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WO2003053356A2 - Inhibiteurs apomorphines de la formation des fibrilles de l'amyloide-$g(b) a$g(b) et leur utilisation dans les maladies derivees de l'amyloidose - Google Patents

Inhibiteurs apomorphines de la formation des fibrilles de l'amyloide-$g(b) a$g(b) et leur utilisation dans les maladies derivees de l'amyloidose Download PDF

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Publication number
WO2003053356A2
WO2003053356A2 PCT/US2002/040660 US0240660W WO03053356A2 WO 2003053356 A2 WO2003053356 A2 WO 2003053356A2 US 0240660 W US0240660 W US 0240660W WO 03053356 A2 WO03053356 A2 WO 03053356A2
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Prior art keywords
apomoφhine
amyloid
derivative
analog
apomorphine
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WO2003053356A3 (fr
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Hilal A. Lashuel
David J. E. Callaway
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Cytokine Pharmasciences Inc
Picower Institute for Medical Research
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Cytokine Pharmasciences Inc
Picower Institute for Medical Research
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Priority to CA002479263A priority patent/CA2479263A1/fr
Publication of WO2003053356A2 publication Critical patent/WO2003053356A2/fr
Publication of WO2003053356A3 publication Critical patent/WO2003053356A3/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/473—Quinolines; Isoquinolines ortho- or peri-condensed with carbocyclic ring systems, e.g. acridines, phenanthridines

Definitions

  • the invention relates to preparations and methods for use in the prophylaxis and treatment of amyloidosis-based diseases, including amyloid encephalopathies such as non-senile dementias, in particular Alzheimer's disease.
  • amyloid encephalopathies such as non-senile dementias, in particular Alzheimer's disease.
  • the invention relates to use of compounds related to apomorphine and oxidation products thereof to inhibit A ⁇ amyloid fibril formation by interfering with aggregation of amyloid- ⁇ peptide (A ⁇ 1-40) into amyloid fibrils.
  • U. S. Patent No. 6,210.680 B 1 to Jia. et al. discloses a method for the prevention and treatment of chronic venous insufficiency using isoquinoline alkaloids, and for purifying and isolating such alkaloids, including aporphine
  • alkaloids from plants.
  • Alzheimer's disease is a progressive neurodegenerative disease that is characterized by the presence of both extracellular (amyloid plaques) as well as intraneuronal (neurofibrillaiy tangles) protein aggregates in the brain of affected patients (1-3). Biochemical analysis reveals that fibrillar aggregates of a 39-42 residue peptide referred to as the A ⁇ protein is the main constituent of amyloid plaques (4).
  • amyloid hypothesis suggests that the aggregation and deposition of the amyloid- ⁇ peptide(s) into fibrils within amyloid plaques precedes neurofibrillaiy tangle formation, and is critical for triggering a cascade of events that contribute directly to the pathogenesis of Alzheimer's disease (1, 5).
  • interfering with the aggregation of ⁇ -amyloid is considered a viable therapeutic strategy for slowing and/or preventing the progression of neurodegeneration in Alzheimer's disease (AD) (6, 7).
  • a ⁇ fibril formation was thought to follow a two-state nucleated polymerization mechanism, where only the amyloidogenic conformational intermediate and amyloid fibrils are significantly populated during fibril formation
  • amyloid-related disorders which are caused by the misfolding and aggregation of a precursor protein from its native (folded, partially folded, or
  • These compounds include dyes such as Congo Red, the antibiotic rifampicin, the anthracycline 4'-iodo-4'-deoxydoxorubicin, among other reported inhibitors (7), imidazopyridoindoles (26), and a large variety of A ⁇ -derived peptides and modified peptides, including peptides homologous to A ⁇ [positions 17-21] with a similar degree of hydrophobicity, but with a very low propensity to adopt a beta-sheet conformation by incorporating proline residues (anti-beta-sheet peptides or beta-sheet inhibitors; 27). Many of the compounds were shown to function by prolonging the nucleation stage associated with the formation of an aggregation competent nucleus, i.e., fibril formation was delayed. Other compounds alter the
  • a ⁇ fibrillogenesis based on the structure of apomorphine (FIG. 1). These compounds strongly interfere with the aggregation of A ⁇ 1-40 into amyloid fibrils in vitro. Sedimentation velocity and electron microscopy studies clearly demonstrate that apomorphine and several of its structural analogues function as A ⁇ fibril formation inhibitors by binding to and stabilizing preamyloid high molecular weight soluble nonfibrillar assemblies. Apomorphine is a long-used drug, which has beneficial effects on Parkinsonian patients experiencing "on” and "off disabling problems after prolonged use. of
  • apomorphine Levodopa (29, 30).
  • one of the limitations of apomorphine is that it is very labile in aqueous solution, where it is highly susceptible to auto-oxidation (31, 32).
  • Apomorphine is oxidized by a multistep reaction that eventually leads to a polymeric melanin-like end product (33).
  • the results presented here demonstrate that auto-oxidation of apomorphine analogs could be critical for their activity as inhibitors of A ⁇ fibril formation. This suggests that one or several of the auto- oxidation products of apomorphine interacts with A ⁇ in a manner that inhibits A ⁇ fibril formation.
  • the present invention relates to methods of inhibiting A ⁇ fibril
  • compositions comprising an apomorphine derivative or analog of the invention are also provided.
  • Apomorphine derivatives and analogs suitable for use in the methods of the invention include aporphine alkaloid compounds that inhibit A ⁇ fibril formation of the following formula I:
  • R j and R 2 are independently selected from the group consisting of H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl or substituted alkenyl;
  • R 3 , R 4 , R 5 , Rg, R 7 and R 8 are independently selected from the group consisting of H, hydroxy, thiol, methoxy, methyl sulfide, methylenedioxy, alkoxy, alkyl sulfide; and pharmaceutically acceptable acid addition salts thereof.
  • Acceptable salts may be selected from the group consisting of chloride, iodide, fluoride, sulfate, phosphate, acetate or carbonate.
  • R 4 and R 5 are hydroxyl groups, and most preferably both R 4 and R 5 are hydroxyl groups.
  • a preferred subgenus of the compounds of formula I, above, comprises apomorphine derivatives or analogs that inhibit A ⁇ fibril formation of the following formula II:
  • R j is H or Cj-C 6 alkyl, and R 2 , R 3 , R 4 and R 5 are independently selected from the group consisting of H,
  • At least one of R, and R 5 is
  • R 4 and R 5 are hydroxyl groups.
  • Additional compounds useful in the invention methods are 1-hydroxy- aporphine derivatives or analogs that inhibit A ⁇ fibril formation of the general formula disclosed and recited in the claims in U.S. Patent No. 4,202,980, the disclosure of which is hereby incorporated herein by reference for its disclosure of the compounds and methods of making same.
  • oxidation products of the above compounds that inhibit A ⁇ fibril formation, particularly such products of auto-oxidation of a catechol moiety (D ring) of the above compounds, are preferred for use in the methods of the invention.
  • the above apomorphine derivatives or analogs or oxidation products thereof that inhibit A ⁇ fibril formation are useful in a method for inhibiting, reducing or delaying A ⁇ fibril formation comprising contacting A ⁇ protein with an amount of such an apomorphine derivative or analog or oxidation product thereof, or pharmaceutically acceptable salt thereof, effective
  • the invention in another aspect relates to inhibiting, reducing or delaying the formation of amyloid or amyloid-like deposits in a subject in need of such treatment, comprising administering to the subject an effective amount of an apomorphine derivative or analog or oxidation product thereof of the invention that inhibits A ⁇ fibril formation.
  • the invention includes a method of
  • a ⁇ fibril formation associated with an amyloidosis-based condition, disease or disease state comprising administering to a subject for treatment or prophylaxis of such a condition or disease an effective amount of an apomorphine derivative or analog or oxidation product thereof of the invention, that inhibits A ⁇ fibril formation.
  • the subject of the above methods is a human subject suspected of being susceptible to or suffering from amyl ⁇ idosis associated with Alzheimer's disease.
  • the invention provides a method of delaying the onset of Alzheimer's disease in individuals predisposed to Alzheimer's disease or for delaying the onset of other amyloidosis-related diseases, comprising administering an effective amount of an apomorphine derivative or analog or oxidation product thereof of the invention to a subject in need thereof to reduce, inhibit or reverse the formation of amyloid fibrils or amyloid or amyloid-like deposits associated with Alzheimer's disease or with other amyloidosis-related diseases. DESCRIPTION OF THE FIGURES
  • FIG. 1 shows the structure of apomorphine which is related to various test compounds disclosed herein.
  • the rectangle highlights a portion of the D ring (catechol moiety) of apomorphine which is thought to be critical for inhibition of
  • FIG. 2 shows an emission spectra of Thioflavin T of solutions of A ⁇ (100 ⁇ M) incubated alone (solid line) and A ⁇ incubated in the presence of apomorphine (100 ⁇ M) (dashed line).
  • Thioflavin T in the presence of aggregates exhibits an emission maximum at 482 nm characteristic of amyloid fibrils (excited at 450 nm). Solutions of A ⁇ containing apomorphine did not exhibit this typical fluorescence in the presence of Thioflavin T.
  • FIG. 3 illustrates sedimentation velocity profiles (A) sample of A ⁇ (100 ⁇ M) after incubation for 3 days in pH 7.4 buffer (50 mM Tris, 150 mM NaCl) in the presence of apomorphine (100 ⁇ M). Scans were acquired at 1-min intervals, for clarity scans every 4 min are shown. The absorbance baseline at 0.6 represents absorbance of unsedimented A ⁇ and excess apomorphine in solution. (B) Analysis of the sedimentation profiles in (A) using the time derivative method. The sedimentation coefficient distribution plot (g(*s)) reveals the presence of heterogeneous distribution of high MW Ab-apomorphine aggregates. The solid line represents three species fit of the experimental data (x).
  • FIG. 4 shows a negatively stained electron micrograph of a sample of A ⁇ 40 (100 ⁇ M) in the presence of apomorphine 50 ⁇ M (A), and 25 ⁇ M (B). At apomorphine concentrations of ⁇ 50 ⁇ M significant fibrils were formed after 3- 6 days of incubation at 37°C.
  • FIG. 5(A) displays sedimentation velocity profiles of A ⁇ (100 ⁇ M) in the
  • FIG. 6 shows structures of apomorphine and apomorphine derivatives used to evaluate the role of the dihydroxy and N-alkyl substitutions in modulating the ability of apomorphine to inhibit A ⁇ fibril formation.
  • FIG. 7 presents a bar graph depicting the extent of A ⁇ fibril formation in the absence and presence of apomorphine and apomorphine derivatives (FIG. 6).
  • FIG. 8 is an electron micrograph of negatively stained quaternary structures deposited from a solution of A ⁇ (100 ⁇ M) at pH 7.4 (50 mM Tris, 150 mM NaCl) after incubation for 9 days at 37°C in the accumulation presence of D030, demonstrating the accumulation of some long fibrils in solution.
  • FIG. 9(A) displays a sedimentation coefficient distribution of a sample of A ⁇ (100 ⁇ M) at pH 7.4 (50 mM Tris, 150 mM NaCl) after incubation for 3 days at 37°C in the presence of D027 (100 ⁇ M) obtained from a 2 species fit (solid line) of the experimental data (x).
  • Sedimentation velocity analysis of the profiles shown in (B) obtained by fitting the data to three-sedimenting species model.
  • FIG. 10 is a bar graph depicting the extent of A ⁇ fibril formation in the absence and presence of apomorphine, D027, D029, and D030 using freshly prepared stock solutions (dark bars), aged stock solutions (gray bars), and stock solution of the compounds containing 1 % (v/v) sodium metabisulphite.
  • FIG. 11 illustrates Thioflavin T fluorescence of samples of A ⁇ (100 ⁇ M) in the presence and absence of apomorphine (dark bars).
  • the gray bar represents the Thioflavin T fluorescence obtained by preincubation of the A ⁇ control sample with apomorphine prior to addition of the Thioflavin T.
  • amyloid- ⁇ deposits in the form of amyloid plaques in the brain parenchyma is thought to play a critical role in the pathogenesis of Alzheimer's disease.
  • a ⁇ amyloid- ⁇
  • Amyloid fibril formation is known to proceed through a nucleation-dependent polymerization mechanism characterized by a slow nucleation phase followed by a rapid fibril growth phase (8). This mechanism presents several stages for possible intervention with the polymerization of A ⁇ into amyloid fibrils.
  • An inhibitor of A ⁇ fibril formation could act by inhibiting the formation of the required nucleation complex or by binding to these nuclei, thus delaying and/or preventing further addition of A ⁇ monomers.
  • An inhibitor that interferes at the nucleation stage is likely to result in a marked delay in the onset of fibrillization and possibly Alzheimer's disease.
  • An alternative strategy could involve the stabilization of non-toxic intermediates (through complex formation with small molecules) on the amyloid fibril formation pathway of A ⁇ resulting in delaying and/or preventing amyloid fibrils from being formed.
  • a delay in amyloid fibril formation may be sufficient to allow for more efficient clearance of toxic preamyloid intermediates as well as preformed amyloid fibrils.
  • the ability to stabilize a non-toxic A ⁇ complex would allow for high A ⁇ load without being detrimental to neurons.
  • Nonfibrillar oligomeric A ⁇ species have been demonstrated to exist in many different forms, including globular, ring structures, and chain-like structures. Many of these oligomeric forms of A ⁇ have been observed mainly as transient species on the fibril formation pathway of A ⁇ as well as other amyloidogenic protein such as ⁇ -synuclein, and transthyretin.
  • the oligomeric intermediates stabilized by apomorphine, D027, D029, D030, and D040 are morphologically distinct from those observed in the case of A ⁇ alone.
  • the size of these oligomers appears to be at least twice that seen for protofibrils of A ⁇ as determined by sedimentation velocity and electron microscopy studies.
  • apomorphine interacts with A ⁇ in a manner that promotes rapid oligomerization into protofibril-like species, but also results in slowed and/or inhibited fibril formation.
  • amyloid fibrils share a common structural feature, namely a beta pleated sheet conformation (43, 44).
  • amyloid hypothesis has implicated amyloid fibrils as the main pathological agent in amyloid diseases. However, it remains to be resolved
  • AD Alzheimer's disease
  • Apomorphine and other compounds capable of stabilizing intermediates on the fibril formation pathway are useful tools for testing such hypotheses.
  • amyloid fibrils are the toxic species then compounds such as apomoiphine or derivatives thereof would yield a potential therapeutic for treating AD.
  • An interesting property of the compounds presented here is that they seem to target a common intermediate (protofibrils) on the fibril formation pathway of A ⁇ . It is possible that these compounds may have the same effect in stabilizing protofibrils of other amyloidogenic proteins and may be a useful tool for the study and treatment of other forms of amyloidosis.
  • apomorphine In addition to its apparent anti-amyloidogenic properties, apomorphine is known to act as a potent antioxidant and protects lipids and proteins from radical damage. This, suggests apomoiphine derivatives and analogs as good therapeutic candidates in Alzheimer's disease, combining both radical scavenging and neurotrophic properties.
  • Samples for fibril formation assay were prepared by diluting the appropriate amount of A ⁇ stock with pH 7.4 Tris-HCl buffer (50 mM Tris, 150 mM NaCl) to afford a final peptide concentration of 100 ⁇ M. The samples were incubated at 37°C without agitation for 1-10 days, depending on the desired experiment. Analytical ultracentrifugation, and electron microscopy were periodically used to ensure that we start our experiments with a hydrodynamically defined A ⁇ monomer, and the absence of any preformed fibrils in solution. Congo Red Binding Studies. A Congo red stock solution was prepared by
  • Thioflavin T Fluorescence Assay for Fibril Formation.
  • Thioflavin T was purchased from Aldrich (70 % purity) and was recrystallized from benzene/ethanol (1 : 1) to afford yellow crystals (36).
  • Thioflavin T binding assays were performed by addition of 50 ⁇ L of 100 ⁇ M A ⁇ solutions, which were incubated at 37°C in the absence and presence of inhibitors, to 450 ⁇ L solution of 10 ⁇ M Thioflavin T in phosphate buffer (10 mM phosphate, 100 mM KC1). The solutions were mixed and fluorescence measurements were recorded in Biosystems model spectrofluorimeter at 25°C using a 1 cm path length quartz cell.
  • A is the absorbance at radius x
  • -o is the absorbance at a reference radius x ⁇ , (usually the meniscus)
  • ⁇ is the partial specific volume of A ⁇ (0.733 ml/g, based on the amino acid sequence of A ⁇ 1-40)
  • p is the density of the solvent (g/mL)
  • ⁇ is the angular velocity of the rotor (radian/sec)
  • E is the baseline error correction factor
  • M is the molecular weight
  • R is the universal gas constant (8.314 x 10 7 erg mole)
  • T is the temperature (Kelvin)!
  • a ⁇ samples (100 ⁇ M) in a pH 7.4 buffer (50 mM Tris-HCl, 150 mM NaCl) were incubated at 37°C for 1-
  • This method has the advantage of allowing for the calculation of the molecular weight of the sedimenting species using equation II since s and D can be obtained from the fitting analysis.
  • the molecular weight of a spherical protein can still be estimated by combining the Svedberg equation and Stokes equation for the frictional coefficient to obtain equation III which utilizes the estimated sedimentation coefficient:
  • Sedimentation velocity was also used to determine the amount of soluble A ⁇ and A ⁇ monomer in solution after sedimentation of A ⁇ aggregates.
  • two radial scans were collected at 3,000 rpm (only fibrils are sedimentable at this speed) and 20,000-
  • Electron Microscopy was used to follow the changes in size and structural mo ⁇ hology of A ⁇ aggregates in the presence and absence of inhibitor at 37°C as a function of time. EM samples were prepared by placing 5 ⁇ L of the A ⁇ solution on a carbon-coated grid and allowing the solution to stand for 2 min before removing excess solution. The grid was then washed once with distilled water and once with 1% uranyl acetate before staining the sample with
  • a ⁇ 1-40 Effect of Apomorphine on Amyloid- ⁇ (A ⁇ 1-40) Fibrillization. Solutions of A ⁇ 1-40 (50-100 ⁇ M) were incubated at 37°C for 1-3 days in the absence and presence of apomo ⁇ hine at equimolar concentration. Within 2-3 days, visible sedimentable aggregates were observed in samples containing A ⁇ 1-40 alone. In strong contrast, A ⁇ 1-40 (100 ⁇ M) solutions incubated under the same conditions, but in the presence of apomo ⁇ hine (100 ⁇ M) did not show any visible sedimentable aggregates.
  • ThT Thioflavin T
  • a ⁇ 1 -40 (100 ⁇ M) solutions incubated in the presence of apomo ⁇ hine at 100 ⁇ M resulted in the accumulation of predominantly soluble, nonfibrillar assemblies.
  • the absence of amyloid fibrils in A ⁇ 1-40 solutions containing apomo ⁇ hine is consistent with the absence of a Thioflavin T signal and of Congo red binding for these samples.
  • FIG. 3 A Analysis of the sedimentation profiles revealed the sedimentation of A ⁇ - apomo ⁇ hine complex(s) with an average sedimentation coefficient of 50.S, indicative of soluble high molecular weight assemblies as the dominant species, FIG. 3B.
  • the sedimentation coefficient distribution plot revealed a heterogeneous distribution of A ⁇ oligomeric species.
  • a ⁇ 1-40 (100 ⁇ M) samples were incubated in the presence of
  • both sedimentation velocity and EM revealed stabilization of soluble oligomeric species with a sedimentation coefficient distribution and structural mo ⁇ hology similar to that observed at 100 ⁇ M of apomo ⁇ hine.
  • both long unbranched fibrils and short nonfibrillar assemblies were observed by electron microscopy (FIG. 4A). However, the nonfibrillar assemblies remained the dominant species in solution ( ⁇ 70-85%) over the incubation period of 3-6 days at 37°C. At concentrations below 50 ⁇ M
  • FIG. 5A Time derivative (dc/dt) analysis of the sedimentation profiles demonstrated the presence of two
  • FIG. 5B At an apomo ⁇ hine concentration of 25 ⁇ M (0.25:1, apomo ⁇ hine: A ⁇ ) approximately 40-50% of the A ⁇ sample sedimented rapidly at 3,000 ⁇ m, reflecting formation of amyloid fibrils. Analysis of the sedimentation velocity profiles of the soluble species rem----- ⁇ ing in solution after three days of incubation at 37°C also revealed two populations of high-molecular weight species with an
  • apomo ⁇ hine The structure of apomo ⁇ hine is characterized by the presence of two adjacent hydroxyl groups on the D ring of the molecule.
  • Several commercially available structural analogues of apomo ⁇ hine were examined to evaluate the role of the dihydroxy substitutions (10, 11) of the D ring, and the N- alkyl group of apomo ⁇ hine in modulating the inhibitory effectiveness of these apo ⁇ hines, FIG. 6.
  • the compounds shown in FIG. 6 were incubated with A ⁇ 1-40 (100 ⁇ M) under conditions that favor fibril formation (pH 7.4, and at 37°C for 3 days).
  • the amount of fibrils formed was evaluated by Thioflavin T fluorescence, and characterization of the mo ⁇ hology and the distribution of aggregated species was evaluated by electron microscopy and sedimentation velocity, respectively.
  • FIG. 7 shows the Thioflavin T binding results of apomo ⁇ hine and related compounds after incubation with A ⁇ 1-40 for three days (37°C).
  • a ⁇ 1-40 solutions did not exhibit any increase in ThT fluorescence, whereas in the presence of bulbocapnine, R (-)- apocodeine, isocrydine, and M121, A ⁇ 1-40 solutions showed ThT binding and ThT fluorescence similar in magnitude to that of A ⁇ 1-40 incubated alone.
  • the compounds were effective at inhibiting A ⁇ 1-40 (100 ⁇ M) fibril formation at 50 and 100 ⁇ M over the incubation period of three days, with the exception of D030, which was shown to inhibit fibril formation only at 100 ⁇ M (1:1, A ⁇ :D030).
  • D030 which was shown to inhibit fibril formation only at 100 ⁇ M (1:1, A ⁇ :D030).
  • apomo ⁇ hine-related alkaloids bulbocapnine, R( - )-apocodeine, isocrydine, and M121 one or both D-ring hydroxyl groups are derivatized (by methyl or methylene).
  • ThT positive samples Examination of these ThT positive samples by EM confirmed the presence of highly ordered fibrils similar to those seen when A ⁇ was incubated alone under the same conditions. Together, these results demonstrate that the hydroxyl groups on the D ring of apomo ⁇ hine and derivatives are required for the inhibitory effectiveness of these
  • a ⁇ fibrils The delayed appearance of A ⁇ fibrils is consistent with the idea that the precedent short nonfibrillar assemblies of A ⁇ -inhibitor are an on-pathway intermediates of amyloid fibril formation.
  • Apomorphine, D027, D029, D030 and D040 Promote A ⁇ Oligomerization, but Inhibit Fibrillization.
  • Incubation of A ⁇ 1-40 at 37°C for 17-20 hr populates a set of protofibril intermediates, but little or no fibril formation occurs. Additional incubation (40-72 h) leads to the disappearance of protofibrils and appearance of fibrils as the predominant species in the sample.
  • FIG.10A shows a negatively stained electron microscopy image of a sample of A ⁇ after incubation for 20 h at 37°C, pH 7.4) revealing protofibrillar aggregates as the predominant species in solution. Sedimentation velocity analysis of this sample indicates that 15-20% of the sample sediments as a broad boundary corresponding to a ladder of protofibrillar species with an average sedimentation coefficient of 20 S. Equilibrium studies revealed that the rem- ⁇ ning 80-85% of A ⁇ in solution (i.e., unsedimented A ⁇ ) exists in its monomeric form.
  • Sedimentation velocity analysis of this sample indicate that the A ⁇ - inhibitor complexes sediment as a broad boundary corresponding to a ladder of sedimenting species with an average sedimentation coefficient of 33, 35, 33, and 31 S in the presence of D027, D029, D030, and D040 respectively.
  • apomo ⁇ hine is unique as an anti-amyloidogenic agent as it acts simultaneously as both a promoter of A ⁇ oligomerization and an inhibitor of A ⁇ fibrillization.
  • Apomorphine and its Structural Analogues Binding to A ⁇ Monomer vs. Oligomeric Intermediates.
  • the sedimentation velocity and EM studies suggest that apomo ⁇ hine and some of its derivatives bind preferentially to early oligomeric intermediates ofA ⁇ and result in a marked delay of fibril formation.
  • Analytical ultracentrifugation methods were also employed to probe the mechanism of interaction of these molecules with A ⁇ . The absorbance of these compounds at 300-340 nm, where A ⁇ does not absorb, enabled us to selectively monitor the sedimentation and estimate the size of the soluble A ⁇ aggregates that were bound to apomo ⁇ hine.
  • Apomo ⁇ hine and its analogs were also evaluated by adding apomo ⁇ hine or one of its analogues to a pre-incubated solutions of A ⁇ , which were determined to contain preformed A ⁇ protofibrils (20-25%).
  • Apomo ⁇ hine, D027, D029, D030, and D040 were added to A ⁇ solutions that were pre-incubated at 37°C for 18-20 hr (mainly protofibrils are formed under these conditions).
  • the samples were further incubated at 37°C for three to nine days, after which the solutions were ex--mined by EM and electron microscopy and sedimentation velocity.

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Abstract

La présente invention concerne une nouvelle classe d'inhibiteurs à petites molécules de l'agrégation de la protéine amyloïde β (Aβ), à base d'apomorphine. Les molécules précitées ciblent la phase de nucléation de l'autoassemblage d'Aβ et entravent efficacement l'agrégation d'Aβ 1-40 dans les fibrilles d'amyloïde in vitro tel que mesuré par le microscope électronique à transmission, la fluorescence par la thioflavine T et la vitesse de sédimentation. Des études de structure-activité dans lesquelles ont été utilisés des analogues de l'apomorphine ont démontré que les substitutions 10,11-dihydroxy du noyau D sont préférées pour l'efficacité inhibitrice des apomorphines, et que la méthylation de ces groupes hydroxyle réduit leur pouvoir inhibiteur. La capacité de ces petites molécules à inhiber la formation des fibrilles de l'amyloïde Aβ semble être liée à leur capacité à subir une auto-oxydation en solution, résultant en un produit d'auto-oxydation tel que l'inhibiteur actif d'Aβ. Des études de la vitesse de sédimentation et par microscope électronique démontrent que l'apomorphine et des analogues de celle-ci facilitent l'oligomérisation d'Aβ en ensembles solubles courts non fibrillaires, mais inhibent la fibrillisation d'Aβ.
PCT/US2002/040660 2001-12-20 2002-12-20 Inhibiteurs apomorphines de la formation des fibrilles de l'amyloide-$g(b) a$g(b) et leur utilisation dans les maladies derivees de l'amyloidose Ceased WO2003053356A2 (fr)

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AU2002357911A AU2002357911A1 (en) 2001-12-20 2002-12-20 APOMORPHINE INHIBITORS OF AMYLOID-Beta (ABeta) FIBRIL FORMATION AND THEIR USE IN AMYLOIDOSIS BASED DISEASE
CA002479263A CA2479263A1 (fr) 2001-12-20 2002-12-20 Inhibiteurs apomorphines de la formation des fibrilles de l'amyloide-.beta. (a.beta.) et leur utilisation dans les maladies derivees de l'amyloidose

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WO2007105823A1 (fr) * 2006-03-16 2007-09-20 Kyushu University, National University Corporation Agent prophylactique/thérapeutique contre la maladie d'alzheimer
WO2010046710A1 (fr) * 2008-10-24 2010-04-29 University Of Sheffield Agents thérapeutiques contre des troubles neurologiques

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US5472954A (en) * 1992-07-14 1995-12-05 Cyclops H.F. Cyclodextrin complexation
US5744476A (en) * 1994-06-27 1998-04-28 Interneuron Pharmaceuticals, Inc. Dopamine D1 agonists for the treatment of dementia

Cited By (2)

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WO2007105823A1 (fr) * 2006-03-16 2007-09-20 Kyushu University, National University Corporation Agent prophylactique/thérapeutique contre la maladie d'alzheimer
WO2010046710A1 (fr) * 2008-10-24 2010-04-29 University Of Sheffield Agents thérapeutiques contre des troubles neurologiques

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