INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED IN ELECTRONIC FORM

Incorporated herein by reference in its entirety is the Sequence Listing submitted via EFS-Web as a text file named 6517US02_SequenceListing.txt, created Jun. 8, 2020 and having a size of 1,280 bytes.

FIELD OF THE INVENTION

The present invention relates to the fields of angiopathy, most notably including cerebral amyloid angiopathy and neurodegenerative disorders, associated with pathogenic fibril formation. More specifically the invention provides compositions and methods useful for the treatment and management of diseases associated with aberrant fibril formation, particularly hereditary cystatin C amyloid angiopathy (HCCAA) and Alzheimer's disease.

BACKGROUND OF THE INVENTION

Several publications and patent documents are cited through the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

Hereditary cystatin C amyloid angiopathy (HCCAA) is a dominantly inherited disease caused by a leucine 68 to glutamine variant of human cystatin C (hCC; L68Q-hCC).1HCCAA is classified as a cerebral amyloid angiopathy (CAA), a group of diseases in which amyloid deposits form on the walls of blood vessels in the central nervous system (CNS). Although HCCAA is rightly classified as a CAA disorder due to its strong cerebral presentation, hCC deposition is systemic and is also found in other internal organs. Most carriers of the mutation suffer micro-infarcts and brain hemorrhages in their twenties leading to paralysis, dementia and death in young adults, with an average life expectancy of 30 years.2-6Post-mortem studies in humans show that hCC is deposited in all brain areas, grey and white matter alike, most prominently in arteries and arterioles.

Human cystatin C, a cysteine protease inhibitor that belongs to the cystatin super-family, is a secretory type 2 cystatin, expressed in all nucleated human cells and found in all tissues and body fluids and at particularly high concentrations in cerebrospinal fluid.2, 7-9hCC inhibits cysteine proteases like papain and legumain by its interaction through multiple binding motifs resulting from the characteristic hCC fold.9-11Its normal conformation is composed of a polypeptide that folds into a five-stranded β-sheet, which partially wraps around a central α-helix. The N-terminal segment and two hair-pin loops build the edge of the protein, which binds into the active site of cysteine proteases and blocks their proteolytic activity.12-14The mutation of leucine 68 to glutamic acid destabilizes the packing between the beta sheets and the alpha helix, allowing the molecule to open. Two such open hCC molecules can interact with each other, with the helix of each molecule interacting with the beta sheet of the other; the resulting dimer is said to be the product of domain swapping.15-17Additionally, through a process called propagated domain swapping, long chains of molecules can be built, in which the free domain of each molecule interacts with a new hCC monomer.18The aggregation of proteins leads to the formation of highly ordered pathogenic fibrillar aggregates, called amyloid fibrils,19, 20which are implicated not only in HCCAA but also in a wide range of neurodegenerative diseases such as Alzheimer's, Parkinson's, Creutzfeldt-Jacob's, Huntington's disease and other CAAs.20

The degree of amyloid maturation observed in cystatin C deposits has been shown to vary between tissues (i.e, less prominent maturation in skin than in brain).21Although deposits in the skin are not comprised of amyloid fibers, quantitative studies on hCC deposition within the skin of mutant carriers showed that symptomatic carriers had significantly higher levels of hCC immunoreactivity in their skin than asymptomatic carriers. The fact that the quantity of hCC deposition in skin was associated with the progression of the disease in the CNS shows that skin biopsies could be used to assess disease progression and could, therefore, be of use in the evaluation of therapeutic interventions.22

Protein oligomers of different pathogenic amyloidogenic proteins precede the fibril formation stage in HCCAA and other diseases, although for HCCAA it is unclear if such oligomers lead directly to pathogenic fibrils, or if assembly of fibrils occurs most rapidly from monomers.23Drugs reducing aggregation of amyloid-producing proteins have the potential to reduce the formation of toxic oligomers known to occur in several types of amyloidosis.24, 25Previous investigations have suggested that preventing domain swapping of hCC might be used for treatment of HCCAA;24Nilsson and colleagues developed variants of WT hCC and L6Q-hCC with intra-chain stabilizing disulfide bonds preventing domain swapping that could form either dimers or amyloid fibrils.26These results suggest that the knowledge of the molecular mechanism causing the transition of physiologically normal and soluble proteins to toxic oligomers and insoluble fibrils is essential for the development of treatment strategies.

Östner et al. have previously attempted to prevent polymerization of hCC monomers, or disrupt or remove multimeric species, through various approaches.24As mentioned, modified, stabilized hCC monomers have been used to demonstrate that preventing domain swapping prevents aggregations. Antibodies can be raised specifically against the domain swapped, dimeric form of hCC; those antibodies were able to specifically remove dimers of hCC, and not monomers, from patient plasma using size exclusion chromatography.27A high-throughput screen of compounds has been pursued using the US Drug Collection (comprised of 1040 FDA approved compounds; (found on the world wide web at .msdiscovery.com/usdrug.html) in an effort to find molecules that prevent dimerization.24Although promising, this approach required large amounts of purified hCC protein produced in bacteria, and the compounds that were identified as inhibiting dimer formation were for the most part used at concentrations too high to be considered therapeutic in an organism.

Clearly, there is a need for improved methods and compositions for treating HCCAA.

SUMMARY OF THE INVENTION

In accordance with the present invention, a method for treating amyloid deposit disease comprising delivering an effective amount of at least one antioxidant to a patient, said antioxidant disrupting said amyloid deposit, thereby alleviating disease symptoms. Amyloid deposit diseases, include for example, hereditary cystatin C amyloid angiopathy (HCCAA), Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jacob's disease, Huntington disease and other forms of cerebral amyloid angiopathies such as the Dutch form of the disease. In certain embodiments, the amyloid disease is HCCAA caused by mutated cystatin C. In other embodiments, the mutated cystatin C comprises a L68Q cystatin C. Preferred antioxidants for use in the method above, include, without limitation, glutathione, N-acetyl cysteine or a derivative thereof. In certain embodiments, the derivatives are selected from NAC-amide, NAC-ethyl ester and zinc mercaptide N-acetyl cysteine carboxylate salt.

In another aspect, a method for treatment of hereditary cystatin C amyloid angiopathy (HCCAA) in a human subject in need thereof is provided. An exemplary method comprises administration of an effective amount of N-acetyl cysteine or functional derivative thereof in a pharmaceutically acceptable carrier to the subject, the administration being effective to reduce amyloid-cystatin protein aggregates, thereby alleviating symptoms of HCCAA. In certain embodiments, the NAC derivative is selected from NAC-amide, NAC-ethyl ester and zinc mercaptide N-acetyl cysteine carboxylate salt. The method can optionally entail performing a skin biopsy on said subject following treatment to assess reduction in amyloid-cystatin protein aggregates in skin or measuring cystatin C monomer, dimer or oligomer in serum or plasma or the amount of monomer excreted in the urine.

In another aspect, the method can entail administration of additional agents which alleviate amyloid deposit symptoms. These include without limitation, one or more ionophores, one or more anti-inflammatory agents and one or more proteases. In other embodiments, siRNA directed to cystatin C coding sequences are administered to selectively block the mutated allele.

In another aspect of the invention, a method for treatment of a neurodegenerative disorder associated with pathogenic fibrillation protein aggregates in a human subject in need thereof is disclosed. An exemplary method comprising administration of an effective amount of N-acetyl cysteine or functional derivative thereof in a pharmaceutically acceptable carrier to the subject wherein the administration is effective to reduce said protein fibril aggregates, thereby alleviating symptoms of the neurodegenerative disorder. In certain embodiments, the disorder is selected from Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jacob's disease, Huntington disease and other forms of cerebral amyloid angiopathy (CAA) such as the Dutch form.

In certain embodiments, the methods above comprise monitoring said patient for amyloid deposit levels.

In yet another aspect of the invention, a method for identifying therapeutic agents which alter amyloid-cystatin protein aggregate formation is provided. An exemplary method comprising providing cells expressing a nucleic acid encoding a mutant hCC protein, said mutant causing formation of amyloid-cystatin protein aggregates; and providing cells which express a hCC protein which lacks the hCC mutation. Both populations of cells are contacted with a test agent and assessed to determine whether the agent alters amyloid-cystatin protein aggregate formation of cells expressing the mutant relative to those expressing the wild type protein, thereby identifying agents which alter amyloid-cystatin protein aggregation. Agents so identified should have efficacy for the treatment of HCCAA or other disorders associated aberrant fibril formation.

Also provided is a pharmaceutical composition comprising an effective amount of an agent which acts as an antioxidant and, or a reducing agent for the treatment of amyloid deposit disease in a pharmaceutically acceptable carrier. Diseases to be treated with the composition, include for example, HCCAA, Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jacob's disease, Huntington disease and other CAAs. In one embodiment, the agent is glutathione, N-acetyl cysteine or a derivative thereof. In a preferred embodiment, the agent is a derivative and is selected from NAC-amide, NAC-ethyl ester and zinc mercaptide N-acetyl cysteine carboxylate salt. The inventive compositions of the invention can also comprise one or more of an ionophore, an anti-inflammatory agent or a protease.

