DISEASES

Field of the invention

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. Indeed, 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).

Amyloid diseases are associated with an inflammatory element, as discussed below.

Two examples of quite different but medically important amyloid diseases are Hereditary ATTR V30M amyloidosis and Alzheimer’s disease.

Hereditary ATTR V30M amyloidosis

Hereditary 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). Even though there are more than 100 mutations causing ATTR amyloidoses, the first discovered and by far the most common is ATTR V30M, which is due to a substitution of a valine with methionine in position 30 (Val30Met) of the TTR protein (Ando et al., 2005).

There is considerable variability in the age of onset and penetrance of hereditary ATTR V30M in different countries. Epigenetic and genetic factors are believed to contribute to this variability. Complement cascade components have been shown to co-precipitate with amyloid in various forms of amyloidotic neuropathy (Hafer-Macko et al., 2000).

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). Activation of the C5a receptor in the CNS could have a detrimental role leading to neurotoxicity or a neuroprotective role through phagocytosis (Fonseca et al., 2009). However, the effects of C5a receptor activation by the C5a anaphylatoxin in ATTRV30M amyloidosis have not been elucidated yet.

Alzheimer’s Disease

Another important amyloid disease is Alzheimer’s Disease (AD). 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].

The main hallmark of AD is the extracellular deposition of Amyloid-b plaques caused by the proteolytic cleavage of the amyloid precursor protein (APP). 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]. In AD, 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].

Brief summary of the invention

Despite the negative association between inflammation and amyloid disease status, the present inventors have surprisingly found that activation of the C5a receptor has significant beneficial effects, including a reduction in the amount of amyloid deposits in established models of both the neurodegenerative Alzheimer’s disease and systemic ATTR amyloidosis. It is considered therefore that treatment with C5a receptor activators, or agonists, is widely beneficial to the treatment and/or prevention of all amyloid diseases.

In the first instance 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 effect of the activator on amyloid plaques in the brain, and the distal effects of an oral dose of activator could not have been predicted from the positive effects of the activator on ATTR amyloidosis. Taken together however it is considered that the activator will be effective against any protein aggregate, for example any amyloid or protein aggregate since both the ATTR aggregates and the Alzheimer’s aggregates are from different initial pre-cursor proteins. It is also considered that the activator will work on any amyloid in any tissue, whatever the route of administration.

Detailed description of the invention

The invention is as set out in the claims. In a first aspect, 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. For example, in one embodiment 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.

The skilled person is well equipped with the means to identify C5a receptors in other species, for example through the use of annotated databases or through BLAST searches, for instance. For example, the term C5a receptor is intended to include the receptors with at least all of the following accession numbers:

Human genomic sequence: NC_000019.10

Human mRNA sequence: NM_001736.3

Human protein sequence: NP_001727.1

Mouse genomic sequence: NC_000073.6

Mouse mRNA variant 1 sequence: NM_007577.4

Mouse mRNA variant 2 sequence: NM_001 173550.1

Mouse protein variant 1 sequence: NP_031603.2

Mouse protein variant 2 sequence: NP_001 167021.1

The skilled person will understand that the term agonist is used to refer to a substance that activates the particular entity that it agonises. For example, the C5a receptor agonist includes substances or agents that are known to activate the C5a receptor.

By 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 skilled person will understand that 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. In one embodiment it is preferred that 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. to determine whether the agonist results in phagocytes exhibiting G-protein coupled signalling of the protein kinase pathways through mitogen activation with various downstream effects. For example, the skilled person can contact the C5a receptor agonist to a population of phagocytes and determine whether G-protein coupled signalling has occurred.

Other 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. In other examples, for example in humans, amyloid load in the brain can be monitored using PET scanning using the Pittsburgh compound B. In addition, in 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.

Accordingly, 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.

To be clear, there is no reason to suppose that an agonist that is active against the C5a receptor on one cell type will not be active against the C5a receptor on another cell type.

It is considered that at least 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. Accordingly one embodiment 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 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. For example, 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.

If 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.

In preferred embodiments 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.

However, to be clear, 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. For example, neutrophils are considered to likely not be well tolerated in any tissue, for example and in particular brain tissue. Accordingly, where the disease characterised by abnormal protein aggregation results in deposits of abnormal protein aggregates accumulating in brain tissue (for example AD and Parkinson’s), it is considered that it may be preferential, though not essential, that 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. In one embodiment therefore the agonist binds to the C5a receptor. However it will also be appreciated that a given agent can have agonistic properties without actually binding to the corresponding receptor. For example, 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.

By the term 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.

In a preferred embodiment, 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. For example 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.

In a preferred embodiment the C5a receptor agonist is a polypeptide. By polypeptide we include the meaning of a series of any number of amino acids joined by peptide bonds. Thus the term polypeptide is not intended to exclude those polypeptides that may otherwise be termed proteins or peptides. For instance, the term 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. In other embodiments 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

It will be appreciated that the C5a receptor agonist may also be a peptidomimetic compound. Thus, by“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. Such 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.

Similarly, 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.

It will be appreciated that the C5a receptor agonist polypeptide may conveniently be blocked at its N- or C-terminus so as to help reduce susceptibility to exoproteolytic digestion.

Thus, it will be appreciated that 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.

In a preferred embodiment, 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). By 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:

SEQ ID NO: 1

TLQKKIEEIAAKYKHSVVKKCCYDGACVNNDETCEQRAARISLGPRCIKAFTECCWASQ

LRANISHKDMQLGR

The C-terminal domain of human C5a protein is considered to have the following sequence:

SEQ ID NO: 2

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.

Accordingly, in one embodiment 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. In preferred embodiments the agonist is not the full length C5a molecule, as will be apparent to the skilled person.

The skilled person will understand that there is generally some degree of allowable variation within a polypeptide (or nucleic acid) sequence that still allows the polypeptide to function, either to the same degree, to a higher degree, or to a lower degree. Accordingly, in one embodiment 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. By performing tests, for example those outlined above, 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 C5a molecule, for example may consist of or comprise less than 10 amino acids of the C-terminal region of the C5a molecule for example may consist of or comprise less than 9 amino acids of the C-terminal region of the C5a molecule, for example may consist of or comprise less than 8 amino acids of the C-terminal region of the C5a molecule, for example may consist of or comprise less than 7 amino acids of the C-terminal region of the C5a molecule, for example may consist of or comprise less than 6 amino acids of the C-terminal region of the C5a molecule, for example may consist of or comprise less than 5 amino acids of the C-terminal region of the C5a molecule, for example may consist of or comprise less than 4 amino acids of the C-terminal region of the C5a molecule, for example may consist of or comprise less than 3 amino acids of the C-terminal region of the C5a molecule. In a preferred embodiment the fragment of the C-terminal region at least comprises the C- terminal domain, for example at least comprises SEQ ID NO: 2. As discussed above, 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. As discussed above by performing simple routine tests the skilled person will be able to determine whether such a fragment of a protein is suitable for use with the invention.

In particular embodiments, 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)

wherein Xaa is a modified proline residue or a residue substitution for proline, and Xaa2 is leucine or N-methyl leucine,

optionally wherein Xaa is a trifluoromethylated pseudoproline;

optionally wherein Xaa is a pseudoproline;

optionally wherein Xaa is a trifluoromethylated azetidine 2-carboxylic acid and/or homoserine;

optionally wherein Xaa is an oxetanyl-containing peptide; d) A1-Ser-Phe-Lys-A2-A3-A4-A5-A6-A7(SEQ ID NO: 6) wherein:

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)

wherein MeL is N-methyl Leu;

(f) Ty r-S er- P h e- Ly s- P ro- M et- P ro- Le u- ( D-AI a )-Arg ; (SEQ ID NO: 8)

(g) Tyr-Ser-Phe-Lys-Asp-Ala-Pro-Leu-(D-Ala)-Arg; (SEQ ID NO: 9)

(h) Tyr-Ser- Phe-Lys-Asp-Met-Pro-Leu-(D-Ala)-Arg (SEQ ID NO: 10)

(i) Tyr-Ser-Phe-Lys-Asp-Met- Pro-Leu-Gly-Arg; (SEQ ID NO: 1 1 )

(j) Tyr-Ser-Phe-Lys-Asp-Ala-Pro-Leu-Gly-Arg; (SEQ ID NO: 12)

(k) Tyr-Ser-Phe-Lys-Asp-Cys-Pro-Leu-Gly-Arg; (SEQ ID NO: 13)

(L) Tyr-Ser- Phe-Lys-Asp-Met-Pro-Leu-(D-Pro)-Arg; (SEQ ID NO: 14)

(m) Tyr-Ser-Phe-Lys-Asp-Met- Gln-Leu-(D-Ala)-Arg; (SEQ ID NO: 15)

(n) Tyr-Ser-Phe-Lys-Asp-Met-Gln-Leu-Gly-Arg; (SEQ ID NO: 16)

(o) Tyr-Ser-Phe-Lys-Asp-Met-Gln-Pro-Gly-Arg; (SEQ ID NO: 17)

(p) Tyr-Ser-Phe-Lys-Asp-Met-Pro-Leu-Aib-Arg; (SEQ ID NO: 18)

(q) Tyr-Ser-Phe-Lys-Gly- Met-Pro-Leu-Gly-Arg; (SEQ ID NO: 19)

(r) Tyr-Ser-Phe-Lys-Gly-Leu-Leu-Leu-Gly- Arg (SEQ ID NO: 20)

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. In one particular embodiment 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 skilled person will appreciate that 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). The skilled person will be aware of suitable routine tests that can be performed to identify whether a particular agonist is also an anaphylatoxin.

In another preferred embodiments, the C5a receptor agonist is capable of oral absorption. Preferably 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. Preferably 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.

In the same or an additional embodiment 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. In one embodiment 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.

In other embodiments administration of the C5a receptor agonist results in a localised activation of the C5a receptor.

It is considered that in one embodiment the C5a receptor agonist acts, or partly acts, through stimulation of the complement pathway at the sites of the protein aggregate. Accordingly, in one embodiment 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.

Ultimately, 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 term“reduced”, or“reduces” as used herein is intended to encompass both: a) the situation wherein a subject already has some accumulation of abnormal protein aggregates and the agonist

i) reduces or prevents a further increase the amount of the abnormal protein aggregates. In this instance 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

ii) actually reduces the amount of aggregate relative to the amount of aggregate that was present prior to administration of the agonist. Accordingly the term“reduces” also includes in the meaning“removes”. Accordingly, in some embodiments 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

ii) prevents the accumulation of abnormal aggregates to at least some degree.

Any prevention of accumulation of protein aggregates is considered to beneficial. In a preferred embodiment 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.

Accordingly the terms reduces, prevents and removes are to be used interchangeable depending on the context. For example, the term 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 .

In one preferred embodiment, 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.

In one embodiment 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. For example, the skilled person would know that 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. In one embodiment of the invention 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.

In one embodiment 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. For example 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. For example 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%.

The agonist is considered to be useful if it reduces the level protein aggregation by any amount, and in any organ. For example 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%.

The greater the reduction in the level of the aggregated protein the more beneficial the C5a receptor agonist is considered to be.

The C5a receptor agonist is considered to be useful if it results in a reduction in the level of aggregated protein in any tissue. For example, if 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.

In some embodiments, the C5a receptor agonist can reduce the level of aggregated protein in any organ in the body. For example, in one embodiment 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. In one embodiment 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%.

In one embodiment 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. For example, in one embodiment the healthy individual is considered to have no abnormal protein aggregation at all in the relevant tissues. Accordingly in one embodiment 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.

In one embodiment the subject is treated before they develop one or more symptoms of the disease.

In one embodiment the subject is treated before they exhibit accumulations of amyloid deposits.

It is considered that 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.

Accordingly, in a preferred embodiment, 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. The skilled person will understand which diseases are considered to be amyloid diseases. Examples of 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.

In one embodiment the disease is a systemic amyloid disease. A non-exhaustive list of 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.

In other embodiments the disease is not a systemic amyloid disease. In a specific embodiment the disease is not ATTR amyloidosis. In the same or different embodiments the disease is not a disease that involves abnormal protein aggregation in the stomach and/or digestive tract.

For example, in one embodiment the disease is any disease characterised by abnormal protein aggregation, or any amyloid disease, other than ATTR amyloidosis. In a further embodiment the disease is any disease characterised by abnormal protein aggregation, or any amyloid disease, other than a systemic amyloid disease.

In a further specific embodiment, 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. For example, in one embodiment 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.

In one embodiment 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.

In other embodiments where the disease is characterised by abnormal protein aggregate deposits, for example amyloid aggregate deposits, in a particular organ 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.

In one preferred embodiment, the disease is a neurodegenerative disease that is characterised by abnormal protein aggregation, for example amyloid deposits in the brain. Such diseases are considered to include Alzheimer’s Disease, Parkinson’s Disease, motor neuron diseases, polyglutamine disorders, Huntington’s disease, familial amyloid polyneuropathy (FAP).

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.

Other systemic and localised amyloidoses are also considered to be suitable for treatment with the C5a receptor agonist of the present invention.

In one embodiment, 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.

In an alternative embodiment, 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. For example, in one embodiment the disease it not a transmissible disease and is not considered to be a prion disease.

In one embodiment the amyloidoses are not considered to be prion diseases.

In one embodiment, the disease is Alzheimer’s disease and the C5a receptor agonist results in a reduction of the levels of amyloid aggregates in the brain. In a particular embodiment treatment with the C5a receptor agonist results in a reduction of the levels of both ab40 and ab42ίh the brain.

In another particular embodiment 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.

It will be appreciated that the C5a receptor of the invention may be administered by any route. For example, 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.

It is however not considered that the surprising effects of the present invention are restricted to an oral administration route. The skilled person will appreciate that once a particular drug is shown to be effective in treating or preventing a different disease, various formulations can be devised which allow the particular drug to be administered via any route. For example, although it is considered that the demonstration of the beneficial effects of an oral administration of the C5a receptor agonist as described herein in distal parts or organs of the body, for example the brain, it is considered that those beneficial effects will equally be obtained through, for example, topical, or subcutaneous administration. The oral route of administration is simply more convenient for the subject. However, there may be particular diseases characterised by abnormal protein aggregates wherein a different route of administration is preferred, for example a topical or sub cutaneous administration. Accordingly all routes of administration are considered to be useful and are encompassed by the present invention.

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. Depending upon the disorder and subject to be treated, as well as the route of administration, the agents may be administered at varying doses.

Preferably, 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.

Accordingly 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.

It will be appreciated that 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. In a further embodiment 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, for example EP67, 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.

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. For aqueous suspensions and/or elixirs, 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.

For oral and parenteral administration to human subjects, 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.

For veterinary use, 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.

It will be appreciated that the term administration is not restricted to a one time administration. The term 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.

In one embodiment, 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).

In another embodiment the C5a receptor agonist is administered to the subject following the deposition of the abnormal protein aggregate.

In one embodiment 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.

In the same or different embodiment the C5a receptor agonist is administered to the subject periodically. By periodically we include the meaning of regular intervals. For example, in one embodiment 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.

In another embodiment, 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.

As discussed above, in one embodiment 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.

In one embodiment 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 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 1 1 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 1 1 months, or 12 months, between doses, optionally at least 1 year, between doses.

The above period may be repeated any number of times, for example may be repeated for the rest of the life of the subject. For example, in one embodiment 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.

In one embodiment 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. For example, 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.

In one embodiment the subject has not been diagnosed as having a disease characterised by abnormal protein aggregation.

In another embodiment the subject has been diagnosed as having a disease characterised by abnormal protein aggregation.

In one embodiment it is considered that 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.

In another embodiment 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. For example, 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.

It is considered that 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. Early accumulation of amyloid could be detected by amyloid PET scans, for example 5-yearly amyloid PET scans to detect amyloid deposits in the brain and be treated accordingly. In a preferred embodiment, the subject is treated with the C5a receptor agonist prior to detection of amyloid deposits in order to prevent the deposition of amyloid aggregates.

Accordingly, in one embodiment 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.

In one embodiment 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.

It will be appreciated in view of the above disclosure that 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.

It will also be appreciated that 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.

Similarly, 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.

The skilled person will also appreciate that the present invention relates to various 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. For example 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.

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Figure Legends

Figure 1

Amyloid deposition: amyloid plaques were quantified through Thioflavin-S staining. (A) All groups exhibited significant difference from one another, with the PMX53 treated mice having the greatest amount of deposition and the full agonist treated group having the lowest recorded amount n = 6/group, data presented as mean ±1 SD. ^* p < 0.05, ^** p < 0.01 , ^*** p < 0.001. (B) Amyloid plaque in the stomach that stains with Congo red and exhibits apple green birefringence (Bi,ii). The same plaque stains Thioflavin-S positive (Biii) and is composed of human transthyretin (TTR; Biv). The area of co-localization of Thioflavin-S and TTR labelling appears yellow (Bv) and morphometric measurements are carried out with the ImageJ software (Bvi). Scale bar = 150 pm.

Figure 2

Amount of hTTR found in the serum and stomach: (A) TTR in the serum was quantified using enzyme-linked immunosorbent assay (ELISA). Results show that no statistical difference in the amount of circulating TTR was recorded between the four groups of mice n = 4/group, data presented as mean ± 1 SD. (B) hTTR levels in the stomach were measured via immunoblotting. Significant decrease was observed in the mice treated with the two agonist molecules when compared to the untreated control mice n = 6/group. Data presented as mean ± 1 SD. Indicative images from each group shown in (Bi-iv). ^* p < 0.05, ^** p < 0.01 , ^*** p < 0.001.

Figure 3

Expression of phagocytic cell markers in stomach tissue: (A) The expression of CD88 was measured by immunoblotting indicating a decrease in the marker in both the untreated group and the group treated with the PMX53 antagonist, while the two groups treated with the agonists exhibited the greatest level of expression. Similar effects were observed with Neutrophil elastase (B), Ly6G (C) and IL-36g (D). Immunoblots for the macrophage specific markers F4/80 (E) and CD68 (F) however indicate their overexpression in the group of mice treated with the modified agonist molecule EP67, while the group treated with the PMX53 antagonist exhibited the lowest levels of expression for both markers n = 6/group data presented as mean ± 1 SD. ^* p < 0.05, ^** p

< 0.01 , ^*** p < 0.001.

Figure 4

Amyloid plaque infiltration by macrophages and neutrophils: serial sections stomach sections from mice representing the four groups of mice were stained with Thioflavin-S, a-TTR, the pan-macrophage marker a-CD68 and the neutrophil marker a-Neutrophil elastase (ELANE). Immunofluorescence on the stomach tissue from the untreated mouse indicates the complete absence of neutrophils from the plaque, while there is some co-expression with CD68 (A). A similar pattern was observed with the mouse treated with the PMX53 antagonist molecule (B), while complete co-localization with both CD68 and ELANE was observed in the mouse treated with the full agonist molecule (C). Immunofluorescence on the mouse treated with the modified agonist EP67 however revealed complete plaque co-localization with CD68 and the complete absence of ELANE from the region (D). Scale bar = 75 pm.

Figure 5

Expression of complement markers: the expression of the classical complement pathway protein C1 q was measured by immunoblotting in stomach tissues of all the animals from the four groups of mice (A). The group treated with the full agonist molecule exhibited the highest levels of C1q which was statistically significant when compared to the rest of the groups, while the mice with the modified agonist also exhibited a significant increase when compared to the untreated control group. Similar effects were observed via immunofluorescence. Properdin, the alternative complement pathway marker, also presented an analogous pattern so that the group of mice treated with the two agonists also expressed the greatest amount of the protein (B). n = 6/group, data presented as mean ± 1 SD. B&D Scale bar = 150 pm. ^* p < 0.05, ^** p < 0.01 , ^*** p < 0.001.

Figure 6

Expression of stress markers: the expression of BiP was measured by immunoblotting

(A) in the stomach tissues of all animals from the four groups. While the animals from the groups treated with the two agonists and the untreated group displayed comparable levels of BiP, the group treated with the PMX53 antagonist exhibited significantly greater amounts of the cellular stress marker. Comparable effects were observed via immunostaining. Similarly, immunoblotting using the apoptotic marker Caspase-3 displayed a significantly increased expression in the group of mice treated with PMX53

(B). n = 6/group, data presented as mean ± 1 SD. B&D Scale bar = 150 pm. ^* p < 0.05,

^** p < 0.01 , ^*** p < 0.001.

Figure 7

Amyloid plaque co-localization with the lysosomal marker: immunofluorescence of serial stomach sections from the group treated with the full agonist molecule (A) and the PMX53 antagonist (B) show the complete co-expression of the amyloid plaque with Lamp-1 (Ai) in the mouse treated with the agonist as opposed to the antagonist treated mouse which presents complete absence of the lysosomal marker in the vicinity of the plaque (Bi). Sections were co-stained with Thioflavin-S, a-Lamp-1 and a-CD68 (Ai-iv and Bi-iv) and Thioflavin-S, a-hTTR and a-ELANE (Av-viii and Bv-viii). Scale bar = 75 ±m.

Figure 8

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.

Figure 9

Phagocytic cell and complement related markers obtained through LC-MS/MS.

Figure 10

Cellular stress and apoptosis related markers obtained through LC-MS/MS Figure 1 1

Proteins involved with macrophage activation Figure 12

Proteins involved with complement activation Figure 13

Proteins involved with apoptosis Figure 14

Proteins involved with inflammation mediated by cytokines and chemokines (1/2)

Figure 15

Proteins involved with peptidase Activity (1/4)

Figure 16

Amyloid deposition (a) 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. Similarly an increase is observed between the 3 month old 5XFAD mice that received a single dose of EP67 ( vii-ix ) and the 6 month old mice which received four separate doses of EP67 ( xvi-xviii ). Scale bar: 300pm.

Elisa for Ab (b,c) Homogenates from the left cerebral hemispheres of wild type, 5XFAD control and EP67 treated 5XFAD mice at ages 3 and 6 months were used to measure the amount of Ab40 ( b ) and Ab42 (c). The amounts of both peptides appear to decrease following treatment with EP67 at both ages. Wild animals of both 3 months and 6 months of age were included in the analysis but did not register a reading for Ab40 or Ab42 and were thus excluded from the graph. n=6 for each 3 month old group and n=8 for each 6 month old group. Data presented as mean ± 1SD.

Figure 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 ). n=6 for each of the 3 month old 5XFAD control group and EP67 treated 5XFAD group of mice, n=8 for each of the 6 month old 5XFAD control group and EP67 treated 5XFAD group of mice , n=4 for each age group of wild mice. Data presented as mean ± 1SD.

Figure 18

Evaluation of neuronal markers. 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 synaptic marker synaptophysin and Thioflavin-S (a). Representative images reveal a dramatic decrease in the expression of the marker in 5XFAD control animals at both 3 and 6 months of age (aii & av) when compared to wild mice ( ai&aiv ). 5XFAD animals treated with either 1 dose of EP67 or 4 doses of EP67 display similar levels of synaptophysin to wild mice ( aiii&avi ) and well above those of 5XFAD control mice. These observations were also corroborated via immunoblotting ( b ). Sections were labelled with antibody against the neuronal marker NeuN and co-stained with Thioflavin-S (c). Again a severe decrease in the expression of the marker was observed in the 5XFAD control mice at 3 months and 6 months of age (c/7 & cv) when compared to wild mice (c/ & civ). EP67 treated 5XFAD mice with either one or four doses exhibit similar levels of NeuN as the wild mice (ciii & cvi). Immunoblotting was again used to corroborate these observations ( d ). Scale bar: 150pm. n=6 for each of the 3 month old 5XFAD control and EP67 treated 5XFAD groups, n=8 for each of the 6 month old 5XFAD and EP67 treated 5XFAD groups, n=4 for each of the age groups of wild mice. Data presented as mean ± 1 SD.

Figure 19

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 ). Labelling with an antibody against the mouse specific microglia and macrophage marker F4/80 and Thioflavin-S revealed no staining in wild mice ( ci&civ ). Some staining was observed around plaques in 5XFAD control mice at 3 and 6 month old of age (c/7 & cv) but even greater staining was observed in EP67 treated 5XFAD mice ( ciii&cvi ) with close proximity between microglia and amyloid (cvi inset). Similar results were also obtained via immunoblotting ( d ). Scale bar: 150pm. n=6 for each of the 3 month old 5XFAD control and EP67 treated 5XFAD groups, n=8 for each of the 6 month old 5XFAD and EP67 treated 5XFAD groups, n=4 for each of the age groups of wild mice. Data presented as mean ± 1 SD.

Figure 20

Neutrophils in the brain. Sagittal cortex sections from 6 month old 5XFAD control and EP67 treated 5XFAD mice were co-stained with Thioflavin-S and an antibody against the neutrophil marker ELANE. The section from the 5XFAD control mice exhibits weak co- localization of the neutrophil marker with the Thioflavin-S positive plaques (a-c) The sections obtained from the 5XFAD mouse treated with EP67 however display a higher expression of ELANE and co-localisation with the plaques ( d-f ). Scale bar: 40pm.

Figure 21

Amino acid sequence and structure of C5a. Human 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. Manthey, H.D., Woodruff, T.M., Taylor, S.M., and Monk, P.N. (2009). Complement component 5a (C5a). The international journal of biochemistry & cell biology 41 , 2114-2117.

Examples

Example 1

MATERIALS AND METHODS

Animals and Tissue Handling

The previously published mouse model of ATTR V30M neuropathy (Kohno et al., 1997) was kindly donated by Dr. M. Saraiva. These animals are knockouts for murine TTR and have been bred to carry the human V30M mutated cDNA in the form of a transgene in a homozygous state (mTTR-/- hTTRMet30+/+ ). Real time PCR was used to ensure that all animals included in the experiments had the same copy number of human TTR transgenes. All animals used for the experiments were 13-14 months old since at this age amyloid deposits are well established.

All animals were kept in a regular 12-h light-12 h dark cycle and were given free access to water and food, under SPF conditions. Animals were separated in cages depending on the molecule they were treated with. Four groups of animals were included for study; one group was treated with PMX53 (kindly provided by Cephalon USA), one group with the full C5a receptor agonist molecule (purchased from Anaspec), one group with the modified C5a receptor agonist, EP67 (Sanderson et al., 2012) and finally there was a control group. Each group was comprised of six animals while the treatment period lasted for 1 week, during which each molecule was added to the animals’ water source at 20 pg/ml. The control group animals only received water without the addition of any other compound. All animal involving experiments were carried out in accordance to the 86/609/EEC Directive. Also, a project license was obtained from the Cyprus Veterinary Services approving the project and methodology (License Number: CY/EXP/P.L6/2010).

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.

Genotypinq

Animals 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.

Amyloid Plaque Visualization and Quantification Thioflavin S staining combined with TTR immunofluorescence were used to identify TTR specific amyloid deposits in paraffin sections obtained from stomach tissue. Paraffin sections were deparaffinized and hydrated to distilled water. Sections were then stained with Mayer’s hemeatoxylin for 5 min, washed further with distilled water and then stained with aqueous 1% Thioflavin S solution (T1892-25G) for a further 5 min and finally differentiated in 50% ethanol before been rinsed with distilled water and then mounted using the DAKO Fluorescence Mounting Medium (S3023). Thioflavin S positive deposits were further confirmed to be amyloid by Congo Red (Figures 1 Bi,ii). 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 Elisa Method

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.

Western Blots and Densitometry

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 (Santa Cruz SC-2004 1/5000).

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).

Immunohistochemistry

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).

Mass Spectrometry-Based Proteomics

Frozen stomach tissue samples from three animals from two groups of animals (full agonist treated and PMX53 treated) 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. The supernatant was collected and proteins were precipitated in tenfold excess volume of ice-cold acetone overnight at -20 _° C and subsequently resuspended in urea buffer (8 M urea, 50 mM ammonium bicarbonate). Protein concentration was determined using BCA protein assay. For each sample, 100 pg of protein was transferred to a new tube, reduced with DTT (10 mM final concentration) for 30 min at 60 _° C and alkylated with iodoacetamide (15 mM final concentration) for 15 min in dark at room temperature followed by fourfold dilution in 50 mM ammonium bicarbonate. 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.

Statistical Analyses

Statistical analysis was performed using GraphPad Prism version 5.00 for Windows (GraphPad software, San Diego, CA, USA) where one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was carried out. Using this information, graphical charts representing the data were prepared.

RESULTS

We have administered two C5a receptor agonists, a full C5a receptor agonist and EP67 (a response-selective C5a receptor agonist deprived of C5a-like anaphylatoxin activity), and a C5a receptor antagonist (PMX53) and examined disease phenotype in ATTR V30M mice after 1 week. We show that treatment with the C5a receptor agonists significantly ameliorated amyloid deposition while C5a receptor antagonist PMX53 exacerbated amyloid deposits. We have also carried out mass spectrometry-based proteomic analysis, comparing the proteome of animals with the highest amount of amyloid and the lowest amount of amyloid. This analysis has shown substantial phagocytic cell activation, as well as the increased expression of proteolytic peptidases accompanying the reduction in amyloid deposition.

Amyloid Deposition in the Stomach

Following administration of all three agents for 1 week, all animals were sacrificed (including untreated, age-matched control hTTRV30M animals) and amyloid deposition was examined by combined Thioflavin S staining and TTR immunofluorescence (Figure 1A). There was a 160% increase in amyloid load following the administration of PMX53 for a week when compared to the control hTTRV30M mice. Administration of the C5a receptor agonist EP67 resulted in a 42% decrease in deposited amyloid. Further amyloid reduction was recorded following administration of the full receptor agonist (65%). hTTR Levels in Serum and Stomach

The levels of human V30M TTR were measured in the serum of all mice participating in the study using the ELISA at the end of the treatment period. Our results indicate that the amount of hTTR found in the serum remains unaffected for all four groups (Figure 2A). The amount of pre-fibrillar hTTR was measured in stomach tissue of all animals (Figure 2B). Our results indicated significantly less hTTR between the two groups treated with the agonists and the control animals. The PMX53 group did not significantly differ from the control group.

Phagocytic Cell Markers in Stomach Tissue

The PMX53 molecule is a known C5a receptor (CD88) inhibitor, while the other two molecules used are agonists for the receptor. Even though C5a receptors (C5R1 , CD88) are ubiquitously expressed on a variety of cells, they are most prominently expressed on the surface of neutrophils and macrophages (Monk et al., 2007), therefore, identifying markers for these two cell types were used to establish their presence in the experimental animals. 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 (ELANE) 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). Our results show that ELANE is highest in the group of mice treated with the full agonist molecule, which retains its anaphylactic properties, while it is lower in the group treated with the agonist EP67 which activates C5aR receptors on macrophages but less so on neutrophils (Figure 3B). The PMX53 treated animals were found to express the lowest levels of ELANE even though the change was not statistically significant when compared to the untreated control group.

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 (IL-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,

1981 ), 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). However, the mice treated with EP67 display the greatest levels of macrophages, even when compared to the group treated with the full agonist. Results yielded from the liquid chromatography— tandem mass spectrometry (LC- MS/MS) analysis comparison between the group with the highest amyloid deposition (PMX53 treated) and the group with the lowest amount of amyloid (full agonist treated) revealed a number of macrophage and neutrophil related markers which were highly expressed in the animals with the lowest amount of amyloid (Figure 9).

Immunofluorescence with Neutrophil and Macrophage Markers

In order to examine the co-expression of the macrophage and neutrophil markers with the amyloid plaques serial deparaffinized stomach sections were immunostained with antibodies specific for CD68, neutrophil elastase/ELANE, hTTR and Thioflavin-S. The untreated group displays minimal co-localization of 0CD68 with the Thioflavin- S/hTTR positive plaques, while there is no co-localization with a-ELANE (Figure 4A).

The sections from the mice treated with the C5a antagonist PMX53 exhibit an even lower level of 0CD68 co-localization, probably indicative of the fact that there’s a decreased recruitment due to inhibition of the receptor (Figure 4B).

In the group treated with the full agonist there was complete plaque co-localization with both a-CD68 and a-ELANE (Figure 4C). The EP67 treated group however exhibited mainly co-localization with 0CD68 positivity with a much less neutrophil recruitment (Figure 4D).

Complement Markers in Stomach Tissue The complement system has been previously shown to be involved with amyloidogenesis and the pathogenesis of the disease. Therefore, markers for both the classical and alternative complement system were analyzed using immunoblotting.

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. On the contrary, 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.

Our results indicate the greatest amount of properdin in the animals treated with the full agonist, followed by the EP67 treated group (Figure 5B). The PMX53 treated mice express the same amount of properdin as the control group.

These results were also corroborated through the LC-MS/MS analysis, where the complement cascade proteins, along with properdin, were found to be higher in the group of mice treated with the full agonist molecule when compared to the group of mice treated with the PMX53 molecule (Figure 9).

Stress and Apoptosis Markers in Stomach Tissue

The presence of extracellular amyloid deposits and pre-fibrillar hTTR species have been shown to increase endoplasmic reticulum stress through the activation of the classical unfolded protein response pathways in tissues not specialized in hTTR synthesis (Teixeira et al., 2006; Macedo et al., 2007). Our results indicate that the 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).

Immunostaining of nerve biopsies from hereditary V30M patients carried out with an activated caspase-3 specific antibody has shown that expression of this apoptotic marker increases as the disease progresses (Sousa et al., 2001 ). We have also observed a significant increase in Caspase-3 in the group with the highest amyloid load (PMX53 treated) as compared to the other groups, even though no significant difference was recorded between the agonists treated groups and the control group (Figure 6B).

The LC-MS/MS analysis has revealed a great number of ER related stress markers which appear greatly increased in the PMX53 treated group of mice, as well as a number of Caspases and apoptotic markers (Figure 10).

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.,

2000). During the formation and maturation of the phagosome, 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. On the contrary, the PMX53 treated mouse does not exhibit any co-localization with ELANE, CD68, or LAMP- 1 (Figure 7A). On the contrary, sections from an animal treated with PMX53 display no plaque co-localization with Lamp-1 (Figure 7B).

Liquid Chromatography-Tandem Mass Spectrometry Analysis

Considering that the animals treated with the full agonist molecule exhibited the least amount of deposited amyloid, and the animals treated with the PMX53 molecule had the highest amount of amyloid recorded, these two groups were compared using label-free mass spectrometry-based proteomic approach. Overall, a total of 3154 quantifiable proteins were identified. The expression of most of the markers examined using immunoblotting, as well as other related markers, was also confirmed through this technique (Figure 9 and 10). Furthermore, the data obtained through the LC-MS/MS technique was further analyzed using the Panther tool in order to identify clusters of proteins involved with specific functions in the two extreme groups of mice (Thomas et al., 2003; Mi et al., 2010). The most relevant results from this analysis have been summarized in Figure 8, while the specific proteins can be found in Figures 11-15. Also, the full list of obtained proteins along with their confidence scores can be found in Supplementary Data Sheet 1. A greater number of proteins involved with macrophage and complement activation were found to be highly expressed in the mice treated with the full agonist molecule. Similarly, a greater number of proteins associated with inflammation mediated by cytokines and chemokines, as well as peptidases were also found in the group treated with the full agonist molecule. However, the animals treated with PMX53 appeared to express a greater number of apoptosis related proteins when compared to the full agonist treated group.

DISCUSSION

Currently there is a variety of approaches, either in the clinic or in clinical trials, for treating ATTR V30M amyloidotic neuropathy; liver transplantation, TTR stabilizers (Tafamidis, Diflunisal), inhibitors of TTR translation (anti-sense oligonucleotides, silencing RNAs), drugs that interfere with amyloid dynamics at the tissue level (doxycycline/TUDCA). There is however an unmet need that arises from the fact that already formed amyloid deposits continue to form foci for further deposition of normal TTR giving rise to further organ damage such as the heart and the kidneys. In addition, 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. More recently, 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).

Our results show that while administering the PMX53 compound results in a 160% increase of fibrillar amyloid deposition (both the full agonist and EP67 result in the decrease of amyloid deposition by 65% and 42%, respectively) following 1 week of oral administration through the animals’ drinking water. Therefore, inhibition of the C5a receptor (CD88) results in an increase in amyloid deposition, whereas enhancing the action of the receptor induces a considerable decrease. This decrease is even more substantial following the administration of the full agonist molecule which recruits both macrophages and neutrophils (Baik et al., 2014).

The fact that the amount of circulating hTTR protein in the serum remains unchanged in all four groups is expected since liver production of TTR is not expected to be affected, while the changes in tissue handling of TTR as a result of the administration of the three types of molecules for only a week would be too short to influence serum levels. However, there was a significant decrease in the amount of prefibrillar hTTR detected in the stomach tissue of animals receiving the C5 receptor agonists when compared to control presumably due to increased phagocytosis of prefibrillar hTTR. 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.

The marked decrease in amyloid load observed with the animals treated with the C5a receptor agonists is accompanied by a remarkable increase in neutrophil and macrophage markers. EP67 predominantly targets the macrophage populations, while the full agonist, which also targets neutrophils, produces significantly more IL-36y perhaps indicative of phagocytic capacity (Sanderson et al., 2012; Hanke et al., 2013). The extra 23% decrease in amyloid observed in the full agonist treated mice is probably a direct effect of neutrophil activation and recruitment. Immunofluorescence labeling of the plaques shows no co-localization with neutrophils except in mice treated with the full C5a receptor agonist (Figure 4C). Interestingly, this is the group which exemplifies the greatest reduction in amyloid load, indicating a capacity of neutrophils to clear amyloid (Baik et al., 2014). Moreover, 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. Additionally, 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. Therefore, 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 ). Furthermore, 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 PMX53 treated mice however did not appear to produce less properdin than the control group of animals, probably a response to the increased amyloid deposition where several complement components co-localize (Reichwald et al., 2009). This evidence is also corroborated by the LC-MS/MS analysis, which indicates the massive up-surge of both the classical and alternative complement cascade in relation to the PMX53 treated group (Figure 9).

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. Evidence from work carried out on amyloid b peptides in Alzheimer’s brains, identifies peptidase and protease mediated cleavage as a possible clearance mechanism in the catabolism of amyloid plaques (Matsumoto et al., 1995; Ogawa et al., 2000; Hersh, 2003; Malito et al., 2008). 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. We present data, via immunoblotting and LC-MS/MS analysis demonstrating the significant increase of IL-36y in animals treated with the C5a receptor agonists vs. the animals treated with the PMX53 inhibitor.

Both apoptosis and cellular stress have been shown to increase along with extracellular TTR amyloid deposition (Macedo et al., 2007). We observe both by immunoblotting and LC-MS/MS analysis, that the groups of animals treated with the C5a receptor agonists do not have lower levels of apoptosis or cellular stress when compared to the control despite a reduction in amyloid load. This is perhaps partly due to the presence of macrophages and neutrophils per se since both are involved in inflammatory pathways which do evoke the ER stress response pathway (Gotoh et ai, 201 1 ). Similarly, 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).

In summary our data show that while inhibition of the C5a receptor results in the significant increase of amyloid load, activation of the C5a receptor results in a substantial reduction of amyloid deposits. Measurable effects were seen with only 1 week oral intake and with no visible side-effects on the mice. The full C5a receptor agonist molecule, retaining its full ability to activate neutrophils, had a greater impact in reducing the amyloid load.

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Example 2 - An orally administered C5aR agonist enhances phagocytosis of Ab amyloid and preserves memory in the 5XFAD animal model of Alzheimer disease

EP67 reduces amyloid plaque formation

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— . Particularly, 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. In order to investigate whether administration of EP67 affects amyloid deposition in the brain, 14 (7 male and 7 female) three month old 5XFAD mice (previously generated and characterised by Oakley et al.,) were given 20 pg/ml of EP67 in their drinking water for one week. At the same time 14 age/sex matched 5XFAD mice and 8 wild (B6SJLF1/J) animals were kept as controls on plain drinking water. The time point of three months was chosen since cerebral Ab40 and Ab42 levels were previously shown to increase exponentially in 5XFAD mice after the age of 2 months—. Six 5XFAD EP67 treated mice were sacrificed following their one week treatment at the age point of 3 months. Similarly 6 control 5XFAD mice were also sacrificed. Considering the steep increase in cerebral Ab peptides in the 5XFAD mice, the remaining 8 animals were treated with EP67 for one week at the end of every month until they reached their 6 ^th month of age, when they received their final treatment. In total the animals sacrificed at the age of 6 months and one week were given 4 treatments of EP67, while 8 control 5XFAD mice maintained on plain drinking water were also sacrificed. During this time wild type B6SJLF1/J mice were also sacrificed at the age of 3 and 6 months (n=4 for each age point, 2 male and 2 female).

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—. At the 6 month age point 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. It should be noted that the 5XFAD mice exhibit Ab accumulation by 3 months of age and as the mice age this is predominantly Ab42 rather than Ab40— . In regards to 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.

These observations suggest a cumulative effect in the action of EP67 where by 6 months of age the levels of Ab42 were nearly halved and Ab40 was decreased by approximately 25%.

EP67 protects against short-term spatial working memory loss

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. Our results indicate a cognitive decline in 5XFAD control mice when compared to the wild mice and this decrease appears to intensify with age (Fig 17a). EP67 treated 5XFAD mice exhibit a marked preservation in short-ferm spatial working memory when compared to their untreated control 5XFAD counterparts. It is interesting to note that the mice treated with EP67 achieved similar results as the wild mice. In addition, the total number of arm entries was comparable for all groups of mice signifying no impairment in motor function which would have affected the mice’ explorative ability (Fig 17c). Therefore our results suggest that EP67 appears to protect against Ab induced hippocampal associated short- term spatial memory loss.

EP67 prevents synaptic and neuronal loss

Evidence from human brains indicates that amyloidogenic Ab species have been found to accumulate within neurons—. In the specific model used here intraneuronal Ab accumulation has been shown as early as at 1.5 months of age—. This early accumulation of neurotoxic Ab has been hypothesized to be one of the initial triggers leading to neurodegeneration 22. In order to investigate this neuronal loss we used antibodies against the synaptic marker synaptophysin (Fig 18a and b) and the post mitotic neuronal marker NeuN (Fig 18c and d). Immunohistochemical evaluation for synaptophysin of wild type, control 5XFAD and EP67 treated 5XFAD mice revealed that control 5XFAD mice exhibit severe decrease in synaptophysin staining at both age points (Fig 18a ii,v) when compared to wild type and 5XFAD animals treated with EP67 (Fig 18a iii,vi and 18a i,iv). Further investigation with immunoblotting confirmed these findings (Fig 18b). It is noteworthy that EP67 treated 5XFAD animals compared favourably with wild animals.

Similar results were obtained with neuronal antibody against NeuN (Fig 18c and d) where both the immunohistochemical evaluation and subsequent quantification with immunoblotting exhibited a dramatic decrease in NeuN expression in the 5XFAD animals at both age points when compared to wild and EP67 treated 5XFAD mice. Again EP67 treated 5XFAD mice appeared to possess similar levels of expression of the neuronal marker as the wild mice.

EP67 reduces astrocvtosis

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). Immunohistochemical analysis of 3 month old 5XFAD mice double stained with Thioflavin-S and an antibody against GFAP revealed a great number of astrocytes surrounding amyloid plaques (Fig 19a ii) while very limited staining was observed in 3 month old EP67 treated 5XFAD sections (Fig 19a iii). Similarly, in 6 month old animals the same pattern was again observed between untreated and treated 5XFAD animals (Fig 19a v and vi respectively). GFAP expression in the EP67 5XFAD animals did increase in the older animals as compared to their 3 month old counterparts. No staining was observed in wild type animals at the 3 month old or 6 month old age point (Fig 19a i and iv respectively). Quantification of GFAP through immunoblotting corroborated these observations (Fig 19b). Both the immunohistochemical and immunoblotting analysis showed that expression of the astrocyte marker GFAP is significantly decreased following treatment with EP67.

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—.

We carried out analysis macrophage evaluation through the mouse specific marker F4/80 (Fig 19c and d), as well as Iba1 and CD68 (results not shown). Comparable results were obtained with both macrophage and microglia specific markers. We were able to observe a general increase in the expression of F4/80 in control 5XFAD mice at both age points when compared to the wild type controls; however an even greater increase was observed in the EP67 treated 5XFAD mice (Fig 19d). While some co- localisation of F4/80 with Thioflavin-S was observed in the 5XFAD animals (Fig 19c ii and iii), in the EP67 we observed amyloid material almost completely surrounded by F4/80 positive microglia-like structures (Fig 19c vi - white circle). Our observations indicate a general increase in the presence of phagocytic cells.

EP67 recruits neutrophils to amyloid plaques

Considering that 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).

Discussion

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 .

According to the amyloid hypothesis of AD the deposition of prefibrillar and fibrillar Ab peptide sets off pathogenic cascades of neuroinflammation and neurodegeration that lead to synaptic and neuronal loss and cognitive decline. Various approaches to reduce amyloid load by reducing prefibrillar production of ab peptide (secretase inhibition) or enhance amyloid clearance (anti-ab peptide antibody mediated clearance) has proven unsuccessful in clinical trials——.

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 . In general activated astrocytes are heavily implicated in the inflammatory response observed in AD through the secretion of cytokines and proinflammatory factors—.

The central nervous system (CNS) 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. In AD brains however, activated 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— . The actual effect of microglia in response to brain amyloidosis however is highly controversial. Microglia have been proposed to have a very limited capacity in actually carrying out phagocytosis of Ab fragments—. However, other reports have shown that microglia not only enhance the clearance of amyloid plaques but also induce the remodelling of the plaques so that they may be broken up ^{2 1} . In spite of the conflicting evidence on the particular function of these cells, it is generally accepted that microglia become activated following Ab extracellular deposition and subsequent aggregation ⁴² .

Our data show that microglia/macrophage specific markers increase following treatment with the C5a receptor agonist which would indicate that the decrease in amyloidogenic peptides may be the result of increased phagocytosis incurred by both microglia and infiltrating phagocytes. The fact that neutrophils have also been observed to co-localise with amyloid plaques indicates a contribution from peripheral leukocytes in breaking up the amyloid. A neutrophil/monocyte marker (Ly6C/G) has been found on cells migrating towards b-amyloid plaques in an AD mouse model—. Materials and Methods

Animals and tissue handling

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. During each treatment cycle EP67 was added to the animals’ water source at 20□g/ml. Furthermore, two groups of untreated control 5XFAD animals were also kept, the first group was comprised of six animals sacrificed at 3 months old and will be referred to as the 3 month 5XFAD group and the second group was comprised of eight 6 month old 5XFAD untreated animals and will be referred to as the 6 month 5XFAD group. The 5XFAD control group animals only received water without the addition of any other compound. In order to allow for proper data interpretation four 3 month and four 6 month old wild type B6SJLF1/J mice were also included.

All animal experiments were carried out in accordance to the 86/609/EEC Directive. Also, a project license was obtained from the Cyprus Veterinary Services approving the project and methodology (License Number: CY/EXP/P.L6/2010).

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.

Amyloid plaque visualisation and quantification

Thioflavin S staining combined with P-Amyloid immunofluorescence were used to identify Ab derived amyloid deposits in paraffin sections obtained from sagittal brain sections. Paraffin sections were deparaffinised and hydrated to distilled water. Sections were blocked with 5% BSA solution in PBS for 1 hour at room temperature and then incubated with the b-Amyloid antibody (Santa Cruz sc-28365 1/500) overnight at 4°C. The slides were then washed and incubated with an anti-mouse Alexa Fluor 555 secondary antibody for 1 hour at room temperature (A-31570). Finally, DAPI staining was used to label the cells’ nuclei (Sigma Aldrich D9542). 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 alternation Y-maze

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).

Immunoblotting

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.

Immunohistochemistry

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). The appropriate Invitrogen Alexa Fluor 555 fluorescence secondary antibodies were used: anti-rabbit (A-21428 1/2000) and anti-mouse (A-31570 1/2000).

Statistical analyses

Statistical analysis was performed using GraphPad Prism version 5.00 for Windows (GraphPad software, San Diego California USA) where one-way ANOVA followed by Tukey’s post-hoc test was carried out. Using this information, graphical charts representing the data were prepared.

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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. For example 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.