COMPOUND AND COMPOSITIONS FOR MULTITARGET TREATMENT OF TAU PROTEIN-RELATED DISORDERS

Abstract

Tau protein misfolding, aggregation and accumulation in the nervous system is an abnormal event which is responsible for widespread synaptic loss and neurodegeneration; it can occur spontaneously or can be triggered by misfolding of amyloid β protein. The present invention concerns the use of hexapeptide Aβ1-6.sub.A2V(D) in the treatment of a family of neurodegenerative diseases related to tau pathology. In the present studies, Aβ1-6.sub.A2V(D) is shown capable of interfering with protein tau at different levels. This property, in association with additional neuroprotective activities of Aβ1-6.sub.A2V(D), offers as a whole an excellent therapeutic asset against tau protein-related diseases. Moreover, the intranasal administration of Aβ1-6.sub.A2V(D) obtains remarkable levels of permeation of this peptide through the blood-brain barrier, with widespread distribution thereof among the most important brain areas involved in tau pathology.

Claims

1-13. (canceled)

14. Method of treating a tau protein-related disease, comprising intranasally administering a therapeutically effective amount of Peptide Aβ1-6.sub.A2V(D) to a patient in need thereof.

15. Method according to claim 14, being effective in inhibiting each of the following: binding of Aβ1-42 oligomers to tau protein, polymerization of full-length tau protein; β-secretase activity.

16. Method according to claim 14, being effective in inhibiting oxygen radicals production induced by β-amyloid peptides and synaptic dysfunction.

17. Method according to claim 14, wherein said tau protein-related disease is selected from the group consisting of: Aging-related tau astrogliopathy, Alzheimer's Disease, Argyrophilic grain disease, Chronic traumatic encephalopathy, Corticobasal degeneration, Diffuse neurofilament tangles with calcification, Frontotemporal dementia, Globular glial tauopathies, Parkinsonism linked to chromosome 17, Pick disease, Postencephalitic parkinsonism, Primary age-related tauopathy and Progressive supranuclear palsy.

18. Method according to claim 14, wherein said peptide is administered as a unit dose comprised between 1 and 7,000 mg.

19. Method according to claim 14, wherein said peptide is administered at daily dose ranging from 0.1 to 100 mg/kg.

20. Method according to claim 14, wherein said peptide is formulated as a solution or spray or aerosol, for intranasal administration.

21. Method according to claim 14, wherein said intranasally administered peptide accumulates in brain areas including cortex, hippocampus, caudate putamen, cerebellum.

22. Method according to claim 14, wherein said treatment does not require daily administrations, being effective for up 48 hours after administration.

23. Pharmaceutical composition comprising the peptide Aβ1-6.sub.A2V(D) in form suitable for intranasal administration.

24. Pharmaceutical composition according to claim 23, in the form of intranasal instillable solution, intranasal spray, intranasal powder, intranasal aerosol.

25. Pharmaceutical composition according to claim 23, wherein said peptide Aβ1-6.sub.A2V(D) is present at a concentration ranging from 10 to 100 mg/ml.

26. Pharmaceutical composition according to claim 23, in form of a unit dose containing from 1 to 7,000 mg of peptide Aβ1-6.sub.A2V(D).

Description

DESCRIPTION OF THE FIGURES

[0010] FIG. 1. Time-course of Aβ1-42 aggregation. Aβ1-6A2V(D) binds selectively to Aβ oligomers as determined by thioflavin T dye (ThT) fluorimetric assay and hinders Aβ1-42 aggregation. Aβ1-42 oligomers were prepared according to Beeg et al.sup.11.

[0011] FIG. 2. Human β-Secretase Inhibition by Aβ1-6.sub.A2V(D).

[0012] FIG. 3. Mouse brain distribution of Aβ1-6.sub.A2V(D) peptide after intranasal administration. Mice were sacrificed 4 (A), 24 (B) and 48 (C) hours after the last administration.

[0013] FIG. 4. Mouse brain distribution of Aβ1-6.sub.A2V (D) peptide 2 hours after the last intranasal administration: A) Control and B) Treated Mice.

[0014] FIG. 5. Effect of Aβ1-6.sub.A2V(D) on Aβ oligomer formation in the brain: biochemical assessment. APPSwe/PS1DE9 were treated with Aβ1-6.sub.A2V(D) at 50 mg/kg for five months. Control group was treated intranasally with PBS. A: Levels of Aβ aggregates in soluble and insoluble brain fractions after 2.5 months of treatment. B: Levels of Aβ aggregates in soluble and insoluble brain fractions after five months of treatment. * p<0.01; *** p<0.001.

[0015] FIG. 6. Effect of Aβ1-6.sub.A2V(D) on Aβ deposits in mouse brain: neuropathological assessment. A: APPSwe/PS1DE9 were treated with Aβ1-6.sub.A2V(D) daily for five months at 50 mg/kg. Control group (CTRL) was treated intranasally with PBS. Amyloid burden measured by plaque count. ** p<0.01; *** p<0,001. B: Immunohistochemistry with the 4G8 anti-Aβ antibody.

[0016] FIG. 7. Effect of Aβ1-6.sub.A2V(D) on synaptopathy. Subcellular fractionation of hippocampal homogenates from mice treated with Aβ1-6.sub.A2V(D) (right columns) or PBS (left columns). Western blot analysis with antibodies against synaptic receptors (NMDA-2A, GluN2B, GluA1 and GluA2) and scaffold protein (PSD95). The treatment with Aβ1-6.sub.A2V(D) preserved synaptic integrity.

[0017] FIG. 8. Binding of different Aβ1-42 assemblies to Immobilized Tau

[0018] FIG. 9. Aβ1-6.sub.A2V(D) inhibits the binding Aβ1-42 oligomers to Tau. The IC50 obtained in two independent experiments were 360.2 and 387.6 μM.

[0019] FIG. 10. Tau aggregation was triggered by heparin in the tau/EPA ratio of 4:1 (w/w) in the presence of 5 mM dithiothreitol (DTT).

[0020] FIG. 11. Time course of WT full-length tau aggregation in the absence and presence of Aβ1-6.sub.A2V(D) as determined by atomic force microscopy (AFM). Tau alone forms fibres whose numbers and size increase with time of incubation. Following the co-incubation with Aβ1-6.sub.A2V(D), the number of fibres is significantly reduced, and an increase of granular structure was evident. Color scale: 0-150 mV.

[0021] FIG. 12. Time course of full-length tau P301L aggregation in the absence and presence of Aβ1-6.sub.A2V(D) as determined by AFM. Tau P301L alone shows an increased fibrils formation with time. In the presence of Aβ1-6A2V(D) the number of fibres is significantly reduced, and an increase of granular structure was evident. Color scale: 0-150 mV.

[0022] FIG. 13. The DEPMO-OH-spin trap assay for the detection of ROS formation during the Cu(II)- or Fe(III)-Aβ-catalyzed processes. Aβ1-40.sub.WT spontaneously produces OH radicals in PBS buffer containing traces of Cu (II) and Fe (III). When co-incubated with Aβ1-6A2V(D) the production of OH radicals is significantly reduced.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The peptide Aβ1-6.sub.A2V(D) used in the present invention is known as such.sup.2, 4: it is the all-D-isomer synthetic peptide corresponding to the first six amino acids of the N-terminal sequence of the A2V-mutated Aβ; its amino acid sequence, DVEFRH, and its method of synthesis are disclosed by EP 2 220 251; all these publications are incorporated herein by reference.

[0024] The object of the present invention is the hexapeptide Aβ1-6.sub.A2V(D) for use in a multi-targeted method of treating diseases related to tau protein activity. A further object is the use of hexapeptide Aβ1-6.sub.A2V(D) in the manufacturing of a medicament for a multi-targeted method of treating diseases related to tau protein activity. A further object of the invention is a method of multi-target treating diseases related to tau protein activity, comprising administering the hexapeptide Aβ1-6.sub.A2V(D) to a patient in need thereof.

[0025] The terms “multi-targeted treatment” or “multi-target treatment” mean herein the effectiveness of Aβ1-6.sub.A2V(D) to interfere simultaneously at different levels with the process of tau protein misfolding and accumulation; such targets, i.e. effects of Aβ1-6.sub.A2V(D), include: inhibiting the binding Aβ1-42 oligomers to tau protein, hindering polymerization of full-length tau protein and inhibiting β-secretase activity; these effects are advantageously associated with other neuroprotective effects of Aβ1-6.sub.A2V(D), also found herein for the first time, namely: inhibiting oxygen radicals production by β-amyloid peptides and inhibiting synaptic dysfunction. The combination of these effects in a single drug makes available a potent therapeutic asset for the treatment of diseases related to tau pathology.

[0026] There is no limitation to the specific diseases related to tau protein which can be an object of the present treatment. These are typical of neurodegenerative type; among them, the following can be mentioned: Ageing-related tau astrogliopathy, Alzheimer's Disease, Argyrophilic grain disease, Chronic traumatic encephalopathy, Corticobasal degeneration, Diffuse neurofilament tangles with calcification, Frontotemporal dementia, Globular glial tauopathies, Parkinsonism linked to chromosome 17, Pick disease, Postencephalitic parkinsonism, Primary age-related tauopathy, Progressive supranuclear palsy. The present invention enables to treat with the same drug, Aβ1-6.sub.A2V(D), the group of tau protein-related diseases which either had no prior treatment or were addressed separately, based on their specific symptoms to be cured.

[0027] The term “treatment” used herein includes any way of administering the hexapeptide Aβ1-6.sub.A2V(D) to a patient in need thereof to obtain a therapeutic effect on a tau protein-related disease. The disease needs not necessarily to be ongoing at the time of administration of Aβ1-6.sub.A2V(D), i.e. the patient to be treated may either be affected or be at risk of being affected by one or more of said diseases, obtaining respectively a curative or a preventive effect on such diseases.

[0028] To practice the present treatment, the hexapeptide Aβ1-6.sub.A2V(D) may be administered by any known administration routes, e.g. orally, personally, buccally, intravenously, intrathecally, intramuscularly, topically, transdermally, inhalatorily, intranasally, intraocularly, etc. A particularly effective administration route, representing a preferred embodiment of the present invention, is the intranasal one: in fact, as reported in the experimental sections, the intranasal administration of hexapeptide Aβ1-6.sub.A2V(D) had obtained very high levels of brain penetration of this peptide: this represents a key unexpected advantage, since this peptide does not permeate through the blood-brain-barrier when administered outside the brain; this effect is particularly important considering that tau protein-related diseases arise mainly within the central nervous system; further the brain penetration is herein obtained without need of using special carriers designed to enable the blood-brain-barrier passing: avoiding the use of these carriers results in lower production costs, positively impacting on the final drug product, also offering a better therapeutic profile by avoiding any unnecessary accumulation of carrier moieties in the brain. All pharmaceutical forms allowing intranasal delivery are usable in this embodiment: reference can be made to, e.g. nasal drops, nasal ointments, nasal inserts, nasal sprays, aerosols, inhalable powders, etc.

[0029] In line with the above, the term “pharmaceutical composition in form suitable for intranasal administration”, refers herein and is limited to a commercial pharmaceutical product (i.e. packaged medicament) which is both formulated and explicitly presented to the user for the administration via the intranasal route: the formulation involves the choice of active and non-active ingredients being qualitativelty and quantitatively chosen to be compatible with contact with the intranasal mucosa, while the explicit presentation to the user is evident in the product as such by means of directions to administer the composition intranasally.

[0030] The present uses and treatments may be performed within a wide range of dosages of Aβ1-6.sub.A2V(D), correlated to the disease type and stage and patient's age and conditions. Dosages are conveniently chosen by the skilled clinician taking the about factors into account; non-limitative daily dosages ranging from 0.1 and 100 mg/Kg. Advantageously when performed intranasally, the present treatment with Aβ1-6.sub.A2V(D) does not require contiguous daily administrations: in the present experiments, effective concentrations of this peptide were found in the brain even 48 hours after administration.

[0031] Pharmaceutical dosage units allowing the administration of said dosages, via one or more administrations during the day, can be produced by standard pharmaceutical techniques; reference, non-limitative amounts of hexapeptide Aβ1-6.sub.A2V(D) in said dose units ranges between 1 and 7,000 mg. Depending on the chosen administration route, the hexapeptide Aβ1-6.sub.A2V(D) can be formulated in all possible pharmaceutical forms, for examples tablets, pills, capsules, microcapsules, dragees, lozenges, powders, granulates, microgranules, pellets, solutions, suspensions, emulsions, infusions, drops, aerosols, liposomes, creams, ointments, gels, bioadhesive films, bioadhesive patches, enemas, suppositories, etc.

[0032] The invention is now disclosed using the following non-limitative examples.

EXPERIMENTAL SECTION

[0033] The present experiments were aimed at providing new insights into the biochemical effects of Aβ1-6.sub.A2V(D), looking for unknown biochemical effects and exploring ways of enhancing the bioavailability of Aβ1-6.sub.A2V(D) in the body districts affected by tau protein-related diseases, with particular reference to the central nervous system. The following results were found.

A. Aβ1-6.SUB.A2V.(D) Selectively Binds Aβ Oligomers

[0034] We have clarified that Aβ1-6.sub.A2V(D) selectively binds to oligomers, as reported in FIG. 1. This feature underlines the presence of a peculiar and selective mechanism of action that hinders the neurotoxicity of Aβ oligomers.

B. Human β-Secretase (BACE1) Inhibition.

[0035] The major component of the amyloid plaques is aggregated Aβ which derived from sequential proteolytic cleavage of the amyloid precursor protein (APP), a transmembrane protein, by the action of β- and γ-secretases. The β-secretase cleavage is the first step in the generation of Aβ peptides, and the β-secretase 1 or β-amyloid-converting enzyme 1 is the major β-secretase; misfolding of the generated Aβ peptides leads to tau protein aggregation accompanied by synaptic loss and neurodegeneration.

[0036] FIG. 2 shows that Aβ1-6.sub.A2V(D) inhibits in a dose-dependent manner the activity of the human recombinant β-secretase with an IC.sub.50 value of 5.2±0.2 μM.

C. In Vivo Effectiveness of Intranasally Administered Aβ1-6.SUB.A2V.(D)

[0037] We studied if intranasal delivery of Aβ1-6.sub.A2V(D), not conjugated with any carrier, could provide therapeutically effective levels of this peptide in the brain. Aβ1-6.sub.A2V(D) was dissolved in saline at the concentration of 125 mg/ml, and each mouse received 12 μl of this solution.

[0038] To this end, preliminarily wild-type mice were treated once a week for 4 weeks at a dose of 16.7 mg/kg. Animals were sacrificed 4, 24, 48 hours from the last intranasal delivery. We observed a consistent diffusion of the peptide in brain tissue involving areas critically implicated in amyloid deposition—such as the hippocampus—as revealed by MALDI-TOF imaging mass spectrometry (FIG. 3). High levels of the peptide are present in the main brain areas (cerebral cortex, hippocampus, caudate putamen and cerebellum) after intranasal administration and as shown in the same figure, the peptide can be still observed in the cerebral cortex 48 hours after the last treatment.

[0039] This observation prompted us to carry out a more comprehensive treatment schedule comprising MALDI-TOF imaging experiments for the determination of Aβ1-6.sub.A2V(D) levels in the brain following intranasal administration for 5 months at 50 mg/kg every 48 hours. Following this approach, we treated the same transgenic mice used in the previous in vivo studies and obtained consistent anti-amyloidogenic effects even in the long-term treatment (5 months). In particular, the intranasal treatment with Aβ1-6.sub.A2V(D)—without use of any blood-brain-barrier passing carrier, resulted in: [0040] consistent diffusion of the peptide in brain tissue involving areas critically implicated in amyloid deposition occurring in AD—such as the hippocampus—as revealed by MALDI-TOF imaging mass spectrometry (FIG. 4); [0041] inhibition of oligomer generation and amyloid accumulation (FIGS. 5 and 6); [0042] prevention of synaptic dysfunction (FIG. 7) even in the long-term treatment schedule.

[0043] In conclusion, the intranasal treatment with Aβ1-6.sub.A2V(D)—without using any brain-targeting carriers—resulted in: [0044] inhibition of oligomer generation and amyloid accumulation; [0045] prevention of synaptic dysfunction even in the long-term treatment schedule.

[0046] Most importantly, very recent studies in our laboratories provided evidence in favour of the ability of the Aβ1-6.sub.A2V(D) peptide of hindering the tau-related pathology in AD by two main molecular mechanisms:

[0047] 1. Inhibition of the binding of Aβ oligomers to full-length wt tau

[0048] 2. Inhibition of the aggregation of tau protein.

D. Aβ1-6.SUB.A2V.(D) Inhibits Aβ1-42 Oligomers Binding to Tau Protein

[0049] The ability of Aβ oligomers to bind tau monomers may be a source of toxicity, e.g. by decreasing the population of tau needed to regulate microtubule dynamics. We used Surface Plasmon Resonance (SPR) to investigate whether or not Aβ1-6.sub.A2V(D) can inhibit the binding of Aβ1-42 oligomers to tau (2N4R-Tau) monomers. First, we found that tau binds Aβ1-42 oligomers but not Aβ1-42 monomers or fibrils.

[0050] FIG. 8 shows the binding behaviour of different assemblies formed during aggregation of 100 μM Aβ1-42 in PBS to tau immobilized on the SPR chip surface. The negligible binding signal was observed injecting the solution at t=0 (monomers) or incubated for 24 h (fibrils). The significant binding was found injecting the solution preincubated for one hour. We previously demonstrated that this solution is enriched with small, toxic oligomers.sup.12, 13.

[0051] Then we demonstrated that Aβ1-6.sub.A2V(D) inhibits Aβ1-42 oligomers binding to tau. For this, oligomers were diluted into PBST containing various concentrations of Aβ1-6.sub.A2V(D) and equilibrated for one hour at room temperature, before injecting into the SPR apparatus. The oligomer-dependent SPR binding signal was inhibited by Aβ1-6.sub.A2V(D) in a concentration-dependent manner (FIG. 9).

E. Aβ1-6.SUB.A2V.(D) Hinders the Polymerization of Full-Length Tau

[0052] This peptide, when incubated with wt full-length tau or P301L tau, significantly reduces the capacity of these proteins to polymerize as deduced by the ThT test (FIG. 10). This was also confirmed by atomic force microscopy analysis, as reported in FIGS. 11 and 12.

F. Aβ1-6.SUB.A2V.(D) Hinders the Reactive Oxygen Generation by Aβ-Catalyzed Redox Processes.

[0053] This peptide, when incubated with Aβ1-40WT, inhibits the spontaneous formation of OH radicals in a buffer containing traces of Cu (II) and Fe (III)

[0054] These data confirm the “multitargeting nature” of the present Aβ1-6.sub.A2V(D) based strategy and open the way to the therapeutic use of this approach for the treatment and/or prevention of neurodegenerative tauopathies.

REFERENCES

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