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WO2019154515A1 - Procédés et compositions pour le traitement de maladies amyloïdes - Google Patents

Procédés et compositions pour le traitement de maladies amyloïdes Download PDF

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Publication number
WO2019154515A1
WO2019154515A1 PCT/EP2018/053362 EP2018053362W WO2019154515A1 WO 2019154515 A1 WO2019154515 A1 WO 2019154515A1 EP 2018053362 W EP2018053362 W EP 2018053362W WO 2019154515 A1 WO2019154515 A1 WO 2019154515A1
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optionally
receptor agonist
disease
days
weeks
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Theodoros KYRIAKIDES
Elena PANAGIOTOU
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Cyprus Foundation For Muscular Dystrophy Research
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Cyprus Foundation For Muscular Dystrophy Research
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Priority to PCT/EP2018/053362 priority Critical patent/WO2019154515A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • 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

Definitions

  • the present invention relates to the field of amyloid diseases and other diseases that involve an accumulation of abnormally folded proteins, and the treatment and prevention thereof.
  • Amyloid diseases cover a range of diseases, all involving the build-up of abnormally folded proteins that fall into the category of amyloid proteins.
  • Amyloid deposits contain extremely insoluble protein fibrils that share similar morphological features (40- to 200-A fibrils) but comprise many different proteins with no obvious sequence similarity.
  • Amyloid diseases include neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease as well as including systemic amyloid diseases, for example transthyretin related amyloidosis (ATTR) and cardiac amyloidosis.
  • AD Alzheimer’s disease
  • Parkinson Parkinson’s disease
  • systemic amyloid diseases for example transthyretin related amyloidosis (ATTR) and cardiac amyloidosis.
  • abnormal protein aggregation characterizes many, if not all, neurodegenerative disorders, not just AD and Parkinson’s disease, but also Creutzfeldt-Jakob disease, motor neuron diseases, the large group of polyglutamine disorders, including Huntington’s disease, as well as diseases of peripheral tissue like familial amyloid polyneuropathy (FAP).
  • FAP familial amyloid polyneuropathy
  • Amyloid diseases are associated with an inflammatory element, as discussed below.
  • amyloid diseases Two examples of quite different but medically important amyloid diseases are Hereditary ATTR V30M amyloidosis and Alzheimer’s disease.
  • ATTR V30M amyloidosis is a life-threatening progressive sensory-motor and autonomic peripheral neuropathy.
  • ATTR V30M amyloidosis is caused by extracellular accumulation of misfolded transthyretin (TTR), subsequently creating insoluble aggregates of amyloid fibrils (Saraiva et al., 1984).
  • TTR misfolded transthyretin
  • ATTR V30M is due to a substitution of a valine with methionine in position 30 (Val30Met) of the TTR protein (Ando et al., 2005).
  • C1q Polymorphisms in C1q have been found to correlate with age of onset in a Cypriot cohort of ATTR V30M patients suggesting that complement C1q may be a modifier gene (Dardiotis et al., 2009). C1q has also been shown to modulate beta-amyloid induced complement activation and neuronal loss in Alzheimer’s disease (Fonseca et al., 201 1 ) as well as modulating phagocytosis of soluble pre-amyloid aggregates (Pisalyaput and Tenner, 2008).
  • a dual role for complement has been proposed, a protective effect from early components of complement (C1q opsonizes foreign material and phagocytes) and a detrimental effect from late components such as C3a and C5a exacerbating neuroinflammation (Fonseca et al., 2011 ).
  • Late component C5a is produced following the activation of any of the three pathways of the complement cascade system.
  • the C5 complement system factor is cleaved by C5 convertases producing the C5a and C5b molecules.
  • C5a acts as an anaphylactic molecule by attracting C5a receptor bearing cells including macrophages and neutrophils leading to a pro-inflammatory response (Mathern and Heeger,2015).
  • AD Alzheimer’s Disease
  • AD is a progressive neurodegenerative/neuroinflammatory disease of the brain causing either sporadic or familial dementia. AD is by far the commonest dementia; the sixth leading cause of death in the general population and the fifth leading cause of death in persons over the age of 65 [1]. In 2016, worldwide, more than 44 million people were diagnosed with Alzheimer’s or another related dementia [1].
  • AD amyloid-b plaques caused by the proteolytic cleavage of the amyloid precursor protein (APP).
  • APP amyloid precursor protein
  • the pathophysiology of AD has long been explained using the amyloid cascade hypothesis.
  • the hypothesis essentially states that genetic mutations in certain genes (APP and PSEN1/2) or age related mis-metabolism of APP results either in excess production or reduced clearance of Ab, thus causing the deposition of amyloid-b oligomers and plaques in the brain which then imposes“aggregation stress” [2]
  • the complement cascade has been shown to be activated from the very early stages of Alzheimer disease [7].
  • the complement component C5a is thought to bind C5a receptors on phagocytic cells such as microglia and thus augment phagocytosis of Ab amyloid and prefibrillar Ab aggregates [8].
  • the inventors found that oral administration of a C5a receptor activator ameliorated amyloid aggregates in the stomachs of mice with ATTR amyloidosis. Whilst this was an important finding in itself, it was possible that the effect was restricted to ATTR. However, the inventors proceed to surprisingly find that an oral dose of the activator also results in a significant decrease in the amyloid plaques associated with Alzheimer’s Disease in brain tissue (and concomitant improvement in behaviour), indicating that not only is the effect of the activator not restricted to stomach tissue since it also works in the very different brain tissue, but also that the effects of an oral dose of the activator are not restricted to the stomach or gastro-intestinal tract, but are effective at distal sites.
  • the invention provides a C5a receptor agonist for use in the treatment and/or prevention of a disease in a subject wherein the disease is characterised by abnormal protein aggregation.
  • the C5a receptor is a well characterised protein that is also known as C5AR1 or CD88 and is found throughout a wide range of species. It is considered that the present invention is suitable for use in any species that has a C5a receptor, for example a receptor that corresponds functionally and/or in sequence to the human and/or mouse C5a receptor.
  • the subject is a subject that has a C5a receptor, optionally the subject is a mammal, for example a human, a dog, a cat, a horse, a cow, a sheep, a pig, or a fish. In a preferred embodiment the subject is a human.
  • C5a receptor is intended to include the receptors with at least all of the following accession numbers:
  • the term agonist is used to refer to a substance that activates the particular entity that it agonises.
  • the C5a receptor agonist includes substances or agents that are known to activate the C5a receptor.
  • agonises the C5a receptor or activates the C5a receptor we include the meaning of activating any of the functions of the C5a receptor, and by activating any of the functions of the C5a receptor we include the meaning of causing the C5a receptor to begin a particular function, or to increase the rate of a particular function.
  • the activation of the C5a receptor can result in the activation of one or more functions, depending on which cell the C5a receptor is located.
  • the C5a receptor agonist results in activation of the C5a receptor that is located on the surface of various phagocytes for example macrophages.
  • a preferred effect is that the C5a receptor agonist results in G-protein coupled signalling of the protein kinase pathways through mitogen activation with various downstream effects.
  • the skilled person will be aware of standard tests that can be performed that will allow the skilled person to determine whether a particular C5a receptor agonist is suitable for use with this embodiment, i.e.
  • the skilled person can contact the C5a receptor agonist to a population of phagocytes and determine whether G-protein coupled signalling has occurred.
  • C5a receptor agonist is suitable for use in the present invention
  • methods that can be used by the skilled person to determine a particular C5a receptor agonist is suitable for use in the present invention can involve the use of animal experiments using antibody assays to document a quantitative reduction of amyloid as a result of phagocytosis.
  • amyloid load in the brain can be monitored using PET scanning using the Pittsburgh compound B.
  • peripheral nerve amyloidosis magnetic resonance neurography could be used to monitor amyloid load. A decrease of any amount in amyloid load as a result of the C5a receptor agonist would indicate that that agonist is suitable for use in the present invention.
  • one embodiment of the invention provides a C5a receptor agonist for use in the treatment and/or prevention of a disease in a subject wherein the disease is characterised by abnormal protein aggregation and wherein the agonist results in G- protein coupled signalling of the protein kinase pathways through mitogen activation on phagocytes.
  • one of the mechanisms by which the present invention is effective is through the recruitment of macrophages and neutrophils to the site of the protein aggregate.
  • a C5a receptor agonist for use in the treatment and/or prevention of a disease in a subject wherein the disease is characterised by abnormal protein aggregation and wherein the C5a receptor agonist results in the recruitment of macrophages and neutrophils to the site of the protein aggregate. Examples of methods to allow the skilled person to determine whether a particular C5a receptor agonist does result in the recruitment of macrophages and neutrophils to the site of the protein aggregate are set out in the Examples and are non- limiting.
  • the presence of macrophages can be detected by assaying for the presence of CD68, whilst the presence of neutrophils can be determined by assaying for the presence of ELANE, both of which are known and can be detected through immunohistochemistry, ELISA and/or Western blotting.
  • the C5a receptor agonist is found to result in the recruitment of macrophages and neutrophils to the site of the protein aggregate it is considered to be suitable for use in the present invention.
  • the C5a receptor agonist results in the recruitment of predominantly macrophages rather than neutrophils to the site of the protein aggregate.
  • a particular C5a receptor agonist, EP67 (SEQ ID NO: 4) has been shown to result in the recruitment of predominantly macrophages rather than neutrophils to the site of the protein aggregate and has been shown to be useful in the present invention. Accordingly, as one simple test, the skilled person can compare the degree of preferential recruitment of macrophages rather than neutrophils by EP67 (SEQ ID NO 4) to the degree of preferential recruitment of macrophages rather than neutrophils by a test C5a receptor agonist according to the claims. In one embodiment a C5a receptor agonist that preferentially recruits macrophages rather than neutrophils to the same degree as EP67 (SEQ ID NO: 4) is considered suitable for use in the present invention.
  • the preferential recruitment of macrophages rather than neutrophils by the activation of the C5a receptor by the C5a receptor agonist is not considered to be essential, but in some situations may be preferable.
  • neutrophils are considered to likely not be well tolerated in any tissue, for example and in particular brain tissue.
  • the C5a receptor agonist is one that results in the preferential recruitment of macrophages rather than neutrophils to the site of the protein aggregation, as considered to be the case for EP67.
  • Agonists of receptors may typically bind to the corresponding receptor.
  • the agonist binds to the C5a receptor.
  • a given agent can have agonistic properties without actually binding to the corresponding receptor.
  • a given agent may activate the receptor, for example the C5a receptor, by modification of the receptor, for example phosphorylation of the receptor.
  • Such agents are also intended to be encompassed by the present invention.
  • bind we include the meaning of any degree of association between the C5a receptor and the agonist.
  • the ability of a particular agonist to bind to the C5a receptor can be readily determined by the skilled person. For example, co-immunoprecipitation assays are commonly used to identify the binding partners of a receptor.
  • the agonist is specific for the C5a receptor, i.e. does not have a significant effect on any other receptor or other protein. Accordingly the agonist must have affinity for the C5a receptor and also efficacy in activation of the receptor, to result in the desired effects. Methods of determining the affinity and efficacy of a particular agonist to the C5a receptor are discussed in Strange 2003, for example.
  • the C5a receptor agonist may be any substance, agent or entity that results in activation of the C5a receptor, for example results in G-protein coupled signalling of the protein kinase pathways through mitogen activation; and/or recruits macrophages, or macrophages and neutrophils, to the site of the protein aggregate.
  • the C5a receptor agonist may be a protein, or it may be a nucleic acid, or a small molecule, or a lipid, or a carbohydrate.
  • the C5a receptor agonist is a polypeptide.
  • polypeptide we include the meaning of a series of any number of amino acids joined by peptide bonds.
  • polypeptide is not intended to exclude those polypeptides that may otherwise be termed proteins or peptides.
  • polypeptide is intended to include those peptides, polypeptides or proteins that are between 2 amino acids and 2000 amino acids in length, for example between 10 amino acids and 1500 amino acids in length, for example between 20 amino acids and 1000 amino acids in length, for example between 30 amino acids and 500 amino acids in length, for example between 40 amino acids and 300 amino acids in length, for example between 50 amino acids and 150 amino acids in length, for example 100 amino acids in length.
  • the polypeptide may be made by any means and may be comprise any number of modifications to any number of the amino acid residues.
  • the length of the polypeptide may be around 50 amino acids, or may be less than 50 amino acids, or may be less than 45 amino acids, or may be less than 40 amino acids, or may be less than 35 amino acids, or may be less than 30 amino acids, or may be less than 25 amino acids of the C-terminal domain of the C5a molecule, or may be less than 20 amino acids, or may be less than 15 amino acids, or may be less than 10 amino acids
  • the C5a receptor agonist may also be a peptidomimetic compound.
  • polypeptide we include not only molecules in which amino acid residues are joined by peptide (-CO-NH-) linkages but also molecules in which the peptide bond is reversed.
  • retro-inverso peptidomimetics may be made using methods known in the art, for example such as those described in MJziPre et al (1997) J. Immunol. 159, 3230-3237. This approach involves making pseudopeptides containing changes involving the backbone, and not the orientation of side chains. MJziPre et al (1997) show that, at least for MHC class II and T helper cell responses, these pseudopeptides are useful.
  • Retro-inverse peptides which contain NH-CO bonds instead of CO-NH peptide bonds, are much more resistant to proteolysis.
  • the peptide bond may be dispensed with altogether provided that an appropriate linker moiety which retains the spacing between the Cl atoms of the amino acid residues is used; it is particularly preferred if the linker moiety has substantially the same charge distribution and substantially the same planarity as a peptide bond.
  • the C5a receptor agonist polypeptide may conveniently be blocked at its N- or C-terminus so as to help reduce susceptibility to exoproteolytic digestion.
  • a C5a receptor agonist polypeptide may be a peptidomimetic compound.
  • the polypeptide may activate the C5a receptor, for example result in G-protein coupled signalling of the protein kinase pathways through mitogen activation; and/or recruit macrophages, or macrophages and neutrophils, to the site of the protein aggregate.
  • the polypeptide may be an antibody, for instance.
  • the C5a receptor agonist is a polypeptide, and is or comprises a fragment of the C5a molecule, for example is or comprises the C-terminal region of the C5a molecule or is or comprises a fragment of the C-terminal region of the C5a molecule (but is not the full length C5a molecule).
  • C-terminal region of the C5a molecule we include the meaning of the C-terminal half of the region which includes the C-terminal domain defined below by SEQ ID NO: 2.
  • the C5a molecule is a protein fragment released from cleavage of complement component C5 by protease C5- convertase into C5a and C5b fragments.
  • the sequence of the human C5a protein is as follows:
  • the C-terminal domain of human C5a protein is considered to have the following sequence:
  • DMQLGR (residues 69-74 of SEQ ID NO: 1 )
  • the human C5a polypeptide comprises 74 amino acids and is 1 1 KDa.
  • the molecule is known to contain four helices connected with three disulphide bonds between helix IV and II, III.
  • C5a can be metabolised by the serum enzyme carboxypeptidase to its 72 amino acid form referred to as C5a des-Arg.
  • Agonist activity is found strictly in the C- terminus. It is considered that the C5a receptor agonist activity of the C5a molecule residues in residues 69-74 of the full length C5a molecule, which are attached to the main helical core via the four residue loop and which assumes the elongated 1.5 turn helix required for receptor activation.
  • the agonist comprises or consists of the C-terminal domain of the C5a molecule, for example comprises or consists of a polypeptide having the sequence of SEQ ID NO: 2.
  • the agonist is not the full length C5a molecule, as will be apparent to the skilled person.
  • the C5a receptor agonist is a polypeptide that comprises or consists of SEQ ID NO: 2 or comprises or consists of a sequence with at least 70% homology or sequence identity to SEQ ID NO: 2, for example at least 75% homology or sequence identity to SEQ ID NO: 2, for example at least 80% homology or sequence identity to SEQ ID NO: 2, for example at least 85% homology or sequence identity to SEQ ID NO: 2, for example at least 90% homology or sequence identity to SEQ ID NO: 2, for example at least 95% homology or sequence identity to SEQ ID NO: 2, for example 100% homology or sequence identity to SEQ ID NO: 2.
  • the skilled person will be able to determine whether a particular agent is able to agonise the C5a receptor, for example to result in G- protein coupled signalling of the protein kinase pathways through mitogen activation; and/or recruit macrophages, or macrophages and neutrophils, to the site of the protein aggregate.
  • the C5a receptor agonist may also be a fragment of the C terminal region of the C5a molecule.
  • the C5a receptor agonist may consist of or comprise, for example, less than 50 amino acids of the C-terminal region of the C5a molecule (SEQ ID NO: 2), for example may consist of or comprise less than 45 amino acids of the C-terminal region of the C5a molecule, for example may consist of or comprise less than 40 amino acids of the C-terminal region of the C5a molecule, for example may consist of or comprise less than 35 amino acids of the C-terminal region of the C5a molecule, for example may consist of or comprise less than 30 amino acids of the C-terminal region of the C5a molecule, for example may consist of or comprise less than 25 amino acids of the C- terminal region of the C5a molecule, for example may consist of or comprise less than 20 amino acids of the C-terminal region of the C5a molecule, for example may consist of or comprise less than 15 amino acids of the C-terminal region of the C
  • the fragment of the C-terminal region at least comprises the C- terminal domain, for example at least comprises SEQ ID NO: 2.
  • the residues of the fragment of the C terminal domain of the C5a molecule may not show 100% homology or sequence identity to the sequence of SEQ ID NO: 2, but may show, for example, at least 70% homology or sequence identity to the relevant portion of SEQ ID NO: 2, for example at least 75% homology or sequence identity to the relevant portion of SEQ ID NO: 2, for example at least 80% homology or sequence identity to the relevant portion of SEQ ID NO: 2, for example at least 85% homology or sequence identity to the relevant portion of SEQ ID NO: 2, for example at least 90% homology or sequence identity to the relevant portion of SEQ ID NO: 2, for example at least 95% homology or sequence identity to the relevant portion of SEQ ID NO: 2, for example 100% homology or sequence identity to the relevant portion of SEQ ID NO: 2.
  • the skilled person will be able to determine whether such a fragment of a protein
  • the C5a receptor agonist is a polypeptide that has any one of the following formulae: a) (N-Methyl-Phe)-Lys-Pro-d-Cha-Cha-d-Arg-C02 H) (SEQ ID NO: 3); or [Full agonist] b) Tyr-Ser-Phe-Lys-Asp- Met-Pro-(MeLeu)-D-Ala-Arg (SEQ ID NO: 4) [EP67]; or c) Tyr-Ser-Phe-Lys-Asp- Met-Xaa-(Xaa2)-D-Ala-Arg (SEQ ID NO: 5)
  • Xaa is a modified proline residue or a residue substitution for proline
  • Xaa2 is leucine or N-methyl leucine
  • Xaa is a trifluoromethylated pseudoproline
  • Xaa is a pseudoproline
  • Xaa is a trifluoromethylated azetidine 2-carboxylic acid and/or homoserine;
  • Xaa is an oxetanyl-containing peptide
  • A1 is Tyr, Trp, or N-acetyl derivatives of Tyr or Trp;
  • A2 is Asp, Gly, Pro or N-methyl derivatives of Asp or Gly;
  • A3 is Ala, Cys, Leu, Met or N-methyl derivatives of Ala, Cys, Leu or Met;
  • A4 is Gin, Leu, Pro or N-methyl derivatives of Gin or Leu;
  • A5 is Pro, Leu, a-methyl Leu or N-methyl Leu
  • A6 is D-Ala, Gly, D-Pro, Aib [aminoisobutyric acid (Aib)] or N-methyl derivatives of D-Ala or Gly; and
  • A7 is Arg or N-methyl Arg; e) Tyr-Ser-Phe-l_ys-Asp-Met-Pro-Mel_-(D-Ala)-Arg (SEQ ID NO: 7)
  • MeL is N-methyl Leu
  • the C5a receptor agonist may also include any of the C5a receptor agonists described in any one or all of WO 2016145365; WO 2012/006149; and WO 9606629 the teachings of which are specifically incorporated by reference in this regard.
  • the C5a receptor agonist comprises or consists of the sequence Tyr-Ser-Phe-Lys-Asp- Met-Pro-(Mel_eu)-D-Ala-Arg (SEQ ID NO: 4) [EP67]
  • the C5a protein is an anaphylatoxin and can cause an undesired smooth muscle contraction, vasodilation, histamine release from mast cells, and enhanced vascular permeability. Accordingly, in one embodiment of the invention the C5a receptor agonist is also considered to be an anaphylatoxin. However, in a preferred embodiment the C5a receptor agonist is not an anaphylatoxin and does not act as an anaphylatoxin.
  • An example of such a C5a receptor agonist is the EP67 polypeptide (SEQ ID NO: 4).
  • SEQ ID NO: 4 An example of such a C5a receptor agonist is the EP67 polypeptide (SEQ ID NO: 4).
  • the skilled person will be aware of suitable routine tests that can be performed to identify whether a particular agonist is also an anaphylatoxin.
  • the C5a receptor agonist is capable of oral absorption.
  • the C5a receptor agonist is capable of oral absorption without any loss of function of the a C5a receptor agonist from for example contact with gastric acid or other bodily fluids.
  • the orally administered C5a receptor agonist is able to pass past the liver, enter the systemic circulation and penetrate the blood-brain barrier. Oral formulations are well known in the art.
  • the C5a receptor agonist is active in all bodily parts, for example the C5a receptor agonist is capable of agonising the C5a receptor systemically, for example is capable of agonising the C5a receptor in the organs and the brain.
  • the C5a receptor agonist is capable of oral absorption such that the effects of the agonist, i.e. the activation of the C5a receptor occurs throughout the body, i.e. systemically.
  • administration of the C5a receptor agonist results in a localised activation of the C5a receptor.
  • the C5a receptor agonist acts, or partly acts, through stimulation of the complement pathway at the sites of the protein aggregate.
  • the C5a receptor agonist is an agonist that results in an increase level of expression of C1q at the site of protein aggregation. Any level of increase in the expression level of C1q is considered to be beneficial.
  • the level of expression of C1q following administration of the C5a receptor agonist can be increased relative to the level of expression of C1q before administration of the C5a receptor agonist .
  • the level of expression of C1 q can also be assessed relative to a standard control, for example the level of C1 q from a subject that has not been administered the C5a receptor agonist . Methods to determine the level of expression of C1 q are detailed in the Examples.
  • the beneficial effects of the C5a receptor agonist are considered to reside in the ability of the C5a receptor agonist to reduce the amount of aggregated protein, or prevent the accumulation of aggregated protein in the first place.
  • the amount of aggregate may still increase but increases at a slower rate than it would otherwise do in the absence of treatment with the C5a receptor agonist , i.e. the rate of deposition of protein aggregates is reduced; or the amount of aggregate may not increase at all, i.e. the C5a receptor agonist prevents further deposition of protein aggregates; and/or
  • the C5a receptor agonist results in the removal of at least some, preferably all, existing aggregates; and b) the situation wherein a subject does not already have any accumulation of abnormal protein aggregates and the C5a receptor agonist
  • i) reduces the rate at which protein aggregates are accumulated compared to a subject that is not receiving treatment with the C5a receptor agonist . i.e. the subject still develops the disease characterise by abnormal protein aggregation, but does so at a slower rate than a subject that has not been administered an agonist of the invention. Accordingly, disease progression is considered to be slower when a subject has been treated with an agonist of the invention, compared to a subject that has not been so treated; and/or
  • the C5a receptor agonist results in a total and complete prevention of the accumulation of protein aggregates, for example such that a subject that does not already have an accumulation of protein aggregates never has an accumulation of protein aggregates, following administration of an agonist of the invention.
  • reduces prevents and removes are to be used interchangeable depending on the context.
  • reduces is intended to encompass the ability of the C5a receptor agonist to result in the prevention and/or removal of protein aggregates; and is also intended to encompass the meaning of reduces relative to the level of protein aggregates in that subject prior to treatment, and also to mean reduces relative to the level of protein aggregates in a subject that has not been treated with the C5a receptor agonist .
  • the C5a receptor agonist is considered to act through macrophages to result in a physical removal of protein aggregates. This can result in an apparent reduction in the overall rate of deposition of protein aggregates.
  • the C5a receptor agonist results in a reduced amount of aggregated protein compared to the expected or normal amount of aggregated protein that would be found in a subject with the same disease and which subject has not been treated with the agonist of the invention.
  • a subject suffering from a disease characterised by abnormal protein aggregation would generally have a certain degree of aggregated protein, for example a subject suffering from AD would be expected, by the skilled person, to have a certain amount of amyloid deposits in the brain at a certain stage in the progression of the disease.
  • administration of the C5a receptor agonist reduces the amount of the protein aggregate that is deposited in the brain, and/or reduces the amount of the protein aggregate that has already been deposited, i.e. removes pre-existing protein aggregates, relative to a subject that has not been administered the agonist of the invention.
  • the C5a receptor agonist causes a reduction in the amount of aggregated protein in a subject compared to the amount of aggregated protein in the subject prior to administration of the agonist, and/or causes a reduction in the amount of aggregated protein in a test system compared to the amount of aggregated protein in the test system prior to administration of the agonist.
  • suitable test systems may include model organisms, for example established models of both the neurodegenerative Alzheimer’s disease and systemic ATTR amyloidosis as described in the Examples.
  • the C5a receptor agonist is considered to be useful if it reduces the level protein aggregation by any amount, and in any organ.
  • the agonist is useful if it reduces the level of protein aggregation by more than 2%, for example more than 5%, for example more than 15%, for example more than 20%, for example more than 25%, for example more than 30%, for example more than 35%, for example more than 40%, for example more than 45%, for example more than 50%, for example more than 55%, for example more than 60%, for example more than 65%, for example more than 70%, for example more than 75%, for example more than 80%, for example more than 85%, for example more than 90%, for example more than 95%, for example 100%.
  • 2% for example more than 5%, for example more than 15%, for example more than 20%, for example more than 25%, for example more than 30%, for example more than 35%, for example more than 40%, for example more than 45%, for example more than 50%, for example more than 55%, for example more than 60%, for example more than 65%, for example more than 70%, for example more than 75%, for example more than 80%, for example more than 85%, for
  • the agonist is considered to be useful if it reduces the level protein aggregation by any amount, and in any organ.
  • the agonist is useful if it reduces the level of protein aggregation by more than 2%, for example more than 5%, for example more than 15%, for example more than 20%, for example more than 25%, for example more than
  • the C5a receptor agonist is considered to be useful if it results in a reduction in the level of aggregated protein in any tissue.
  • the disease is a systemic disease and the agonist results in a reduction in the level of aggregated protein only in the stomach, or the liver, or the lungs, the agonist is still considered to be beneficial and useful.
  • the C5a receptor agonist can reduce the level of aggregated protein in any organ in the body.
  • the C5a receptor agonist can reduce the level of aggregated protein in any one or more of or all of the central nervous system, the brain; the peripheral nervous system; the heart; the kidneys; liver; the lungs, the spleen; the stomach; the digestive tract; the colon; the bladder; and the pancreas.
  • the C5a receptor agonist can reduce the level of aggregated protein in any one or more of or all of the central nervous system, the brain; the peripheral nervous system; the heart; the kidneys; liver; the lungs, the spleen; the stomach; the digestive tract; the colon; the bladder; and the pancreas by any amount, for example by more than 2%, for example more than 5%, for example more than 15%, for example more than 20%, for example more than 25%, for example more than 30%, for example more than 35%, for example more than 40%, for example more than 45%, for example more than 50%, for example more than 55%, for example more than 60%, for example more than 65%, for example more than 70%, for example more than 75%, for example more than 80%, for example more than 85%, for example more than 90%, for example more than 95%, for example 100%.
  • the C5a receptor agonist results in the same level of aggregated protein as is found in healthy individuals, i.e. subjects that do not have the disease characterised by abnormal protein aggregation.
  • the healthy individual is considered to have no abnormal protein aggregation at all in the relevant tissues.
  • the C5a receptor agonist results in the subject having no abnormal protein aggregation. If the subject is administered the C5a receptor agonist before the onset of any protein aggregation, the agonist may prevent the formation of the aggregated protein deposits, such that the subject never develops the disease, or symptoms of the disease.
  • the subject is treated before they develop one or more symptoms of the disease.
  • the subject is treated before they exhibit accumulations of amyloid deposits.
  • the C5a receptor agonist is useful in the treatment or prevention of any disease that is characterised by abnormal protein aggregation since, and without intending to be bound by any theory, it is considered that administration of the C5a receptor agonist results in removal of the protein aggregates by phagocytes such as macrophages. Accordingly, any disease where abnormal protein aggregation is considered to be involved in the aetiology of the disease is considered to be a disease suitable for treatment with the agonist of the invention. It will be apparent to the skilled person as to what these diseases are. In one embodiment the abnormal protein aggregates that characterise the disease are amyloid aggregates.
  • the disease is considered to be any amyloid disease, i.e. a disease in which the abnormal protein aggregates are amyloid aggregates or amyloid plaques.
  • amyloid diseases i.e. a disease in which the abnormal protein aggregates are amyloid aggregates or amyloid plaques.
  • diseases are considered to be amyloid diseases.
  • amyloid diseases which are considered to be suitable for treatment with the agonist of the invention include Alzheimer’s disease (AD), ATTR, Parkinson’s disease, Huntington’s disease and Amyotrophic Lateral Sclerosis, Non- neuropathic systemic amyloidoses such as Amyloid light chain (AL) amyloidosis, Amyloid A (AA) amyloidosis, Senile systemic amyloidosis, Lysozyme amyloidosis; and Non- neuropathic localized amyloidoses including Diabetes Type II; ATTR and other type of amyloidoses.
  • the disease is a systemic amyloid disease.
  • systemic diseases that are considered to be suitable for treatment with the agonist of the invention are the Non-neuropathic systemic amyloidoses such as Amyloid light chain (AL) amyloidosis, Amyloid A (AA) amyloidosis, Senile systemic amyloidosis, Lysozyme amyloidosis; and Non-neuropathic localized amyloidoses including Diabetes Type II; ATTR and other type of amyloidoses.
  • the disease is not a systemic amyloid disease.
  • the disease is not ATTR amyloidosis.
  • the disease is not a disease that involves abnormal protein aggregation in the stomach and/or digestive tract.
  • the disease is any disease characterised by abnormal protein aggregation, or any amyloid disease, other than ATTR amyloidosis.
  • the disease is any disease characterised by abnormal protein aggregation, or any amyloid disease, other than a systemic amyloid disease.
  • the disease is not a disease that involves abnormal protein aggregation in the stomach and/or the digestive tract, and wherein the activator is administered in an oral form.
  • the disease is any disease characterised by abnormal protein aggregation, or any amyloid disease, other than a disease that involves abnormal protein aggregation in the stomach and/or the digestive tract.
  • the disease is characterised by abnormal protein aggregate deposits, for example amyloid aggregate deposits, in a particular organ. Any organ may be affected. In one embodiment the affected organ is the brain, and the subject has abnormal protein aggregates, for example amyloid aggregates, in the brain tissue. In a further embodiment the abnormal protein aggregates, for example amyloid aggregates, are deposited in the central nervous system. In yet a further embodiment the abnormal protein aggregates, for example amyloid aggregates, are deposited in the peripheral nervous system.
  • abnormal protein aggregate deposits for example amyloid aggregate deposits, in a particular organ. Any organ may be affected. In one embodiment the affected organ is the brain, and the subject has abnormal protein aggregates, for example amyloid aggregates, in the brain tissue. In a further embodiment the abnormal protein aggregates, for example amyloid aggregates, are deposited in the central nervous system. In yet a further embodiment the abnormal protein aggregates, for example amyloid aggregates, are deposited in the peripheral nervous system.
  • the affected organ is the central nervous system; the peripheral nervous system; the heart; the kidneys; liver; the lungs, the spleen; the stomach; the digestive tract; the colon; the bladder; and the pancreas.
  • the disease is a neurodegenerative disease that is characterised by abnormal protein aggregation, for example amyloid deposits in the brain.
  • diseases are considered to include Alzheimer’s Disease, Parkinson’s Disease, motor neuron diseases, polyglutamine disorders, Huntington’s disease, familial amyloid polyneuropathy (FAP).
  • FAP familial amyloid polyneuropathy
  • the inventors have found that the treatment of these neurodegenerative diseases and other diseases in which abnormal protein aggregates are deposited in the brain requires the agonist to cross the blood-brain barrier, see for example the Examples.
  • the ability of the C5a receptor agonist to cross the blood-brain barrier was unknown and accordingly the effective treatment of these diseases was even more surprising. This is particularly so in view of an oral route of administration of the agonist.
  • the disease is an infectious or transmissible disease and is considered to be suitable for treatment with the C5a receptor agonist of the invention.
  • An example of transmissible diseases that are considered to be suitable for treatment with the agonist of the invention are the prion diseases.
  • the disease is an infectious disease and is not considered to be suitable for treatment with the C5a receptor agonist of the invention.
  • An example of transmissible diseases that are not considered to be suitable for treatment with the C5a receptor agonist of the invention are the prion diseases.
  • the disease it not a transmissible disease and is not considered to be a prion disease.
  • amyloidoses are not considered to be prion diseases.
  • the disease is Alzheimer’s disease and the C5a receptor agonist results in a reduction of the levels of amyloid aggregates in the brain.
  • treatment with the C5a receptor agonist results in a reduction of the levels of both ab40 and ab42 ⁇ h the brain.
  • the C5a receptor agonist is for use in treating ATTR amyloidosis and the C5a receptor results in a reduction of the levels of amyloid aggregates in the stomach.
  • the C5a receptor of the invention may be administered by any route.
  • the C5a receptor can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications.
  • the agonist of invention may also be administered via intracavernosal injection.
  • the inventors have surprisingly found that oral administration of the C5a receptor results in advantageous effects distal to the site or route of administration, and even more surprisingly results in the reduction of protein aggregates in the brain. Accordingly, in a preferred embodiment, the C5a receptor is administered orally.
  • Administration of the C5a receptor can take the form of a pharmaceutical formulation comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form.
  • the agents may be administered at varying doses.
  • the formulation is a unit dosage containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of the agent or active ingredient.
  • the C5a receptor agonist for example EP67, can be administered alone but will generally be administered in admixture with a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the invention also provides a pharmaceutical composition comprising a C5a receptor agonist as defined herein.
  • a further embodiment provides a pharmaceutical composition comprising a C5a receptor agonist as defined herein for use in treating or preventing a disease characterised by abnormal protein aggregates.
  • the C5a receptor agonist may be administered to the subject along with other beneficial therapeutic agents, for example additional agents that are Accordingly one embodiment provides a composition comprising a C5a receptor agonist and a further therapeutic agent that is useful in treating or preventing the disease characterised by abnormal protein aggregates.
  • the composition may be a pharmaceutical composition.
  • the invention provides a composition comprising a C5a receptor agonist and a further therapeutic agent that is useful in treating or preventing the disease characterised by abnormal protein aggregates for use in in treating or preventing the disease characterised by abnormal protein aggregates.
  • the composition may be a pharmaceutical composition.
  • Tablets comprising the C5a receptor agonist may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included. Capsules or tablets may also be enteric coated to enhance gastric stability.
  • excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine
  • disintegrants such as starch (preferably corn, potato or tapi
  • Solid compositions of a similar type may also be employed as fillers in gelatin capsules.
  • Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols.
  • the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
  • the C5a receptor agonist can also be administered parenterally, for example, intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intra-muscularly or subcutaneously, or they may be administered by infusion techniques.
  • the C5a receptor agonist may be administered in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • the aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.
  • the preparation of suitable parenteral Formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.
  • the daily dosage level of the C5a receptor agonist (and/or further therapeutic agents) will usually be from 1 to 5000 mg per adult, administered in single or divided doses.
  • the C5a receptor agonist (and/or further therapeutic agents) is administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.
  • administration is not restricted to a one time administration.
  • administration is taken to cover all of, but not limited to, a single dose administration, multiple administrations over a period of time, variable dosage administrations over a period of time, variable means of administration over a period of time, administration in conjunction with one or more further therapeutic agents.
  • Administration can be by any means known in the art and includes, but is not limited to, oral, intravenous, topically direct to the tumour, sublingually or suppository.
  • the C5a receptor agonist is administered to the subject prior to the deposition of the abnormal protein aggregate. Routine clinical tests are able to determine whether a subject has, or does not have, deposits of the relevant protein aggregate(s).
  • the C5a receptor agonist is administered to the subject following the deposition of the abnormal protein aggregate.
  • the C5a receptor agonist is administered to the subject for 1 week, for example is administered every day for 1 week, for example every day for 7 days.
  • the C5a receptor agonist is administered to the subject periodically.
  • the C5a receptor agonist is administered at regular intervals with at least 1 day between doses, or for example at least 2 days, or at least 3 days, or at least 4 days, or at least 5 days, or at least 6 days, or at least 7 days, or at least 1 week, or at least 2 weeks, or at least 3 weeks, or at least 4 weeks, or at least 5 weeks, or at least 6 weeks, or at least 7 weeks, or at least 8 weeks, or at least 9 weeks, or at least 10 weeks, or at least 11 weeks, or at least 12 weeks between doses, optionally at least 1 month, or 2 months, or 3 months, or 4 months, or 5 months, or 6 months, or 7 months, or 8 months, or 9 months, or 10 months, or 11 months, or 12 months, between doses, optionally at least 1 year, between doses.
  • the C5a receptor agonist is administered to the subject every day for at least 2 days, optionally at least 3 days, optionally at least 4 days, optionally at least 5 days, optionally at least 6 days, optionally at least 7 days, optionally at least 8 days, optionally at least 9 days, optionally at least 10 days, optionally at least 1 1 days, optionally at least 12 days, optionally at least 13 days, optionally at least 14 days, optionally at least 1 week, optionally at least 2 weeks, optionally at least 3 weeks, optionally at least 4 weeks, optionally at least 5 weeks, optionally at least 6 weeks, optionally at least 7 weeks, optionally at least 8 weeks.
  • the C5a receptor agonist is administered periodically. It is considered that the periodic or intermittent administration of the C5a receptor agonist which is considered to stimulate cycles of phagocytosis and which prevents prolonged stimulation of the C5a receptor may be beneficial.
  • a period in a period administration regime may comprise the following administration regime: a) administration of the C5a receptor agonist every day for at least 2 days, optionally at least 3 days, optionally at least 4 days, optionally at least 5 days, optionally at least 6 days, optionally at least 7 days, optionally at least 8 days, optionally at least 9 days, optionally at least 10 days, optionally at least 11 days, optionally at least 12 days, optionally at least 13 days, optionally at least 14 days, optionally at least 1 week, optionally at least 2 weeks, optionally at least 3 weeks, optionally at least 4 weeks, optionally at least 5 weeks, optionally at least 6 weeks, optionally at least 7 weeks, optionally at least 8 weeks; followed by: b) at least 1 dose-free day, optionally at least 2 days, or at least 3 days, or at least 4 days, or at least 5 days, or at least 6 days, or at least 7 days, or at least 1 week, or at least 2 weeks, or at least 3 weeks, or at least 4 weeks, or or at
  • the above period may be repeated any number of times, for example may be repeated for the rest of the life of the subject.
  • the subject is administered the agonist for 7 days followed by 4 weeks where the agonist is not administered, followed by administration of the agonist for 7 days, followed by 4 weeks where the agonist is not administered, and so on.
  • the administration of the C5a receptor agonist is such that the number of days of administration and/or number of dose-free days varies between periods.
  • the administration may comprise administering the agonist for 7 days followed by 4 weeks where the agonist is not administered, followed by administration of the agonist for 14 days, followed by 3 weeks where the C5a receptor agonist is not administered, etc.
  • the administration regime may comprises a series of different periods, or two or more alternating periods.
  • the subject has not been diagnosed as having a disease characterised by abnormal protein aggregation.
  • the subject has been diagnosed as having a disease characterised by abnormal protein aggregation.
  • any subject that is at risk of developing one or more disease characterised by abnormal protein aggregation is particularly suitable for treatment with the C5a receptor agonist as described herein.
  • the subject is a subject that has been determined to carry a particular gene or gene mutation that is indicative of an increased likelihood of developing a disease characterised by abnormal protein aggregation.
  • subjects that are known to be gene carriers of a particular amyloidoses and so are expected to develop the disease are considered to be particularly suitable subjects for treatment with the agonist of the invention. These subjects can be treated in a timely manner before they begin to accumulate amyloid deposits. Subclinical accumulations of amyloid deposits can be detected by laboratory testing prior to the appearance of symptoms.
  • elderly patients are also particularly suitable for treatment with the C5a receptor agonist of the invention, since this population is at increased risk of developing amyloid diseases, particular for example the neurodegenerative amyloid diseases such as Alzheimer’s disease.
  • amyloid PET scans for example 5-yearly amyloid PET scans to detect amyloid deposits in the brain and be treated accordingly.
  • the subject is treated with the C5a receptor agonist prior to detection of amyloid deposits in order to prevent the deposition of amyloid aggregates.
  • the subject is aged 50 or over, for example aged 60 or over, for example aged 65 or over, for example aged 70 or over, for example aged 75 or over, for example aged 80 or over, for example aged 85 or over, for example aged 90 or over, for example aged 95 or over, for example aged 100 or over.
  • the C5a receptor agonist can be considered to be a vaccine. Accordingly, one embodiment provides a C5a receptor agonist for use as a vaccine against the development of a disease characterised by abnormal protein aggregation, for example against the development of Alzheimer’s Disease and/or ATTR amyloidosis, wherein the C5a receptor agonist is defined herein.
  • the invention also provides a C5a receptor agonist for use in a method of preventing or reducing neuronal degeneration, wherein the C5a receptor agonist is defined herein.
  • the above disclosure also relates to various methods of treating or preventing a disease characterised by abnormal protein aggregation. Accordingly, the invention also provides a method of treating and/or preventing a disease characterised by abnormal protein aggregation wherein the method comprises administering a C5a receptor agonist as defined herein and wherein the disease is defined herein.
  • the invention also provides the use of a C5a receptor agonist as defined herein in a method of manufacture of a medicament for the treatment and/or prevention of a disease characterised by abnormal protein aggregation, wherein the disease is defined herein.
  • kits for putting the invention into practice for example a kit comprising a C5a receptor agonist of the invention and a further therapeutic agent.
  • Preferences and options for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features and parameters of the invention.
  • the invention provides a C5a receptor agonist of the sequence Tyr-Ser-Phe-Lys-Asp-Met- Pro-Leu-Gly-Arg; (SEQ ID NO: 11 ) for use in the treatment of Parkinson’s disease, wherein the c5a receptor agonist is administered in accordance with a repeating cycle of administration every day for 3 days, followed by 4 days of non-treatment; as well as also providing EP67 for use in the prevention of Alzheimer’s disease wherein EP67 is administered to a subject that has not been diagnosed as suffering from Alzheimer’s disease, nor has been identified as having any amyloid deposits in the brain; as well as also providing a C5a receptor agonist for oral administration to remove abnormal protein aggregates from stomach tissue, for example.
  • Amyloid deposition amyloid plaques were quantified through Thioflavin-S staining.
  • LC-MS/MS clustering protein functional clustering was carried out through the Panther tool between the group of mice treated with the full agonist molecule and the PMX53 antagonist.
  • the group of mice treated with the full agonist displayed an increase in the expression of markers related with macrophage and complement system activation. Furthermore, an upsurge in the expression of peptidases and factors involved with inflammation was observed.
  • the group treated with the antagonist exhibited a greater expression of caspases and other proteins involved in the apoptosis pathways.
  • Phagocytic cell and complement related markers obtained through LC-MS/MS.
  • Amyloid deposition Sagittal sections were taken from 3 and 6 month old wild mice, 5XFAD control mice and 5XFAD mice treated with EP67 (a). The sections were co- stained with Thioflavin-S and an anti-Ab antibody. Representative images of the cortex, thalamus and hippocampus are shown. Wild mice exhibited no amyloid plaques at 3 months or 6 months of age (i-/ ' /7) and (x-x/V). Sections from 5XFAD control mice show an increase in amyloid load between 3 months (iv-vi) and 6 months ( xiii-xv ) in all three regions shown.
  • FIG 17 Spontaneous alternation Y-maze task. Wild, 5XFAD control and 5XFAD EP67 treated mice at 3 and 6 months of age were given the spontaneous alternation behavioural test using a Y maze. Results indicate a significant increase in the percentage of spontaneous alternations in the mice treated with EP67 versus the 5XFAD control mice in both ages, even though no significant increase was recorded against the wild mice (a). The number of arm entries for each group was also recorded and exhibited no significant difference among any group of animals ( b ).
  • Astrocytes and microglia Sagittal cortex sections were taken from the brains of 3 month and 6 month old wild mice, 5XFAD control mice and EP67 treated 5XFAD mice. Sections were co-stained with an antibody against the astrocytic marker GFAP and Thioflavin-S (a). While sections taken from wild mice and EP67 treated with 5XFAD mice at both age groups show very limited staining ( ai&aiv , aiii&avi respectively), 5XFAD control mice exhibit strong staining which is intensified at 6 months of age ( aii&av ). Similar results were obtained through immunoblotting ( b ).
  • C5a is a 74 amino acid glycoprotein (A) consisting of four alpha helices arranged in an anti-parallel orientation (B), connected by peptide loops located at the surface of the molecule and stabilised by three critical disulphide linkages (Cys 21 - Cys 47 , Cys 22 - Cys 54 and Cys 34 - Cys 55 ).
  • the c-terminus contains a four-residue loop, critical for receptor activation.
  • Complement component 5a (C5a). The international journal of biochemistry & cell biology 41 , 2114-2117.
  • mice were anesthetized and then euthanized using Tribromoethanol (Avertin) through IP injection at a dose of 250 mg/Kg. The animals were then exsanguinated via PBS perfusion to reduce the contribution of plasma in tissue measurements. Tissues were processed for immunohistochemistry by carrying out overnight 4% PFA fixation followed by wax embedding or were frozen and kept at -80 ° C for immunoblotting. Amyloid deposition assessment was confined to stomach tissue since this tissue is heavily involved in amyloid deposition at an early age in this particular mouse model of ATTR V30M neuropathy.
  • mice were genotyped using the PCR method.
  • Primers for the mouse TTR gene mTTR F 50 -CTG ACC CAT TTC ACT GAC ATT T-30 & mTTR R 50 -CAA ATG GGA ACC TGG AAC C-30
  • the human mutated transgene hMET30 F 50 -TG CT GAT GAC AC CT G G GAG C-30 and hMET30 R 50
  • TCAGGTTCCTGGTCACTTCC-30 were utilized for screening with annealing temperature at 58 ° C.
  • Plaques positive for both Thioflavin S and hTTR were measured using the ImageJ software set to measure yellow (570-585 nm; Figures 1 Biii— Bvi). TTR amyloid plaques were measured over the entire area of stomach section, a percentage of the surface area occupied by plaques was calculated and an average percentage obtained over five serial sections.
  • Serum human TTR was measured using enzyme-linked immunosorbent assay (ELISA) from four animals from each of the four groups. Blood samples were collected, without sacrificing any of the animals, from the orbital sinus in the absence of anticoagulant. The samples were allowed to stand at room temperature for approximately 30 min to coagulate. They were then centrifuged at 3500 rpm for 10 min and the top layer was collected in order to obtain the serum. Samples were diluted 1/50,000 using the supplied mix diluent from the kit used (Abnova TTR Human ELISA Kit KA0495) and the procedure was carried out as outlined by the supplier. Absorbance was measured at 450 nm using a microplate reader.
  • ELISA enzyme-linked immunosorbent assay
  • Stomach homogenate tissue lysed with RIPA buffer and protease inhibitors under sonication was separated via reducing SDS-PAGE and transferred onto PVDF membranes.
  • the membranes were blocked with 5% BSA for 1 h at room temperature.
  • the membranes were then incubated overnight at 4 ° C with the appropriate primary antibody.
  • Specific antibodies were then visualized using the Super Signal West Femto Maximum Sensitivity Substrate (Thermo Fisher 34095) after incubating with the required HRP conjugated secondary antibody for 1 h at room temperature. Blots were repeated in triplicates and were visualized using the UVP bio-imaging system.
  • the antibodies used for immunoblotting were against: BiP (anti-rabbit Santa Cruz sc-13968 1/350), C1 q (anti-rabbit Santa Cruz sc-27661 1/100), Caspase-3 (anti-rabbit Enzo Life Sciences ALX-210-806-C100 1/1000), CD68 (anti-rabbit Santa Cruz sc-9139 1/150), CD88 (anti-mouse Santa Cruz sc-53795 1/100), ELANE (anti-rabbit Abeam ab68672 1/1000), F4/80 (anti-rabbit Santa Cruz sc-25830 1/100), II_-36g (anti-goat Santa Cruz sc-168163 1/100), Ly6G (anti-mouse Antibodies online ABIN361224 1/1000) and Properdin (anti-rabbit Santa Cruz sc-68367 1/100).
  • the appropriate HRP conjugated secondary antibodies were used, anti-mouse (Santa Cruz SC-2031 1/5000), anti-rabbit
  • ImageJ was used to carry out densitometry calculations, while all bands were normalized against a GAPDH loading control (Santa Cruz sc-25778 1/1000), while the same reference sample was in all westerns to allow cross-gel comparison).
  • Paraffin sections from animals’ stomachs were deparaffinized and hydrated to distilled water. Sections were then blocked with 5% BSA solution in PBS for 1 h at room temperature and then incubated with the appropriate primary antibody overnight at 4 ° C. The slides were then washed and incubated with the appropriate secondary antibody for 1 h at room temperature. Finally, DAPI staining was used to label the cells’ nuclei (Sigma Aldrich D9542) before been mounted using the DAKO Fluorescence Mounting Medium (S3023). Pictures were taken using a Zeiss AXIOIMAGER M2 fluorescence microscope.
  • the primary antibodies used were against: BiP (anti-rabbit Santa Cruz sc-13968 1/100), Caspase-3 (anti-rabbit Santa Cruz sc-7148 1/500), CD68 (anti-goat Santa Cruz sc- 7084 1/50), ELANE (anti-rabbit Abeam ab68672 1/100), Lamp-1 (anti-rabbit Cell signaling 8653 1/800) and human TTR (anti rabbit DAKO A000202 1/500).
  • the appropriate Invitrogen Alexa Fluor 555 and 488 fluorescence secondary antibodies were used, anti-rabbit(A-21428 and A-11008 1/2000) and anti-goat (A-21432 and A-11055 1/2000).
  • Frozen stomach tissue samples from three animals from two groups of animals were incubated in lysis buffer (10 mM Tris-HCI pH 7.4, 150 mM NaCI, 1 mM EDTA, 1 % (v/v) SDS, 1X protease inhibitors) for 30 min on ice, followed by sonication for 30 s (50% pulse) using Model 150VT (Biologies Inc., Virginia, USA). Lysates were clarified by centrifugation at 12,000 rpm for 20 min at 4 ° C.
  • Proteins were digested with 2 pg of proteomics grade trypsin (Roche Diagnostics GmbH, Mannheim, Germany) at 37 ° C for 18 h. Digestion was quenched by addition of TFA to a final concentration of 0.5%. Peptides were desalted and purified using reverse phase solid phase extraction cartridges (Sep-Pak C18, Waters, Vienna, Austria) and eluates were lyophilized using a centrifugal vacuum concentrator. Peptide pellets were re- dissolved in 1 % acetonitrile, 0.1 % formic acid (mobile phase A) to yield an approximate concentration of 200 ng/pL (determined by NanoDrop measurement at 280 nm).
  • the peptide separation was performed on a Waters nanoAcquity UPLC system (Waters Co., Wilmslow, UK). Peptides were loaded onto a C18 column (Acquity UPLC M- Class, Peptide CSH, 75 pm x 250 mm, 1.7 pm, 130 A) and eluted with a linear gradient from 5% mobile phase B (0.1% formic acid in acetonitrile) to 40% mobile phase B over 175-min. Peptides were analyzed on a Waters Synapt G2Si HDMS instrument (Waters Co., Wilmslow, UK) operated in ion mobility mode using the UDMSE approach (Distler et al., 2014). Each sample analyzed in triplicate.
  • Raw mass spectrometry data were analyzed using Progenesis Ql for proteomics software (version 3.0) and were subjected to protein identification against the SwissProt mouse reference proteome database (version July 2016, 16761 sequences plus human TTR, P02766) using the MSe peptide identification method.
  • the searching parameters used were: trypsin digestion, 1 missed cleavage, FDR ⁇ 4%.
  • the identifications were refined using the following parameters: score >5, hits >2, sequence length >6, description not containing probable, predictive, potential or putative.
  • the PMX53 molecule is a known C5a receptor (CD88) inhibitor, while the other two molecules used are agonists for the receptor.
  • C5a receptors C5R1 , CD88
  • CD88 C5a receptors
  • CD88 the ubiquitous C5a receptor was found to be significantly elevated in both the full agonist and EP67 groups when compared to the control animals, whereas the receptor was severely decreased in the animals treated with PMX53 (Figure 3A).
  • Neutrophil elastase and Ly6G are both well characterized markers of neutrophils (Talukdar et al., 2012; Amsalem et al., 2014).
  • ELANE is a chymotrypsin like serine proteinase which is mainly secreted by neutrophils during inflammation in order to induce the clearance of bacteria and host tissue (Belaaouaj et al., 2000).
  • ELANE is also very similar to other immune system cytotoxic serine proteases such as granzymes and cathepsin G (Thomas et al., 2014).
  • Ly6G is a neutrophil specific marker which has been previously used to deplete neutrophils in mice (Pillay et a/., 2013).
  • Our data indicate a similar pattern as with ELANE, where the groups treated with the agonist molecules exhibit the highest level of Ly6G, while the mice treated with the PMX53 antagonist molecule exhibit the lowest amount of Ly6G detected (Figure 3C).
  • Interleukin 36y has been found to be up-regulated in a number of inflammatory diseases and is believed to be expressed by both neutrophils and macrophages (Bozoyan et al., 2015; Kovach et al., 2016; Macleod et al., 2016). Our results also indicate that IL-36y is highest in the group of mice treated with the full agonist and the lowest in the group treated with the PMX53 molecule ( Figure 3D).
  • the murine F4/80 (EGF-like module-containing mucin-like hormone receptor-like 1 , Emr1 homolog) is a well-known marker of murine macrophage populations (Austyn and Gordon,
  • mice While CD68 is found to be expressed on all macrophages (Murray and Wynn, 201 1 ).
  • the PMX53 treated mice exhibited the lowest levels of both macrophage markers, while both groups of mice treated with the agonist molecules displayed greater expression of the two markers when compared to the untreated control ( Figures 3E,F).
  • the mice treated with EP67 display the greatest levels of macrophages, even when compared to the group treated with the full agonist.
  • the classical complement pathway is initiated through the C1q molecule.
  • C1q itself is produced by peripheral tissue phagocytic cells (Petry et al., 1991 ).
  • the group of mice treated with the full agonist molecule express the greatest amount of C1q; even though the mice treated with the EP67 molecule also exhibit elevated amounts of C1q when compared to the untreated control animals.
  • the animals treated with the C5aR inhibitor, PMX53 do not display any significant difference from the control animals ( Figure 5A).
  • Properdin (factor P) is a unique positive regulator of complement activation which functions by stabilizing the alternative pathway convertases (Smith et al., 1984) so that it may be used as an alternative complement pathway marker.
  • mice treated with PMX53 possess the highest levels of BiP which is in accordance with the high levels of amyloid deposits.
  • the groups treated with the agonists exhibit significantly less amount of BiP than the PMX53 mice even though they do not appear to be lower than the control untreated mice ( Figure 6A).
  • Lysosomal Marker in Stomach Tissue Lysosomal-associated membrane protein 1 (Lamp-1 ) is a glycoprotein known to primarily reside across lysosomal membranes (Carlsson and Fukuda, 1989), also Lamp-1 may be expressed on the cell surface following lysosomal fusion with the cell membrane during phagocytosis (Kima et al.,
  • Lamp-1 will specifically become localized on the phagosomes (Sugaya et al., 2011 ), so that, expression of Lamp-1 signifies the final steps of activated phagocytosis.
  • Serial deparaffinized stomach sections from a mouse treated with the full agonist and EP67 exhibit the complete co- localization of a-Lamp-1 with a-CD68 and a-ELANE.
  • the PMX53 treated mouse does not exhibit any co-localization with ELANE, CD68, or LAMP- 1 ( Figure 7A).
  • sections from an animal treated with PMX53 display no plaque co-localization with Lamp-1 ( Figure 7B).
  • amyloidogenesis in certain tissue beds such as the eye and the brain has not yet been effectively addressed partly due to issues of access of the various treatments to the relevant tissue beds.
  • antibodies against serum amyloid P or in the case of ATTR V30M amyloidosis, a cryptic TTR epitope visible only when TTR is in its monomeric form are beginning to be explored as possible treatments (Richards et al., 2015; Hosoi et al., 2016). Both of these antibodies appear to enhance phagocytosis by macrophages. Complement participation has also been demonstrated with the anti- serum amyloid P antibody in vivo.
  • Agonist and antagonists of the C5a receptor molecule are constructed based on the C terminus of the C5a molecule (Higginbottom et al., 2005). Agonists can be used to activate the C5a receptor bearing cells such as macrophages and neutrophils in order to induce the release of proinflammatory agents, thus activating an inflammatory response (Short et al., 1999).
  • the C5a molecule is considered an anaphylatoxin based on its propensity to induce mast cell, basophil and neutrophil degranulation (Lee et al., 2008).
  • the full agonist molecule of the C5a receptor used here ((N-Methyl-Phe)-Lys- Pro-d-Cha-Cha-d-Arg-C02 H) is a peptide analog of the last six C-terminal residues of the C5a molecule (Higginbottom et al., 2005). In essence, this molecule retains its anaphylactic activity by not only activating macrophages but also neutrophils.
  • EP67 Thr-Ser-Phe-Lys-Asp- Met-Pro-(MeLeu)-D-Ala-Arg
  • a conformationally-restricted decapeptide of the last ten amino acids of the C5a molecule contains an altered structure, which is accommodated by C5a receptors expressed on antigen presenting cells such as macrophages (Sanderson et al., 2012; Hanke et al., 2013).
  • the C5a receptor antagonist PMX53 (AcF-[OP(D Cha) WR]) is a small cyclic peptide molecule which binds the C5a receptor suspending its downstream function (Woodruff et al., 2006; Lobato and Rocha, 2012).
  • the PMX53 group mice exhibited no significant rise in prefibrillar hTTR, compared to control, as might be expected due to reduced phagocytosis of prefibrillar hTTR (Misumi et al., 2013; Suenaga et al., 2016). This is most likely explained by a shift to a higher rate of amyloid formation driven by higher prefibrillar hTTR.
  • amyloid plaques in animals treated with the two C5a receptor agonists exhibit increased expression of the lysosomal marker Lamp- 1 , signifying that the recruitment of macrophages and neutrophils does ultimately lead to activated phagocytosis of the amyloid plaque.
  • the PMX53 treated group of animals exhibited the lowest expression of both neutrophil and macrophage markers as might be expected due to the inhibition of the C5a receptor (Brennan et al., 2015; Gupta and Kaplan, The complement pathway is involved in innate immunity and C5a is one of the final effector molecules produced. C5a is imperative in tissue clearance through the recruitment of inflammatory cells. Mice treated with the C5a receptor agonists express a greater amount of C1q than both the control and the PMX53 treated mice. Neutrophils are known to possess C1q receptors which in turn enhance the expression of the CR3 receptor (Eggleton et al., 1994), an integral part of the innate immune response.
  • C1q the significant increase in C1q, observed especially in the group treated with the full C5a receptor agonist, is probably due to the presence of neutrophils.
  • Neutrophils also activate the alternative complement pathway and release C5 fragments, which further amplify the neutrophil pro-inflammatory response, acting in a positive feedback loop (Camous et al., 2011 ).
  • macrophages have also been shown to activate the alternative complement pathway by activating C3 (Schorlemmer et al., 1977), explaining the increase in properdin in the groups treated with the C5a receptor agonists.
  • the LC-MS/MS data reveal that a greater number of peptidases, and more specifically serine proteases, become up-regulated in the full agonist treated group when compared to the PMX53 treated animals which exhibit the greatest amyloid load.
  • Neutrophils are known to release a number of serine proteases which induce chemokine and cytokine release as well as proteolytic cleavage (Kessenbrock et al., 2011 ; Meyer-Hoffert and Wiedow, 201 1 ). Furthermore, neutrophil elastase (ELANE), a serine protease secreted by neutrophils has been shown to preferentially activate IL-36 yielding the three by-products IL-36a, I L-363 and IL-36y triggering further inflammatory response.
  • ELANE neutrophil elastase
  • macrophages have the ability to release Fas ligands, thus increasing extrinsic-signal triggered apoptosis (Brown and Savill, 1999) but also the ability to induce chronic inflammation leading to further apoptosis (Diez-Roux and Lang, 1997; Gregory and Devitt, 2004).
  • Phagocytosis triggers macrophage release of Fas ligand and induces apoptosis of bystander leukocytes. J. Immunol. 162, 480-485.
  • Macrophages induce apoptosis in normal cells in vivo. Development 124, 3633-3638.
  • a serine protease in Alzheimer’s disease cells cleaves a 16K-peptide with flanking residues upstream to b-amyloid-N-terminus as natural substrate.
  • Amyloid fibril protein in familial amyloidotic polyneuropathy Portuguese type. J. Clin. Invest. 74, 104-1 19. doi: 10.1 172/jci1 11390
  • Familial amyloid polyneuropathy receptor for advanced glycation end products-dependent triggering of neuronal inflammatory and apoptotic pathways. J. Neurosci. 21 , 7576-7586. Suenaga, G., Ikeda, T., Komohara, Y., Takamatsu, K., Kakuma, T., Tasaki, M., et al. (2016).
  • Example 2 An orally administered C5aR agonist enhances phagocytosis of Ab amyloid and preserves memory in the 5XFAD animal model of Alzheimer disease
  • EP67 is a response-selective analogue of the biologically active C-terminal region of the human complement component C5a 65-74 ⁇ .
  • This specific receptor agonist molecule however was generated by replacing certain residues and adding a methyl group—— which allowed for differentiation between C5a-like inflammatory and immune enhancing action— .
  • these alterations enable the molecule to act on C5a receptors found on antigen presenting cells (APCs)—. Therefore, the EP67 molecule, while able to activate APCs, lacks the ability to specifically activate neutrophils or retain any neutropenic action.
  • FIG 16a Representative sagittal sections from the cortex, thalamus and hippocampus double stained with a pan Ab antibody and the amyloid plaque stain Thioflavin-S are shown in figure 16a.
  • the wild mice at 3 and 6 months of age exhibit no staining for amyloid-b or Thioflavin-S as expected (Fig 16a i-iii and x-xii).
  • Both the control 5XFAD and 5XFAD EP67 treated mice exhibit similar levels of staining in the thalamus and hippocampus at the age of 3 months (Fig 16a v,vi and viii x respectively), while the cortex of the control 5XFAD mice appears to carry a greater amyloid load than the animals treated with EP67 (Fig 16a iv and vii respectively).
  • the 5XFAD mice accumulate plaques in the deep layers of the cortex and the subiculum before exhibiting deposition in the hippocampus which probably accounts for visible effect of EP67 only in the cortex at the age of three months—.
  • the untreated control 5XFAD mice exhibit greater load in all three regions examined (Fig 61a xiii-xv) when compared to the EP67 treated mice (Fig 16a xvi-xviii).
  • Quantification of both the Ab40 (Fig 16b) and Ab42 (Fig 16c) peptides was carried out using immunoassays against these peptides. While Ab40 levels did not appear to change at the 3 month age point, significant reduction was observed in the 6 month EP67 treated animals.
  • mice exhibit Ab accumulation by 3 months of age and as the mice age this is predominantly Ab42 rather than Ab40— .
  • Ab42 a significant reduction was recorded at both the 3 month and 6 month age points, with a greater change observed following four treatments with EP67.
  • EP67 protects against short-term spatial working memory loss
  • mice Neurotoxic Ab1-42 oligomers have previously been shown in vivo to severely affect both the metabolic and cognitive processes associated with the hippocampus probably a result of diminished synaptic activity caused by impaired insulin signalling 13 ’ 22 .
  • the Y- maze task spontaneous alternation is essentially a short-term spatial working memory index in mice—. This task involves no training and no rewards and mice are allowed to freely explore the 3-armed maze for 8 minutes. During the session all entries are recorded and the percentage of spontaneous alternations is calculated as a ratio of successful triads over the total number of arm entries. All mice participating in this study, including wild type animals were evaluated via the Y-maze task.
  • EP67 prevents synaptic and neuronal loss
  • Astrocytosis has long been recognized as part of the neuroinflammation in AD brains in both humans and animal models of the disease and thought to be a result of amyloid deposition——.
  • the astrocytic marker for the glial fibrillary acidic protein (GFAP) was used to evaluate the distribution of astrocytes in the brains of EP67 treated and control 5XFAD brains (Fig 19a and b).
  • EP67 recruits microglia and macrophages to amyloid plaques
  • Microgliosis is another feature of neuroinflammation 23 ’ 24 .
  • C5a receptors are carried by macrophages and neutrophils and also neurons, microglia and astrocytes within the brain.
  • Monocytes represent up to 10% of peripheral blood leukocytes and differentiate into dendritic cells and tissue-specific macrophages—. Simard et a!., demonstrated that peripheral macrophages can migrate towards Ab plaques, a response elicited by Ab40 and Ab42, in order to initiate clearance of the plaques through phagocytosis—.
  • EP67 recruits neutrophils to amyloid plaques
  • neutrophils have a very short life span they have been detected to infiltrate the AD brain—.
  • Neutrophils have been shown to migrate to the parenchyma through blood vessel adherence and ingress into brain tissue, in both AD human samples and mouse models of the disease——.
  • Immunohistochemical examination using the neutrophil elastase marker, ELANE, in 6 month old control 5XFAD (Fig 20 a-c) and 6 month EP67 treated 5XFAD animals (Fig 20 d-f) did reveal weak ELANE staining, co- localising with amyloid plaques in the 5XFAD animals (Fig 20b), however increased expression and co-localisation was observed in the EP67 treated 5XFAD mice (Fig 20d).
  • the complement cascade is an integral part of the immune system which promotes the clearance of cellular debris, microbes and damaged cells from the organism through both innate and antibody mediated stimulation of phagocytic cells—. Irrespective of the means of its activation, the complement cascade essentially results in the assembly of the terminal attack complex and the recruitment of phagocytic cells through the activation of the C5a receptor 21 .
  • EP67 By introducing the modified C5a receptor agonist, EP67, in the drinking water of 5XFAD mice we were to decrease Ab amyloid, preserve neurons and synapses and ameliorate cognitive impairment.
  • EP67 treated 5XFAD mice exhibited increased numbers of phagocytes and reduced levels of astrocytosis.
  • Neurotoxic Ab peptides have previously been shown to induce astrocytosis in vitro— as well as trigger changes in astrocyte glutamate uptake and metabolism 22 .
  • activated astrocytes are heavily implicated in the inflammatory response observed in AD through the secretion of cytokines and proinflammatory factors—.
  • the central nervous system contains resident macrophages, microglia which serve as the first line of deference for the entire CNS by scanning the tissue for foreign agents and cellular debris.
  • Microglia have been shown to be activated in both the brains of AD patients and transgenic disease mouse models, associated with Ab amyloid plaques and contributing to neuroinflammation. In the absence of a stimulus, microglia remain in a deactivated state while secreting neurotrophic and anti-inflammatory factors.
  • microglia and astrocytes flock around amyloid plaques and secrete several inflammatory molecules such as the major histocompatibility complex (MHC) class II, the monocyte chemoattractant/chemotactic protein (MCP)-1 , tumor necrosis factor (TNF)-a and interleukin 1 b— .
  • MHC major histocompatibility complex
  • MCP monocyte chemoattractant/chemotactic protein
  • TNF tumor necrosis factor
  • interleukin 1 b interleukin 1 b
  • mice The 5XFAD transgenic mouse model (Tg6799) previously described by Oakley et al.— was kindly donated by Dr S. Papacostas. Hemizygous mice were then bred with B6SJLF1/J hybrids (Jackson Laboratories) in order to produce mice exhibiting the 5XFAD phenotype. These mice contain the known FAD mutations APP K670N/M671 L (Swedish), 1716V (Florida), V717I (London) and PS1 L286V and M146L. Standard PCR reactions were carried out to identify the mice expressing all 5 mutations (PCR protocol as indicated by Jackson Laboratories stock #: 006554).
  • All animals were kept in a regular 12-hour light-12 hour dark cycle and were given free access to water and food, under SPF conditions. Animals were separated in cages depending on their age and treatment arm.
  • One group of treated animals was comprised of six 5XFAD animals which were treated with a single dose of the modified C5a receptor agonist, EP67— at 3 months of age for one week; these animals will be referred to as the 3 months EP67 treated group.
  • the second group of treated animals contained eight 5XFAD animals which were treated with four doses of EP67, more specifically for one week at 3 months of age, and another one week after reaching 4 months, 5 months and 6 months of age; these animals will be referred to as the 6 months EP67 treated group.
  • mice were anesthetized and then euthanized using Tribromoethanol (Avertin) though IP injection at a dose of 250 mg/Kg.
  • the whole brain was harvested and the two hemispheres were separated.
  • One hemisphere was used for immunohistochemistry by carrying out overnight 4% PFA fixation followed by wax embedding and the other was kept at -80°C until further processing in order to be used for immunoblotting and immunoassays.
  • Sections were then stained with aqueous 1% Thioflavin S solution (T1892-25G) for a further 5 minutes and finally differentiated in 50% ethanol before been rinsed with distilled water and then mounted using the DAKO Fluorescence Mounting Medium (S3023). Pictures were taken using a Zeiss AXIOIMAGER M2 fluorescence microscope.
  • Amyloid plaques were then quantified via ELISA where A b40 and Ab42 were measured (Novex KMB3481 and KMB3441 ). Whole hemisphere homogenate was processed and lysed according to the manufacturer’s instructions; the outlined procedure was then followed. Absorbance was measured at 450 nm using a microplate reader.
  • Spontaneous alternations scores were obtained for each participating mouse by carrying out the protocol as previously described—. Briefly, at the end of the appropriate treatment for each group of animals and before sacrifice, each mouse was allowed to explore the maze freely for a total of 8 minutes. During each session the sequence of entries was recorded, as well as the total number of entries. The equation used to calculate percentage alternation was: number of triads completed over the number of maximum possible alternations (total number of arms entered-2).
  • Brain homogenates (tissue lysed with RIPA buffer and protease inhibitors followed sonication) were separated via reducing SDS-PAGE and transferred onto PVDF membranes.
  • the membranes were blocked with 5% BSA for one hour at room temperature.
  • the membranes were then incubated overnight at 4°C with the appropriate primary antibody.
  • the specific antibodies were then visualized using the Super Signal West Femto Maximum Sensitivity Substrate (Thermo Fisher 34095) after incubating with the required HRP conjugated secondary antibody for one hour at room temperature. Blots were repeated in triplicates and were visualized using the UVP bio-imaging system.
  • the antibodies used for immunoblotting were against: Synaptophysin (anti-rabbit Abeam ab32127 1/400), NeuN (anti-mouse Millipore MAB377 1/500), GFAP (anti-mouse Sigma G3893 1/500) and F4/80 (anti-rabbit Santa Cruz sc-25830 1/200).
  • the appropriate HRP conjugated secondary antibodies were used: anti-mouse (Santa Cruz SC-2031 1/5,000), anti-rabbit (Santa Cruz SC-2004 1/5000).
  • the Image J image processing program was used to carry out densitometry calculations, while all bands were normalized against a GAPDH loading control (Santa Cruz sc-25778 1/1000), while the same reference sample was used in all westerns to allow cross-gel comparison.
  • Paraffin sections from sagittal brain sections were deparaffinised and hydrated to distilled water. Sections were then blocked with 5% BSA solution in PBS for 1 hour at room temperature and then incubated with the appropriate primary antibody overnight at 4°C. The slides were then washed and incubated with the appropriate secondary antibody for 1 hour at room temperature. Finally, DAPI staining was used to label the cells’ nuclei (Sigma Aldrich D9542) before been mounted using the DAKO Fluorescence Mounting Medium (S3023). Pictures were taken using a Zeiss AXIOIMAGER M2 fluorescence microscope and a Leica TCSL confocal microscope.
  • the primary antibodies used were against: Synaptophysin (anti-rabbit Abeam ab32127 1/200), NeuN (anti-mouse Millipore MAB377 1/300), GFAP (anti-mouse Sigma G3893 1/300), F4/80 (anti-rabbit Santa Cruz sc-25830 1/100) and ELANE (anti-rabbit Abeam ab68672 1/400).
  • Synaptophysin anti-rabbit Abeam ab32127 1/200
  • NeuN anti-mouse Millipore MAB377 1/300
  • GFAP anti-mouse Sigma G3893 1/300
  • F4/80 anti-rabbit Santa Cruz sc-25830 1/100
  • ELANE anti-rabbit Abeam ab68672 1/400
  • the appropriate Invitrogen Alexa Fluor 555 fluorescence secondary antibodies were used: anti-rabbit (A-21428 1/2000) and anti-mouse (A-31570 1/2000).
  • Alzheimer's A. 2016 Alzheimer's disease facts and figures. Alzheimer's & dementia : the journal of the Alzheimer's Association 12, 459-509 (2016).
  • Alzheimer's disease The New England journal of medicine 370, 31 1-321 , doi:10.1056/NEJMoa1312889 (2014).
  • Amyloid-beta peptide decreases glutamate uptake in cultured astrocytes: involvement of oxidative stress and mitogen-activated protein kinase cascades Neuroscience 156, 898-910, doi: 10.1016/j. neuroscience.2008.08.022 (2008).
  • Preferences and options for a given aspect feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features and parameters of the invention.
  • the treatment of Alzheimer’s Disease or Parkinson’s Disease by parenteral administration following a dosage regime of one dose per day for 4 consecutive days followed by 4 dose-free weeks, repeated continually.

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Abstract

La présente invention concerne des procédés et des agents destinés à être utilisés dans le traitement et la prévention de maladies caractérisées par une agrégation de protéines anormale, en particulier dans le traitement de l'ATTR et de la maladie d'Alzheimer.
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WO2023056527A1 (fr) * 2021-10-07 2023-04-13 The University Of Queensland Molécules protéiques et leurs utilisations

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996005226A1 (fr) * 1994-08-16 1996-02-22 Human Genome Sciences, Inc. POLYNUCLEOTIDE CODANT UN RECEPTEUR C5a
WO1996006629A1 (fr) 1994-08-31 1996-03-07 University Of Nebraska Board Of Regents ANALOGUES A TERMINAISON C D'ANAPHYLATOXINE C5a PRESENTANT UNE AFFINITE ELEVEE ET CAPABLES DE PROVOQUER DES REACTIONS BIOLOGIQUES SELECTIVES
WO2012006149A2 (fr) 2010-06-29 2012-01-12 Board Of Regents Of The University Of Nebraska Analogues de c5a et procédés d'utilisation associés
WO2016145365A1 (fr) 2015-03-11 2016-09-15 Board Of Regents Of The University Of Nebraska Analogues à conformation stable de l'agoniste ep67 du fragment c5a à réponse sélective

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996005226A1 (fr) * 1994-08-16 1996-02-22 Human Genome Sciences, Inc. POLYNUCLEOTIDE CODANT UN RECEPTEUR C5a
WO1996006629A1 (fr) 1994-08-31 1996-03-07 University Of Nebraska Board Of Regents ANALOGUES A TERMINAISON C D'ANAPHYLATOXINE C5a PRESENTANT UNE AFFINITE ELEVEE ET CAPABLES DE PROVOQUER DES REACTIONS BIOLOGIQUES SELECTIVES
WO2012006149A2 (fr) 2010-06-29 2012-01-12 Board Of Regents Of The University Of Nebraska Analogues de c5a et procédés d'utilisation associés
WO2016145365A1 (fr) 2015-03-11 2016-09-15 Board Of Regents Of The University Of Nebraska Analogues à conformation stable de l'agoniste ep67 du fragment c5a à réponse sélective

Non-Patent Citations (107)

* Cited by examiner, † Cited by third party
Title
"CCL2 accelerates microglia-mediated Abeta oligomer formation and progression of neurocognitive dysfunction", PLOS ONE, vol. 4, 2009, pages e6197
ALZHEIMER'S, A.: "Alzheimer's disease facts and figures", ALZHEIMER'S & DEMENTIA : THE JOURNAL OF THE ALZHEIMER'S ASSOCIATION, vol. 12, 2016, pages 459 - 509
AMSALEM, H.; KWAN, M.; HAZAN, A.; ZHANG, J.; JONES, R. L.; WHITTLE, W. ET AL.: "Identification of a novel neutrophil population: proangiogenic granulocytes in second-trimester human decidua", J. IMMUNOL., vol. 193, 2014, pages 3070 - 3079
ANDO, Y.; NAKAMURA, M.; ARAKI, S.: "Transthyretin-related familial amyloidotic polyneuropathy", ARCH. NEUROL., vol. 62, 2005, pages 1057 - 1062, XP008179756, DOI: doi:10.1001/archneur.62.7.1057
AUSTYN, J. M.; GORDON, S.: "F4/80, a monoclonal antibody directed specifically against the mouse macrophage", EUR. J. IMMUNOL., vol. 11, 1981, pages 805 - 815
BAIK, S. H. ET AL.: "Migration of neutrophils targeting amyloid plaques in Alzheimer's disease mouse model", NEUROBIOLOGY OF AGING, vol. 35, 2014, pages 1286 - 1292, XP028631623, DOI: doi:10.1016/j.neurobiolaging.2014.01.003
BAIK, S. H.; CHA, M. Y.; HYUN, Y. M.; CHO, H.; HAMZA, B.; KIM, D. K. ET AL.: "Migration of neutrophils targeting amyloid plaques in Alzheimer's disease mouse model", NEUROBIOL. AGING, vol. 35, 2014, pages 1286 - 1292, XP028631623, DOI: doi:10.1016/j.neurobiolaging.2014.01.003
BELAAOUAJ, A.; KIM, K. S.; SHAPIRO, S. D.: "Degradation of outer membrane protein A in Escherichia coli killing by neutrophil elastase", SCIENCE, vol. 289, 2000, pages 1185 - 1188
BODIN, K.; ELLMERICH, S.; KAHAN, M. C.; TENNENT, G. A.; LOESCH, A.; GILBERTSON, J. A. ET AL.: "Antibodies to human serum amyloid P component eliminate visceral amyloid deposits", NATURE, vol. 468, 2010, pages 93 - 97, XP007918427, DOI: doi:10.1038/nature09494
BOZOYAN, L.; DUMAS, A.; PATENAUDE, A.; VALLIERES, L.: "Interleukin-36y is expressed by neutrophils and can activate microglia, but has no role in experimental autoimmune encephalomyelitis", J. NEUROINFLAMMATION, vol. 12, 2015, pages 173
BRENNAN, F. H.; GORDON, R.; LAO, H. W.; BIGGINS, P. J.; TAYLOR, S. M.; FRANKLIN, R. J. ET AL.: "The complement receptor c5ar controls acute inflammation and astrogliosis following spinal cord injury", J. NEUROSCI., vol. 35, 2015, pages 6517 - 6531
BROWN, S. B.; SAVILL, J.: "Phagocytosis triggers macrophage release of Fas ligand and induces apoptosis of bystander leukocytes", J. IMMUNOL., vol. 162, 1999, pages 480 - 485
CAMOUS, L.; ROUMENINA, L.; BIGOT, S.; BRACHEMI, S.; FREMEAUX-BACCHI, V.; LESAVRE, P. ET AL.: "Complement alternative pathway acts as a positive feedback amplification of neutrophil activation", BLOOD, vol. 117, 2011, pages 1340 - 1349
CARLSSON, S. R.; FUKUDA, M.: "Structure of human lysosomal membrane glycoprotein 1. Assignment of disulfide bonds and visualization of its domain arrangement", J. BIOL. CHEM., vol. 264, 1989, pages 20526 - 20531
CAVACO, C. K. ET AL.: "A novel C5a-derived immunobiotic peptide reduces Streptococcus agalactiae colonization through targeted bacterial killing", ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, vol. 57, 2013, pages 5492 - 5499
CHAKRABARTY, P. ET AL.: "Massive gliosis induced by interleukin-6 suppresses Abeta deposition in vivo: evidence against inflammation as a driving force for amyloid deposition", FASEB JOURNAL: OFFICIAL PUBLICATION OF THE FEDERATION OF AMERICAN SOCIETIES FOR EXPERIMENTAL BIOLOGY, vol. 24, 2010, pages 548 - 559
CHAKRABORTY, A.; DE WIT, N. M.; VAN DER FLIER, W. M.; DE VRIES, H. E.: "The blood brain barrier in Alzheimer's disease", VASCULAR PHARMACOLOGY, vol. 89, 2017, pages 12 - 18, XP029920548, DOI: doi:10.1016/j.vph.2016.11.008
DARDIOTIS, E.; KOUTSOU, P.; ZAMBA-PAPANICOLAOU, E.; VONTA, I.; HADJIVASSILIOU, M.; HADJIGEORGIOU, G. ET AL.: "Complement C1 Q polymorphisms modulate onset in familial amyloidotic polyneuropathy TTR Val30Met", J. NEUROL. SCI., vol. 284, 2009, pages 158 - 162
DE FELICE, F. G. ET AL.: "Protection of synapses against Alzheimer's-linked toxins: insulin signaling prevents the pathogenic binding of Abeta oligomers", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 106, 2009, pages 1971 - 1976, XP002580648, DOI: doi:10.1073/PNAS.0809158106
DEWITT, D. A.; PERRY, G.; COHEN, M.; DOLLER, C.; SILVER, J.: "Astrocytes regulate microglial phagocytosis of senile plaque cores of Alzheimer's disease", EXPERIMENTAL NEUROLOGY, vol. 149, 1998, pages 329 - 340
DIEZ-ROUX, G.; LANG, R. A.: "Macrophages induce apoptosis in normal cells in vivo", DEVELOPMENT, vol. 124, 1997, pages 3633 - 3638
DISTLER, U.; KUHAREV, J.; NAVARRO, P.; LEVIN, Y.; SCHILD, H.; TENZER, S.: "Drift time-specific collision energies enable deep-coverage data-independent acquisition proteomics", NAT. METHODS, vol. 11, 2014, pages 167 - 170
DOENS, D.; FERNANDEZ, P. L.: "Microglia receptors and their implications in the response to amyloid β for Alzheimer's disease pathogenesis", JOURNAL OF NEUROINFLAMMATION, vol. 11, 2014, pages 48 - 48, XP021180037, DOI: doi:10.1186/1742-2094-11-48
DOODY, R. S. ET AL.: "A phase 3 trial of semagacestat for treatment of Alzheimer's disease", THE NEW ENGLAND JOURNAL OF MEDICINE, vol. 369, 2013, pages 341 - 350
DOODY, R. S. ET AL.: "Phase 3 trials of solanezumab for mild-to-moderate Alzheimer's disease", THE NEW ENGLAND JOURNAL OF MEDICINE, vol. 370, 2014, pages 311 - 321, XP055183832, DOI: doi:10.1056/NEJMoa1312889
DUYCKAERTS, C.; POTIER, M. C.; DELATOUR, B.: "Alzheimer disease models and human neuropathology: similarities and differences", ACTA NEUROPATHOLOGIES, vol. 115, 2008, pages 5 - 38, XP019562927, DOI: doi:10.1007/s00401-007-0312-8
EGGLETON, P.; GHEBREHIWET, B.; COBURN, J. P.; SASTRY, K. N.; ZANER, K. S.; TAUBER, A. I.: "Characterization of the human neutrophil C1 q receptor and functional effects of free ligand on activated neutrophils", BLOOD, vol. 84, 1994, pages 1640 - 1649
FELLA ELENI ET AL: "Pharmacological Stimulation of Phagocytosis Enhances Amyloid Plaque Clearance; Evidence from a Transgenic Mouse Model of ATTR Neuropathy", FRONTIERS IN MOLECULAR NEUROSCIENCE, FRONTIERS RESEARCH FOUNDATION, CH, vol. 10, 10 May 2017 (2017-05-10), XP009507599, ISSN: 1662-5099, DOI: 10.3389/FNMOL.2017.00138 *
FELLA, E. ET AL.: "Pharmacological Stimulation of Phagocytosis Enhances Amyloid Plaque Clearance; Evidence from a Transgenic Mouse Model of ATTR Neuropathy", FRONTIERS IN MOLECULAR NEUROSCIENCE, vol. 10, 2017
FONSECA, M. I. ET AL.: "Treatment with a C5aR antagonist decreases pathology and enhances behavioral performance in murine models of Alzheimer's disease", JOURNAL OF IMMUNOLOGY, vol. 183, 2009, pages 1375 - 1383, XP002565080, DOI: doi:10.4049/jimmunol.0901005
FONSECA, M. I.; AGER, R. R.; CHU, S. H.; YAZAN, O.; SANDERSON, S. D.; LAFERLA, F. M. ET AL.: "Treatment with a C5aR antagonist decreases pathology and enhances behavioral performance in murine models of Alzheimer's disease", J. IMMUNOL., vol. 183, 2009, pages 1375 - 1383, XP002565080, DOI: doi:10.4049/jimmunol.0901005
FONSECA, M. I.; CHU, S. H.; BERCI, A. M.; BENOIT, M. E.; PETERS, D. G.; KIMURA, Y. ET AL.: "Contribution of complement activation pathways to neuropathology differs among mouse models of Alzheimer's disease", J. NEUROINFLAMMATION, vol. 8, 2011, pages 4, XP021089613, DOI: doi:10.1186/1742-2094-8-4
FREELEY, S.; KEMPER, C.; LE FRIEC, G.: "The ''ins and outs'' of complement-driven immune responses", IMMUNOLOGICAL REVIEWS, vol. 274, 2016, pages 16 - 32
FU, H. ET AL.: "Complement component C3 and complement receptor type 3 contribute to the phagocytosis and clearance of fibrillar Abeta by microglia", GLIA, vol. 60, 2012, pages 993 - 1003
GATE, D.; REZAI-ZADEH, K.; JODRY, D.; RENTSENDORJ, A.; TOWN, T.: "Macrophages in Alzheimer's disease: the blood-borne identity", JOURNAL OF NEURAL TRANSMISSION, vol. 117, 2010, pages 961 - 970, XP019853205
GOTOH, T.; ENDO, M.; OIKE, Y.: "Endoplasmic reticulum stress- related inflammation and cardiovascular diseases", INT. J. INFLAM., 2011, pages 259462
GOURAS, G. K. ET AL.: "Intraneuronal Abeta42 accumulation in human brain", THE AMERICAN JOURNAL OF PATHOLOGY, vol. 156, 2000, pages 15 - 20, XP002258587
GREGORY, C. D.; DEVITT, A.: "The macrophage and the apoptotic cell: an innate immune interaction viewed simplistically?", IMMUNOLOGY, vol. 113, 2004, pages 1 - 14
GUPTA, S.; KAPLAN, M. J.: "The role of neutrophils and NETosis in autoimmune and renal diseases", NAT. REV. NEPHROL., vol. 12, 2016, pages 402 - 413
HAFER-MACKO, C. E.; DYCK, P. J.; KOSKI, C. L.: "Complement activation in acquired and hereditary amyloid neuropathy", J. PERIPHER. NERV. SYST., vol. 5, 2000, pages 131 - 139
HANKE, M. L.; HEIM, C. E.; ANGLE, A.; SANDERSON, S. D.; KIELIAN, T.: "Targeting macrophage activation for the prevention and treatment of Staphylococcus aureus biofilm infections", J. IMMUNOL., vol. 190, 2013, pages 2159 - 2168
HENEKA, M. T.; RODRIGUEZ, J. J.; VERKHRATSKY, A.: "Neuroglia in neurodegeneration", BRAIN RESEARCH REVIEWS, vol. 63, 2010, pages 189 - 211, XP027032611
HERSH, L. B.: "Peptidases, proteases and amyloid β-peptide catabolism", CURR. PHARM. DES., vol. 9, 2003, pages 449 - 454, XP009023732, DOI: doi:10.2174/1381612033391676
HIGGINBOTTOM, A.; CAIN, S. A.; WOODRUFF, T. M.; PROCTOR, L. M.; MADALA, P. K.; TYNDALL, J. D. ET AL.: "Comparative agonist/antagonist responses in mutant human C5a receptors define the ligand binding site", J. BIOL. CHEM., vol. 280, 2005, pages 17831 - 17840
HOHSFIELD, L. A.; HUMPEL, C.: "Migration of blood cells to beta-amyloid plaques in Alzheimer's disease", EXPERIMENTAL GERONTOLOGY, vol. 65, 2015, pages 8 - 15
HOLCOMB, L. ET AL.: "Accelerated Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin 1 transgenes", NATURE MEDICINE, vol. 4, 1998, pages 97 - 100, XP002157991, DOI: doi:10.1038/nm0198-097
HOSOI, A.; SU, Y.; TORIKAI, M.; JONO, H.; ISHIKAWA, D.; SOEJIMA, K. ET AL.: "Novel antibody for the treatment of transthyretin amyloidosis", J. BIOL. CHEM., vol. 291, 2016, pages 25096 - 25105, XP055372389, DOI: doi:10.1074/jbc.M116.738138
KESSENBROCK, K.; DAU, T.; JENNE, D. E.: "Tailor-made inflammation: how neutrophil serine proteases modulate the inflammatory response", J. MOL. MED., vol. 89, 2011, pages 23 - 28, XP019855786, DOI: doi:10.1007/s00109-010-0677-3
KIMA, P. E.; BURLEIGH, B.; ANDREWS, N. W.: "Surface-targeted lysosomal membrane glycoprotein-1 (Lamp-1) enhances lysosome exocytosis and cell invasion by Trypanosoma cruzi", CELL. MICROBIOL., vol. 2, 2000, pages 477 - 486
KOHNO, K.; PALHA, J. A.; MIYAKAWA, K.; SARAIVA, M. J.; ITO, S.; MABUCHI, T. ET AL.: "Analysis of amyloid deposition in a transgenic mouse model of homozygous familial amyloidotic polyneuropathy", AM. J. PATHOL., vol. 150, 1997, pages 1497 - 1508, XP055372532
KOVACH, M. A.; SINGER, B. H.; NEWSTEAD, M. W.; ZENG, X.; MOORE, T. A.; WHITE, E. S. ET AL.: "IL-36y is secreted in microparticles and exosomes by lung macrophages in response to bacteria and bacterial components", J. LEUKOC. BIOL., vol. 100, 2016, pages 413 - 421
LANDLINGER, C. ET AL.: "Active immunization against complement factor C5a: a new therapeutic approach for Alzheimer's disease", JOURNAL OF NEUROINFLAMMATION, vol. 12, 2015, pages 150, XP021228476, DOI: doi:10.1186/s12974-015-0369-6
LEE, H.; WHITFELD, P. L.; MACKAY, C. R.: "Receptors for complement C5a", IMMUNOL. CELL BIOL., vol. 86, 2008, pages 153 - 160, XP009152114, DOI: doi:10.1038/sj.icb.7100166
LIAN, H. ET AL.: "Astrocyte-Microglia Cross Talk through Complement Activation Modulates Amyloid Pathology in Mouse Models of Alzheimer's Disease", THE JOURNAL OF NEUROSCIENCE : THE OFFICIAL JOURNAL OF THE SOCIETY FOR NEUROSCIENCE, vol. 36, 2016, pages 577 - 589
LOBATO, L.; ROCHA, A.: "Transthyretin amyloidosis and the kidney", CLIN. J. AM. SOC. NEPHROL., vol. 7, 2012, pages 1337 - 1346
MACEDO, B.; BATISTA, A. R.; DO AMARAL, J. B.; SARAIVA, M. J.: "Biomarkers in the assessment of therapies for familial amyloidotic polyneuropathy", MOL. MED., vol. 13, 2007, pages 584 - 591
MACLEOD, T.; DOBLE, R.; MCGONAGLE, D.; WASSON, C. W.; ALASE, A.; STACEY, M. ET AL.: "Neutrophil Elastase-mediated proteolysis activates the anti-inflammatory cytokine IL-36 Receptor antagonist", SCI. REP., vol. 6, 2016, pages 24880
MALITO, E.; HULSE, R. E.; TANG, W. J.: "Amyloid β-degrading cryptidases: insulin degrading enzyme, presequence peptidase, and neprilysin", CELL. MOL. LIFE SCI., vol. 65, 2008, pages 2574 - 2585, XP019619993
MAMIYA, T.; UKAI, M.: "Gly(14)]-Humanin improved the learning and memory impairment induced by scopolamine in vivo", BRITISH JOURNAL OF PHARMACOLOGY, vol. 134, 2001, pages 1597 - 1599, XP001153677, DOI: doi:10.1038/sj.bjp.0704429
MANDREKAR-COLUCCI, S.; LANDRETH, G. E.: "Microglia and inflammation in Alzheimer's disease", CNS & NEUROLOGICAL DISORDERS DRUG TARGETS, vol. 9, 2010, pages 156 - 167
MANTHEY, H.D.; WOODRUFF, T.M.; TAYLOR, S.M.; MONK, P.N.: "Complement component 5a (C5a", THE INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY, vol. 41, 2009, pages 2114 - 2117, XP026644018, DOI: doi:10.1016/j.biocel.2009.04.005
MATHERN, D. R.; HEEGER, P. S.: "Molecules great and small: the complement system", CLIN. J. AM. SOC. NEPHROL., vol. 10, 2015, pages 1636 - 1650
MATOS, M.; AUGUSTO, E.; OLIVEIRA, C. R.; AGOSTINHO, P.: "Amyloid-beta peptide decreases glutamate uptake in cultured astrocytes: involvement of oxidative stress and mitogen-activated protein kinase cascades", NEUROSCIENCE, vol. 156, 2008, pages 898 - 910, XP025586161, DOI: doi:10.1016/j.neuroscience.2008.08.022
MATSUMOTO, A.; MATSUMOTO, R.; BABA, H.; FUJIWARA, Y.: "A serine protease in Alzheimer's disease cells cleaves a 16K-peptide with flanking residues upstream to 3-amyloid-N-terminus as natural substrate", NEUROSCI. LETT., vol. 195, 1995, pages 171 - 174
MEYER-HOFFERT, U.; WIEDOW, O.: "Neutrophil serine proteases: mediators of innate immune responses", CURR. OPIN. HEMATOL., vol. 18, 2011, pages 19 - 24, XP055386673, DOI: doi:10.1097/MOH.Ob013e32834115d1
MI, H.; DONG, Q.; MURUGANUJAN, A.; GAUDET, P.; LEWIS, S.; THOMAS, P. D.: "PANTHER version 7: improved phylogenetic trees, orthologs and collaboration with the Gene Ontology Consortium", NUCLEIC ACIDS RES., vol. 38, 2010, pages D204 - D210
MISUMI, Y.; ANDO, Y.; GONGALVES, N. P.; SARAIVA, M. J.: "Fibroblasts endocytose and degrade transthyretin aggregates in transthyretin-related amyloidosis", LAB. INVEST., vol. 93, 2013, pages 911 - 920
MJZIPRE ET AL., J. IMMUNOL., vol. 159, 1997, pages 3230 - 3237
MONK, P. N.; SCOLA, A. M.; MADALA, P.; FAIRLIE, D. P.: "Function, structure and therapeutic potential of complement C5a receptors", BR. J. PHARMACOL., vol. 152, 2007, pages 429 - 448, XP008140616, DOI: doi:10.1038/sj.bjp.0707332
MORGAN, E. L. ET AL.: "Enhancement of in vivo and in vitro immune functions by a conformationally biased, response-selective agonist of human C5a: implications for a novel adjuvant in vaccine design", VACCINE, vol. 28, 2009, pages 463 - 469, XP026791385
MORGAN, E. L.; THOMAN, M. L.; SANDERSON, S. D.; PHILLIPS, J. A.: "A novel adjuvant for vaccine development in the aged", VACCINE, vol. 28, 2010, pages 8275 - 8279, XP027543542
MURRAY, P. J.; WYNN, T. A.: "Protective and pathogenic functions of macrophage subsets", NAT. REV. IMMUNOL., vol. 11, 2011, pages 723 - 737, XP055112087, DOI: doi:10.1038/nri3073
OAKLEY, H. ET AL.: "Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer's disease mutations: potential factors in amyloid plaque formation", THE JOURNAL OF NEUROSCIENCE : THE OFFICIAL JOURNAL OF THE SOCIETY FOR NEUROSCIENCE, vol. 26, 2006, pages 10129 - 10140, XP002688009, DOI: doi:10.1523/JNEUROSCI.1202-06.2006
OGAWA, K.; YAMADA, T.; TSUJIOKA, Y.; TAGUCHI, J.; TAKAHASHI, M.; TSUBOI, Y. ET AL.: "Localization of a novel type trypsin-like serine protease, neurosin, in brain tissues of Alzheimer's disease and Parkinson's disease", PSYCHIATRY CLIN. NEUROSCI., vol. 54, 2000, pages 419 - 426, XP002339998, DOI: doi:10.1046/j.1440-1819.2000.00731.x
OLABARRIA, M.; NORISTANI, H. N.; VERKHRATSKY, A.; RODRIGUEZ, J. J.: "Concomitant astroglial atrophy and astrogliosis in a triple transgenic animal model of Alzheimer's disease", GLIA, vol. 58, 2010, pages 831 - 838
PEARSON-LEARY, J.; MCNAY, E. C.: "Intrahippocampal Administration of Amyloid-β(1-42) Oligomers Acutely Impairs Spatial Working Memory, Insulin Signaling, and Hippocampal Metabolism", JOURNAL OF ALZHEIMER'S DISEASE : JAD, vol. 30, 2012, pages 413 - 422
PETRY, F.; REID, K. B.; LOOS, M.: "Gene expression of the A- and B-chain of mouse C1q in different tissues and the characterization of the recombinant A-chain", J. IMMUNOL., vol. 147, 1991, pages 3988 - 3993
PILLAY, J.; TAK, T.; KAMP, V. M.; KOENDERMAN, L.: "Immune suppression by neutrophils and granulocytic myeloid-derived suppressor cells: similarities and differences", CELL MOL. LIFE SCI., vol. 70, 2013, pages 3813 - 3827, XP035325022, DOI: doi:10.1007/s00018-013-1286-4
PISALYAPUT, K.; TENNER, A. J.: "Complement component C1 q inhibits β-amyloid- and serum amyloid P-induced neurotoxicity via caspase- and calpain-independent mechanisms", J. NEUROCHEM., vol. 104, 2008, pages 696 - 707
REICHWALD, J.; DANNER, S.; WIEDERHOLD, K. H.; STAUFENBIEL, M.: "Expression of complement system components during aging and amyloid deposition in APP transgenic mice", J. NEUROINFLAMMATION, vol. 6, 2009, pages 35, XP021063709, DOI: doi:10.1186/1742-2094-6-35
REITZ, C.: "Alzheimer's disease and the amyloid cascade hypothesis: a critical review", INTERNATIONAL JOURNAL OF ALZHEIMER'S DISEASE, 2012, pages 369808
RICHARDS, D. B.; COOKSON, L. M.; BERGES, A. C.; BARTON, S. V.; LANE, T.; RITTER, J. M. ET AL.: "Therapeutic clearance of amyloid by antibodies to serum amyloid P component", N. ENGL. J. MED., vol. 373, 2015, pages 1106 - 1114, XP055225016, DOI: doi:10.1056/NEJMoa1504942
RODRIGUEZ, J. J.; OLABARRIA, M.; CHVATAL, A.; VERKHRATSKY, A.: "Astroglia in dementia and Alzheimer's disease", CELL DEATH AND DIFFERENTIATION, vol. 16, 2009, pages 378 - 385
RUS, H.; CUDRICI, C.; NICULESCU, F.: "The role of the complement system in innate immunity", IMMUNOLOGIC RESEARCH, vol. 33, 2005, pages 103 - 112
SANDERSON, S. D. ET AL.: "Innate immune induction and influenza protection elicited by a response-selective agonist of human C5a", PLOS ONE, vol. 7, 2012, pages e40303
SANDERSON, S. D.; THOMAN, M. L.; KIS, K.; VIRTS, E. L.; HERRERA, E. B.; WIDMANN, S. ET AL.: "Innate immune induction and influenza protection elicited by a response-selective agonist of human C5a", PLOS ONE, vol. 7, 2012, pages e40303
SARAIVA, M. J.; BIRKEN, S.; COSTA, P. P.; GOODMAN, D. S.: "Amyloid fibril protein in familial amyloidotic polyneuropathy, Portuguese type", J. CLIN. INVEST., vol. 74, 1984, pages 104 - 119
SCHORLEMMER, H. U.; BITTER-SUERMANN, D.; ALLISON, A. C.: "Complement activation by the alternative pathway and macrophage enzyme secretion in the pathogenesis of chronic inflammation", IMMUNOLOGY, vol. 32, 1977, pages 929 - 940
SCHWARTZ, M.; SHECHTER, R.: "Systemic inflammatory cells fight off neurodegenerative disease", NATURE REVIEWS. NEUROLOGY, vol. 6, 2010, pages 405 - 410
SHORT, A. J.; PACZKOWSKI, N. J.; VOGEN, S. M.; SANDERSON, S. D.; TAYLOR, S. M.: "Response-selective C5a agonists: differential effects on neutropenia and hypotension in the rat", BR. J. PHARMACOL., vol. 128, 1999, pages 511 - 514
SIMARD, A. R.; SOULET, D.; GOWING, G.; JULIEN, J. P.; RIVEST, S.: "Bone marrow-derived microglia play a critical role in restricting senile plaque formation in Alzheimer's disease", NEURON, vol. 49, 2006, pages 489 - 502, XP055143620, DOI: doi:10.1016/j.neuron.2006.01.022
SMITH, C. A.; PANGBURN, M. K.; VOGEL, C. W.; MIJLLER-EBERHARD, H. J.: "Molecular architecture of human properdin, a positive regulator of the alternative pathway of complement", J. BIOL. CHEM., vol. 259, 1984, pages 4582 - 4588
SOUSA, M. M.; DU YAN, S.; FERNANDES, R.; GUIMARAES, A.; STERN, D.; SARAIVA, M. J.: "Familial amyloid polyneuropathy: receptor for advanced glycation end products-dependent triggering of neuronal inflammatory and apoptotic pathways", J. NEUROSCI., vol. 21, 2001, pages 7576 - 7586, XP055021746
SPANGENBERG, E. E.; GREEN, K. N.: "Inflammation in Alzheimer's disease: Lessons learned from microglia-depletion models", BRAIN, BEHAVIOR, AND IMMUNITY, vol. 61, 2017, pages 1 - 11, XP029920310, DOI: doi:10.1016/j.bbi.2016.07.003
SUENAGA, G.; IKEDA, T.; KOMOHARA, Y.; TAKAMATSU, K.; KAKUMA, T.; TASAKI, M. ET AL.: "Involvement of macrophages in the pathogenesis of familial amyloid polyneuropathy and efficacy of human ips cell-derived macrophages in its treatment", PLOS ONE, vol. 11, 2016, pages e0163944
SUGAYA, K.; SETO, S.; TSUJIMURA, K.; KOIDE, Y.: "Mobility of late endosomal and lysosomal markers on phagosomes analyzed by fluorescence recovery after photobleaching", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 410, 2011, pages 371 - 375, XP028296506, DOI: doi:10.1016/j.bbrc.2011.06.023
TALUKDAR, S.; OH, D. Y.; BANDYOPADHYAY, G.; LI, D.; XU, J.; MCNELIS, J. ET AL.: "Neutrophils mediate insulin resistance in mice fed a high-fat diet through secreted elastase", NAT. MED., vol. 18, 2012, pages 1407 - 1412
TAYLOR, S. M.; SHERMAN, S. A.; KIRNARSKY, L.; SANDERSON, S. D.: "Development of response-selective agonists of human C5a anaphylatoxin: conformational, biological, and therapeutic considerations", CURRENT MEDICINAL CHEMISTRY, vol. 8, 2001, pages 675 - 684
TEIXEIRA, P. F.; CERCA, F.; SANTOS, S. D.; SARAIVA, M. J.: "Endoplasmic reticulum stress associated with extracellular aggregates", J. BIOL. CHEM., vol. 281, 2006, pages 21998 - 22003
TENNER ANDREA: "THE BIOLOGY OF COMPLEMENT RISK GENES IN ALZHEIMER'S DISEASE", ALZHEIMER'S & DEMENTIA: THE JOURNAL OF THE ALZHEIMER'SASSOCIATION, vol. 13, no. 7, July 2017 (2017-07-01), XP085218742, ISSN: 1552-5260, DOI: 10.1016/J.JALZ.2017.07.486 *
THERIAULT, P.; ELALI, A.; RIVEST, S.: "The dynamics of monocytes and microglia in Alzheimer's disease", ALZHEIMER'S RESEARCH & THERAPY, vol. 7, 2015, pages 41, XP021215784, DOI: doi:10.1186/s13195-015-0125-2
THOMAS, M. P.; WHANGBO, J.; MCCROSSAN, G.; DEUTSCH, A. J.; MARTINOD, K.; WALCH, M. ET AL.: "Leukocyte protease binding to nucleic acids promotes nuclear localization and cleavage of nucleic acid binding proteins", J. IMMUNOL., vol. 192, 2014, pages 5390 - 5397
THOMAS, P. D.; CAMPBELL, M. J.; KEJARIWAL, A.; MI, H.; KARLAK, B.; DAVERMAN, R. ET AL.: "PANTHER: a library of protein families and subfamilies indexed by function", GENOME RES., vol. 13, 2003, pages 2129 - 2141, XP002327041, DOI: doi:10.1101/gr.772403
VOGEN, S. M. ET AL.: "Differential activities of decapeptide agonists of human C5a: the conformational effects of backbone N-methylation", INTERNATIONAL IMMUNOPHARMACOLOGY, vol. 1, 2001, pages 2151 - 2162
WOODRUFF, T. M.; CRANE, J. W.; PROCTOR, L. M.; BULLER, K. M.; SHEK, A. B.; DE VOS, K. ET AL.: "Therapeutic activity of C5a receptor antagonists in a rat model of neurodegeneration", FASEB J., vol. 20, 2006, pages 1407 - 1417, XP002463892, DOI: doi:10.1096/fj.05-5814com
ZENARO, E. ET AL.: "Neutrophils promote Alzheimer's disease-like pathology and cognitive decline via LFA-1 integrin", NATURE MEDICINE, vol. 21, 2015, pages 880 - 886, XP055389839, DOI: doi:10.1038/nm.3913
ZIEGLER-HEITBROCK, L.: "The CD14+ CD16+ blood monocytes: their role in infection and inflammation", JOURNAL OF LEUKOCYTE BIOLOGY, vol. 81, 2007, pages 584 - 592, XP055051239, DOI: doi:10.1189/jlb.0806510

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