human plasma or 10% rat brain homogenate (prepared in PBS). The resulting solution was incubated at 37°C for different times and the reaction was stopped by adding a complete cocktail of protease inhibitors. The bulk of plasma and brain proteins (but none of the peptide) were precipitated in cold methanol (mix/MeOH, 4/5, v/v) during one hour at -20°C. The precipitated proteins were pelleted by centrifugation (lOOOOg, 10 min, 4°C).
  • the activity of the compound of the invention in inhibiting the formation of aggregated fibrils can be tested by following the changes in fluorescence signal of a fluorophore that has an affinity for the amyloid fibrils.
  • Amyloid formation was quantitatively evaluated by the fluorescence emission of Thioflavine T (ThT) bound to amyloid fibrils, as reported by Levine and also Soto et al.
  • Aliquots of A ⁇ l-42 (a synthetic peptide with the same sequence as the one deposited in amyloid plaques in Alzheimer's brain) at a concentration of 0.5 mg/ml prepared in 0.1M Tris, pH 7.4 were incubated for 5 days at 37°C, gently swirled on a rotaiy shaker, in the absence or in the presence of different concentrations of the compounds.
  • 50 mM glycine, pH 9.2 and 2 ⁇ M ThT were added in a final volume of 2 ml.
  • Amyloid fibrils are cytotoxic, inducing cell death by apoptosis. 18
  • the ability of the compounds of the invention in preventing the amyloid formation can be evaluated by measuring the inhibition of the cytotoxicity in a cell assay.
  • the ability of compound of the invention to cross the BBB can be checked by capillary depletion experiments and the kinetics of their penetration into the brain can be measured.
  • Capillary depletion is used to assess the penetration into the brain of compounds of the invention.
  • a "wash-out” step removes blood from the brain so that levels of the compounds of the invention present in the brain parenchyma can be measured.
  • mice of group 1 are sacrificed or blood is removed by injecting 20 ml lactated Ringer's solution (7.19 g/1 NaCl, 0.3 g/1 KCL, 0.28 CaC12, 2.1 g/1 NaHC0 3 , 0.16+ g/1 KH 2 P0 4 , 0.37 g/1 MgCl 2 6H 2 0, 0.99 g/1 D-glucose, 10 g/1 bovine serum albumin, pH 7.4) into the left ventricle of the heart for 30 sec, which removes more than 95 % of the vascular contents of the brain (blood brain washout, group 2).
  • 20 ml lactated Ringer's solution (7.19 g/1 NaCl, 0.3 g/1 KCL, 0.28 CaC12, 2.1 g/1 NaHC0 3 , 0.16+ g/1 KH 2 P0 4 , 0.37 g/1 MgCl 2 6H 2 0, 0.99 g/1 D-glucose, 10 g/1 bovine serum albumin, pH 7.4
  • Brain/serum ratio (cpm/g brain)/(cpm/ ⁇ l serum).
  • the cerebral cortex is weighed and homogenized in a physiological buffer (10 mM HEPES, 140 mM NaCl, 4 raM KC1, 2.8 mM CaCl 2 , 1 mM MgS0 4 , 1 mM Na H 2 P0 ; and 10 mM D-glucose, pH 7.4).
  • Dextran solution 1.6 ml of a 26% solution
  • centrifugation (5,400 g, 15 min, 4°C)
  • brain vasculature (pellet) and parenchyma (supernatant) are separated and the radioactivity determined in each fraction.
  • Blood brain barrier permeability study The kinetics of penetration of compound of the invention into the brain can be evaluated through blood brain barrier permeability experiments. The percentage of injected peptide found in the brain can then be calculated.
  • mice are anaesthetized with i.p. urethane (40%) and the left jugular vein is exposed.
  • 0.2 ml of lactate Ringer's solution (7.19 g/1 NaCl, 0.3 g/1 KCL, 0.28 CaC12, 2.1 g/1 NaHC0 3 , 0.16+ g/1 KH 2 P0 4 , 0.37 g/1 MgCl 2 6H 2 0, 0.99 g/1 D-glucose, 10 g/1 bovine serum albumin, pH 7.4) containing 1% BSA and tritium labelled peptide ("hot") of Example 7 (100 000 cpm/ml of tritium radioactive peptide) is injected.
  • lactate Ringer's solution (7.19 g/1 NaCl, 0.3 g/1 KCL, 0.28 CaC12, 2.1 g/1 NaHC0 3 , 0.16+ g/1 KH 2 P0 4 , 0.37 g/1 MgCl 2 6H 2 0, 0.99
  • the representation of the brain to serum ratio versus time allows the derivation of the influx rate, Ki (slope) and the volume of distribution (Y intercept), Vi.
  • the influx rate Ki, microl (serum)/g (tissue weight)-min
  • the volume of distribution Vi, microl (seram)/g (tissue weight)
  • results are presented in Table 6.
  • the influx rate (Ki), with radioactive peptide, alone was 3.54 +/- 0.29 microl/g-min with a Y intercept of about 50 microl/g (Table 4). This indicates that compound of Example 1 penetrates the brain.
  • HPLC analysis of brain samples showed that 12% of intact peptide was recovered 20 min post i.v. injection.
  • the inhibitory activity the compound of the invention in the formation of amyloid deposits by can be visualized in vivo by staining cerebral tissue sections with anti-A ⁇ - 2 antibodies in the presence and absence of a peptide of the invention.
  • the animal received a bilateral injection of 5.0 nmol A ⁇ 1- 2 into each amygdale by using a Kopf stereotaxic instrument with the incisor bar set at 3.3 mm below the interaural line.
  • Injection coordinates measured from the bregma and the surface of the skull were empirically determined based on the atlas of Paxinos and Watson.
  • a volume of 3.0 ⁇ l of the solution of A ⁇ - 2 at 5.0 nmol was administered over 6 min (flow rate 0.5 ⁇ l/min) using a CMA/100 micro syringe pump.
  • the cannula was left in situ for 2 min following injection, then it was withdrawn 0.2 mm and left for 3 min, and following these 5 min the cannula was slowly withdrawn. Following surgery the animals were placed on a heating pad until they regained their righting reflex. The animals were treated with compound of Example 1 and of Example 8. The peptides, solubilized in a 0.9%o NaCl at the concentration of 4.4 mM were injected s.c (0.5 ml per injection), 4 times a week during 7 and a half weeks. After treatment with the compounds, animals were sacrificed by an overdose of sodium pentobarbital (150 mg/kg, i.p.), perfused transaortically.
  • sodium pentobarbital 150 mg/kg, i.p.

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EP03753576A 2002-07-08 2003-07-07 Beta-sheet breaking peptides Withdrawn EP1532170A2 (en)

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