Modified and unmodified peptidylglycine alpha-amidating monooxygenase (PAM) for use in subjects having increased amyloid-beta and / or phospho-TAU-protein load and / or neuroinflammation

Abstract

The present invention relates to modified or unmodified Peptidylglycine alpha-amidating monooxygenase (PAM) or fragments thereof for use in the treatment of a subject having an increased amyloid-beta and / or phospho-Tau-protein load in the brain and / or for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation, as well as corresponding pharmaceutical formulations and kits.

Description

[0001] Modified and unmodified Peptidylglycine alpha-amidating monooxygenase (PAM) for use in subjects having increased amyloid-beta and / or phospho-Tau-protein load and / or neuroinflammation

[0002] The present invention relates to modified or unmodified Peptidylglycine alpha-amidating monooxygenase (PAM) or fragments thereof for use in the treatment of a subject having an increased amyloid-beta and / or phospho-Tau-protein load in the brain and / or for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation.

[0003] The present invention more particularly relates to modified or unmodified PAM or fragments thereof, and to corresponding pharmaceutical formulations, for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function.

[0004] The present invention more particularly also relates to modified or unmodified PAM or fragments thereof, and to corresponding pharmaceutical formulations, for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation.

[0005] Background of the invention

[0006] Peptidylglycine alpha-amidating monooxygenase (PAM) is the only known enzyme for the conversion of inactive peptide hormones into their active form via C-terminal alpha amidation (Eipper et al., Annu Rev Neurosci 1992, 15: 57-85). More than half of known peptide hormones require this process for their activation. PAM specifically recognizes C-terminal glycine residues in its substrates, cleaves glyoxylate from the peptide’s C-terminal glycine residue in a two-step enzymatic reaction leading to the formation of C-terminally alpha-amidated peptide hormones, wherein the resulting alpha-amide group originates from the cleaved C-terminal glycine (Prigge et al. 2004. Science 304(5672): 864-67). This amidation reaction takes place in the lumen of secretory granules prior to exocytosis of the amidated product (Martinez and Treston 1996. Molecular and Cellular Endocrinol 123: 113-17). Alpha-amidated peptides are for example adrenomedullin (ADM), substance P, vasopressin, neuropeptide Y, Amylin, calcitonin, neurokinin A and others. However, previously it was demonstrated that PAM can also catalyze the formation of alpha-amides from glycinated substrates of non-peptide character, e.g. N-fatty acylglycines, which are converted by PAM to primary fatty acid amides (PFAMs) like oleamide. The identified and purified peptidylglycine amidating activities were shown to be dependent on copper and ascorbate (Emeson et al. 1984. Journal of Neuroscience: 2604-13; Kumar et al. 2016. J Mol Endocrinol 56(4): T63-76; Wand et al. 1985. Neuroendocrinology 41: 482-89).

[0007] In humans, the PAM gene is located at chromosome 5q21.1 having a length of 160 kb containing 25 known exons (Gaier et al. 2014. BMC Endocrine Disorders 14). At least 6 isoforms are known to be generated by alternative splicing (SEQ ID Nos. 30 - 35). The PAM enzyme was found to be expressed at different levels in almost all mammalian cell types, with significant expression in airway epithelium, endothelial cells, ependymal cells in the brain, adult atrium, brain, kidney, pituitary, gastrointestinal tract and reproductive tissues (Chen et al. 2018. Diabetes Obes Metab 20 Suppl 2:64-76; Oldham et al. 1992. Biochem Biophys Res Commun 184(1): 323-29; Schafer et al. 1992. JNeurosci 12(1): 222-34). The precursor protein (1-973 amino acids) of the largest known PAM Isoform 1 (SEQ ID No. 1 relates to PAM isoform 1, SEQ ID No. 30 relates to the prepro-Form of PAM isoform 1) encoded by the PAM cDNA is depicted in Figure 1. The N-terminal signal sequence (amino acids 1-20) assures direction of the nascent PAM polypeptide into the secretory lumen of endoplasmic reticulum and is subsequently cleaved co-translationally.

[0008] Afterwards the PAM-pro-peptide is processed by the same machinery used for the biosynthesis of integral membrane proteins and secreted proteins including cleavage of the pro-region (amino acids 21-30), assuring proper folding, disulfide bond formation, phosphorylation and glycosylation (Bousquet-Moore et al. 2010. J Neurosci Res 88(12):2535-45). The sequences of PAM isoforms 1 to 6 after cleavage of the N-terminal signal sequence (amino acid 1-20) as well as the pro-region (amino acids 21 to 30) are given as SEQ ID No. 1 to 6.

[0009] As depicted in Figure 1, the PAM cDNA further encodes two distinct enzymatic activities. The first enzymatic activity is named peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3), is an enzyme, capable of catalyzing the conversion of a C-terminal glycine residue to an alpha hydroxyglycine. The second activity is named peptidyl-a-hydroxyglycine alpha-amidating lyase (PAL; EC 4.3.2.5) is an enzyme capable of catalyzing the conversion of an alpha hydroxyglycine to an alpha-amide with subsequent glyoxylate release. The sequential action of these separate enzymatic activities results in the overall peptidylglycine alpha amidating activity. The first enzymatic activity (PHM) is located directly upstream of the pro-region (within amino acids 31-494 of isoform 1 (SEQ ID No. 7 and SEQ ID No. 9)). The second catalytic activity (PAL) is located after exon 16 in isoform 1 within amino acids 495-817 (SEQ ID No. 8).

[0010] As depicted in Figure 1, both activities may be encoded together within of one polypeptide as a membrane-bound protein (isoforms 1, 2, 5, 6; corresponding to SEQ ID No. 1, 2, 5 and 6) as well within of one polypeptide as a soluble protein lacking the transmembrane domain (TMD) (isoforms 3 and 4; corresponding to SEQ ID No. 3 and 4). While isoforms 1, 2, 5 and 6 remain in the outer plasma membrane after fusion of secretory vesicles with the plasma membrane with subsequent endocytosis and recycling or degradation, soluble PAM isoforms lacking the TMD (isoforms 3 and 4) (amino acids 864-887) are co-secreted with the peptide-hormones (Wand et al. 1985 Metabolism 34(11): 1044-52). Furthermore, prohormone convertases may convert membrane bound PAM protein into soluble PAM protein by cleavage within the flexible region (exons 25 / 26) connecting PAL with the TMD during the secretory pathway (Bousquet-Moore et al. 2010. J Neurosci Res 88(12):2535-45). The PHM subunit may be cleaved from soluble or membrane bound PAM within the secretory pathway by prohormone convertases that address a double-basic cleavage-site in the exon 16 region. Furthermore, during endocytosis the full-length PAM protein may be also converted into a soluble form due to the action of alpha- and gamma secretases (Bousquet-Moore et al. 2010. J Neurosci Res 88(12):2535-45). Membrane bound PAM from late endosome can be further secreted in form of exosomal vesicles.

[0011] PHM and PAL activities, as well as the activity of the full-length PAM were determined in several human tissues and body fluids. However, the separated PHM and PAL activities in soluble forms will also lead to formation of C-terminally alpha amidated products from C-terminally glycinated substrates when allowed to perform their separate reactions in the same compartment, body-fluid or in vitro experimental setup.

[0012] How the transfer of the PHM hydroxylated product to the PAL takes place is not exactly understood to date. There is evidence that the hydroxylated product is released into solution and is not directly transferred from PHM to PAL (Yin et al. 2011. PLoS One 6(12):e28679). Additionally, the source of PAM in circulation remained unclear.

[0013] The partial reaction of PHM is depicted in Figure 2. PHM is a copper dependent monooxygenase responsible for stereo-specific hydroxylation of the C-terminal glycine at the alpha carbon atom. During the hydroxylation reaction ascorbate is believed to be the naturally occurring reducing agent, while the oxygen in the newly formed hydroxyl group was shown to originate from molecular oxygen. The partial reaction of the PAL is depicted in Figure 2. The catalytic action of PAL involves proton abstraction form the PHM-formed hydroxyglycine by a protein-backbone derived base and a nucleophilic attack of hydroxyl-group oxygen to the divalent metal leading to a cleavage of glyoxylate and formation of a C-terminal amide.

[0014] One example of a substrate of PAM is Adrenomedullin-Gly (SEQ ID No. 14). The Adrenomedullin (ADM) mRNA encodes a preprohormone of 185 amino acids (SEQ ID No. 11), the pre-pro-Adrenomedullin that is enzymatically converted into Proadrenomedullin by cleavage of the N-terminal signal peptide. Proadrenomedullin is then further process by several prohormone convertases to result in four peptides, namely

[0015] PAMP-Gly (SEQ ID No. 12): Proadrenomedullin amino-terminal peptide or Proadrenomedullin N-terminal 20 peptide with a C-terminal glycine residue

[0016] MR-proADM (SEQ ID No. 16): Mid-regional Proadrenomedullin, a stable and inert peptide ADM-Gly (SEQ ID No. 14): C-terminally glycine extended, inactive precursor of biologically active ADM (bio-ADM (SEQ ID No. 15))

[0017] CT-proADM (SEQ ID No. 13): C-terminal Proadrenomedullin or Adrenotensin.

[0018] Cognitive function / cognitive impairment

[0019] Cognitive function refers to the mental processes that allow individuals to receive, select, store, transform, develop, and recover information from external stimuli. Cognition encompasses several well-recognized but often overlapping domains including attention, executive function, memory, language, visuospatial and constructional abilities, sensory-perceptual-motor skills and social cognition (Zhang 2019. ArXiv, 1907.02863). Traditional risk factors for cognitive impairment and factors predictive of progression to dementia include hypertension, hyperglycaemia, hyperlipidaemia, smoking, alcohol, and metabolic syndrome. Emerging factors include atrial fibrillation, inflammation, hyperhomo-cysteinaemia, and heart failure. The cognitive decline is primarily associated with neurodegenerative diseases, including Alzheimer's Disease (AD), MCI (mild cognitive impairment), Frontotemporal Dementia (FTD), Lewy Body Dementia (LBD), Parkinson's Disease with Dementia (PDD), Huntington's Disease (HD), Amyotrophic Lateral Sclerosis (ALS) with cognitive impairment, Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD), Multiple System Atrophy (MSA), Prion Diseases (e.g., Creutzfeldt-Jakob Disease) and many other. These pathologies can lead to a variety of cognitive deficits, which manifest itself in loss of memory, attention, concentration and executive functioning (Scheltens et al. 2016. Lancet 388(10043): 505-17). aphasia (Mesulam 2003. N Engl J Med. 349(16): 1535-42). loss of visuospatial abilities (Mapstone et al. 2008. Brain 131(Pt 6): 1618-29). extreme deviation from the normal socio-accepted behavioral spectra (loss of empathy, lack of judgment, lack of inhibition, compulsive behaviors), increase in anxiety, depression and apathy, as well as social withdrawal (Shany-Ur and Rankin 2011. Curr Opin Neurol. 24(6):550-5).

[0020] Many vascular disorders are important potential causes of cognitive impairment. The list of such vascular disorders include cerebrovascular diseases, such as vascular dementia, Multi-Infarct Dementia, Subcortical Ischemic Vascular Dementia, Strategic Single-Infarct Dementia, Cerebral Amyloid Angiopathy, Hypertensive Encephalopathy, stroke, Transient Ischemic Attack (TIA), Atherosclerosis, Cerebral Emboli, and cardiovascular diseases, such as atrial hypertension, atrial hypertension, myocardial infarction and heart failure (Jianu and Bar san 2019. J Neurol. 18(1): Begum et al. 2009. Int J Geriatr Psychiatry 24(7):701-8). Vascular disorders lead to cognitive impairments through mechanisms of acute or chronic vascular injury to brain regions critical for memory, executive function, and processing speed. The extent and location of the ischemia or hemorrhage significantly influence the nature and severity of the cognitive deficits. Cognitive impairment is also associated with Traumatic Brain Injury (TBI) Related Disorders including, but not limited to Chronic Traumatic Encephalopathy (CTE), Post-Concussion Syndrome, Diffuse Axonal Injury, Second Impact Syndrome, Subdural Hematoma, Epidural Hematoma, Penetrating Brain Injury, Post-Traumatic Amnesia. TBI can result in a wide range of cognitive impairments, from mild deficits in attention and processing speed to severe impairments in memory, executive function, mood disorders, behavioral changes and language, depending on the injury severity and brain areas affected (Gardner and Yaffe 2015. Molecular and Cellular Neuroscience, 66, 75-80).

[0021] Cognitive impairments, which are associated with inflammatory and infectious disorders include Multiple Sclerosis (MS), Sepsis-Associated Encephalopathy (SAE), HIV-Associated Neurocognitive Disorder (HAND), Neurosyphilis, Autoimmune Encephalitis, Meningitis, Encephalitis, Tuberculosis Meningitis, usually affect cognitive function through direct infection, immune-mediated inflammation, or both, leading to impairments in cognitive speed, concentration, memory, and executive functioning (Alves 2022. Frontiers in Neuroscience Vol.15)

[0022] The cognitive disorders related to Endocrine and Metabolic Disorders include Diabetes Mellitus, Hyperglycemia, Hypoglycemia are typically associated with reductions in processing speed, executive function, the ability to learn and retain new verbal information, as well as impaired episodic memory (Kodl and Seaquist (2008). Endocrine Reviews, 29(4), 494-511; Palta, et al. (2014). Journal of the International Neuropsychological Society, 20(3), 278-291). Such cognitive decline is due to fluctuations in glucose levels, which affect brain metabolism and neuronal function.

[0023] The role or influence of PAM in some clinical conditions has been described. The presence of alpha-amidating activity in human circulation was initially proved by Wand et al. (Wand et al. 1985. Neuroendocrinology 41, 482-489). They reported no sex differences but some variations of PAM activity in certain disease states: Plasma PAM activities were increased in hypothyroid adults as well as in patients with medullary thyroid carcinoma. The activity of PAM in tissues of medullary thyroid carcinoma, pheochromocytoma and pancreatic islet tumors were shown to be elevated suggesting increased formation of amidated peptides in endocrine tumor tissues (Gether et al. 1991. Mol. Cell. Endocrinol. 79, 53-63; Wand et al. 1985. Neuroendocrinology 41, 482-489). Patients suffering from multiple endocrine neoplasia type 1 (MEN-1) and pernicious anemia showed a decreased plasma PAM activity in comparison to healthy control subjects. The presence of amidating activity in human cerebrospinal fluid (CSF) was shown by Wand and colleagues. In patients suffering from Alzheimer’s disease (AD) plasma PAM activities were shown to be unaltered when compared to healthy controls, while CSF PAM activities were significantly decreased in comparison to activities from normal specimen (Wand et al. 1987. Neurology 37, 1057-1061). In addition, in WO2015 / 103594 it was proposed that the presence of PAM-Protein in CSF detected by mass spectrometry of AD-patients was reduced compared to healthy controls. Moreover, ADM, one of the hormones amidated by PAM, was shown to be reduced in patients with prevalent and incident Alzheimer’s disease (W02019 / 154900). Direct associations of circulating PAM activities were reported being associated with prediction, diagnosis or progression of AD in WO2021 / 170816A1 and WO2021 / 170752A1. PAM activities of patients suffering from multiple sclerosis (MS) were shown to be increased in CSF, with a significant decrease in serum (Tsukamoto et al. 1995. Intern. Med. 34, 229-232 and W02010 / 005387}. An association between plasma activity of PAM and type-2 -diabetes was described in (WO2014 / 118634}. Further, W02021170816A1 and WO2021170752A1 show an increase of PAM activity in circulation due to acute conditions such as sepsis and shock.

[0024] Neurodegenerative disorders

[0025] Neurodegenerative disorders as a group of conditions are characterized by progressive degeneration of the structure and function of the nervous system. Neurodegenerative diseases can be classified according to primary clinical features (e.g., dementia, parkinsonism, or motor neuron disease), anatomic distribution of neurodegeneration (e.g., frontotemporal degenerations, extrapyramidal disorders, or spinocerebellar degenerations), or principal molecular abnormality (e.g., amyloid beta depositions, tau neurofibrillary tangles, alpha-synuclein aggregates, chronic neuroinflammation, ubiquitin-positive inclusions, prion protein deposits etc.) (Dugger, etal.2017. Cold Spring Harbor perspectives in biology 9 7). The range of disorders is extensive including but not limited to Alzheimer's Disease (AD), Mild cognitive impairment (MCI), Frontotemporal Dementia (FTD), Lewy Body Dementia (LBD), Parkinson's Disease with Dementia (PDD), Huntington's Disease, Amyotrophic Lateral Sclerosis (ALS) with cognitive impairment, Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD), Multiple System Atrophy (MSA), prion diseases and many others, which affect millions worldwide, posing significant challenges to individuals and healthcare systems (Vyawhare etal. 2023. International Journal for Research in Applied Science and Engineering Technology.

[0026] Despite their broad molecular pattern, the most common pathological features of many neurodegenerative disorders are the proteinopathy, including amyloid-B deposition, tau neurofibrillary tangles, and chronic neuroinflammation.

[0027] Neuroinflammation

[0028] One hallmark of neurodegeneration is neuroinflammation. The general term „neuroinflammation“ is an immune response in the brain and spinal cord, involving cell types such as astrocytes, microglia, and peripherally derived cells (Lyman et al. 2014. Neurosci Res. 79:1-12}. The acute neuroinflammatory response includes activation of the resident immune cells resulting in a phagocytic phenotype and the release of inflammatory mediators such as cytokines and chemokines. While an acute insult may trigger oxidative and nitrosative stress, it is typically short-lived and unlikely to be detrimental to long-term neuronal survival, and is shown to have a neuroprotective effect. In contrast, chronic neuroinflammation is a long-standing and often self-perpetuating neuroinflammatory response that persists long after an initial injury or insult. Chronic neuroinflammation includes not only long-standing activation of immune cells, such as microglia, astrocytes, pericytes and other cells, and subsequent sustained release of inflammatory mediators, but also the resulting increased oxidative and nitrosative stress, which subsequently leads to neurodegeneration (Frank-Cannon et al 2009. Mol Neurodegeneration 4:47; DiSabato etal. 2016. JNeurochem. 139 (Suppl 2):136-153). Finally, the onset and progression of several neurodegenerative disorders, including MS, AD, PD, HD, ALS, tauopathies, and age-related macular degeneration (ARMD), have been associated with chronic neuroinflammation (Frank-Cannon et al 2009. Mol Neurodegeneration 4:47}.

[0029] Activation of immune cells is determined by changes in their morphology and function. Under pathological conditions astrocytes undergo morphological and functional changes characterized by cell hypertrophy and excessive release of neurotoxic factors, referred to as reactive astrocytes or astrogliosis. Some key molecular biological hallmarks of astrogliosis include the upregulation of Glial Fibrillary Acidic Protein (GFAP), increased expression of pro-inflammatory cytokines and chemokines (such as interleukin-1β and tumor necrosis factor-a), changes in calcium signaling, and activation of signal transduction pathways by STAT3, NF-kB, and MAPK, which regulate the expression of genes involved in inflammation, scar formation, and cellular proliferation (reviewed in Matusova et al. 2023. Front. Cell. Neurosci. 17:1173200; reviewed in Lawrence et al. 2023. Acta Neuropathol Commun 11, 42). Similar to astrogliosis, microglia activation is marked by a morphological shift from a resting state with branched processes to an amoeboid form, enhancing their phagocytic capabilities. This transformation is often accompanied by the release of pro-inflammatory cytokines (such as TNF-a, IL-1, and IL-6), reactive oxygen species (ROS), and other cytotoxic substances, contributing to neuroinflammation and neuronal damage. Key molecular markers of microglial activation include increased expression of surface receptors like CDllb, CD86, CD206 and ionized calcium-binding adapter molecule 1 (Iba-1), upregulation of inflammation-associated genes, and activation of intracellular signaling pathways such as nuclear factor kappa B (NF-KB), mitogen-activated protein kinase (MAPK), and (signal transducer and activator of transcription 3) STAT3, which regulate the inflammatory response (reviewed in Qin et al. 2023. Front. Neurol. 14: 1103416)

[0030] Also, pericytes undergo activation. Under pathological conditions, a subset of pericytes known as PC2, also referred to as disease-associated pericytes, increases in AD patients compared to healthy controls. This shift is characterized by the development of an a-smooth muscle actin (a-SMA)-rich cytoskeletal structure and enhanced phagocytic capabilities. PC2 cells can secrete cathepsin D, an enzyme that degrades AB peptides, reducing Ap plaque load and mitigating blood-brain barrier (BBB) breakdown. In contrast, physiological pericytes (PCI) often internalize Ap42, leading to cell death and subsequent BBB disruption (Bohannon et al. 2024. Brain Pathol. 27:el3282) Many studies suggest a direct role for neuroinflammation in the production, accumulation, and clearance of amyloid-beta (AP) peptides, known as the neuroinflammatory-amyloid hypothesis. Neuroinflammation can increase A production and accumulation as shown by Paasila et al. 2020, where the clustering of activated microglia occurs before the formation of dystrophic neurites in the evolution of Ap plaques (Paasila et al. 2020. Free Neuropathology 1:1-20}. while the presence of Ap can further stimulate neuroinflammatory responses. This creates a vicious cycle where each process exacerbates the other, highlighting the complexity of pathology progression and the need to address both factors in treatments. The exact mechanism by which Ap induces neurotoxicity is still unclear, but it is thought to involve the dysregulation of the immune response by direct interaction of various Ap complexes with glial expressed pattern recognition receptors. This process involves secretion of pro-inflammatory cytokines, chemokines and generation of reactive oxygen species that, in excess, drive a dysregulated immune response that contributes to neurodegeneration (Minter et al. 2016. Journal of Neurochemistry 136(3), 457-474).

[0031] Below is the summary of some experimental findings indicating the link between neuroinflammation and AB- and tau-related pathology.

[0032] In AD reactive astrocytes are found in proximity to AB-plaques and are implicated in the propagation of inflammatory responses (Kato et al. 1998. Pathol Int. 48(5): 332-40). Excessive production of neurotoxic factors, such as TNF-a and IFN-y, modulates astrocyte APP processing homeostasis and tau phosphorylation, which leads to increased Ap load and toxicity (Garwood et al. 2011. Cell Death Dis.

[0033] 2(6): e167}. Despite their role in Ap clearance, astrocytes can be impaired by prolonged exposure to Ap, leading to a loss of supporting functions (Thai 2012. Experimental Neurology 236: 1-5). Additionally, astrocytes can accumulate and secrete significant quantities of Ap, contributing to overall amyloid burden in the brain and leading to neuronal apoptosis (Frost and Li 2017. Open Biology 7(12): 170228; Söllvander et al. 2016. Molecular Neurodegeneration 11(1):38) In addition, an APP mouse model displays extensive amyloid-induced reactive astrogliosis and advanced astrogliosis was found in samples from individuals diagnosed with Alzheimer’s disease, mixed dementia, and vascular mediated dementia (Kashon et al. 2004. J Alzheimers Dis. 6(6):595-604).

[0034] It has been demonstrated that activated microglia releases inflammatory mediators such as inflammatory cytokines, complement components, chemokines, and free radicals, that contribute to Ap production and accumulation (Cai et al. 2014. International Journal of Neuroscience 124: 307- 321). Reactive microglia, which often localized in the close proximity to AB plaques, is involved in the dynamics of Ap plaque formation and growth by taking up Ap and causing microglial cell death (Baik et al. 2016. Glia 64(12):2274-2290). Upregulation of microglial biomarkers, such as IL-6 and TGFB2, has been observed in the brains of AD patients, particularly in advanced stages, possibly reflecting the chronic neuroinflammation (Chaudhary et al. 2021. Journal of Alzheimer's disease: 82(4): 1487-1497). Detailed description of the invention

[0035] However, nothing is known about PAM for use in the treatment of a subject having an increased amyloid-beta and / or phospho-Tau-protein load in the brain or for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation, or for the treatment or prevention of for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function.

[0036] The examples in the description of the present invention show that administration of Peptidylglycine alpha-amidating monooxygenase (PAM), especially modified PAM (by PEGylation) significantly improved spatial long-term memory, as assessed by the state-of-the-art Morris Water Maze test in in vivo models with induced memory deficits (Example 2). Furthermore, it has been demonstrated that PAM, especially modified PAM (by PEGylation) has been shown to significantly improve spatial shortterm memory and reduce anxiety in an Alzheimer’s transgenic mouse model, as demonstrated in Examples 3 and 4, respectively. This clearly demonstrates that PAM, especially modified PAM (e.g. PEG-PAM) is able to treat or prevent cognitive impairment in a subject in need thereof and improves cognitive function.

[0037] The examples in the description of the present invention furthermore show that administration of PAM, especially modified PAM (by PEGylation), and substantially reduces the number of reactive astrocytes in various brain regions, such as the dentate gyrus, the hippocampus and Stratum Lacunosum-Moleculare (Example 6). Furthermore, it has been demonstrated that PAM, especially modified PAM (by PEGylation) resulted in a significant reduction of the number of Amyloid-plaques in a 3xTG model in mice in several brain regions such as the motor cortex, basolateral amygdala, somatosensory cortex and subiculum region of the hippocampus (Example 9). Additionally, it has been demonstrated that PAM, especially modified PAM (by PEGylation) resulted in a significant reduction of pTau accumulation in brain regions, such as basolateral amygdala and CAI region of hippocampus (Example 9). This clearly demonstrates that PAM, especially modified PAM (e.g. PEG-PAM) is able to treat or prevent neuroinflammation in a subject in need thereof and / or reduces the amyloid-B and pTau load in a subject in need thereof.

[0038] It is a surprising finding of the present invention that PAM, and in particular modified PAM (e.g. by PEGylation) or fragments thereof are suitable for the treatment of a subject having an increased amyloidbeta and / or phospho-Tau-protein load in the brain.

[0039] It is furthermore a surprising finding of the present invention that PAM, and in particular modified PAM (e.g. by PEGylation) is suitable for use in therapy or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation. It is furthermore a surprising finding of the present invention that PAM, especially modified PAM (e.g. PEG-PAM) is suitable for use in therapy or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function.

[0040] Certain embodiments of the present invention relate to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof, wherein said subject has an increased amyloid-beta and / or phospho-Tau-protein load.

[0041] Certain embodiments of the present invention relate to modified or unmodified PAM or fragments thereof for use in therapy or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function, wherein said subject has neuroinflammation.

[0042] Amyloid Beta

[0043] Amyloid beta (AP) deposits are formed through the cleavage of amyloid precursor protein (APP), a 695-770 amino acid, single membrane-spanning protein that is strongly expressed in the nervous system. APP is sequentially cleaved by the enzymes -secretase (or P-amyloid cleaving enzyme (BACE)) and y-secretase, resulting in fragments of Ap proteins of various length, with the most often length being 40 or 42 amino acids (‘Ap40’ and ‘Ap42’). Both fragments are known to contribute to neuronal damage either in form of plaques (Ap42 augment its tendency to self-assemble into oligomers and fibrils, which are found extracellularly in the brain parenchyma, especially in areas such as the hippocampus, cortex, and other regions associated with memory and cognitive functions) or in a form of cerebral angiopathy (Ap40 is more abundant form as a deposition along the large vessels and capillary). AB-proteinopathy is the most common hallmark for AD, but is also found in other neurodegenerative disorders such as CAA, LBD, hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWA-D) and others (reviewed in Walker 2020. Free Neuropathology, 1, 31).

[0044] In clinical practice today, following pathological Ap fragments are used as biomarkers for diagnosing and monitoring neurodegenerative diseases:

[0045] • Ap42: while decreased levels of Ap42 in cerebrospinal fluid (CSF) or blood are associated with disease progression, Ap42 levels in brain tissue increase due to its sequestration into amyloid plaques, reducing its soluble form in the CSF and blood (Spies et al. 2012. Frontiers in bioscience 17: 2024-34).

[0046] • AP42 / AP40 ratio is used to normalize Ap42 measurements and account for individual variations in Ap production. A decreased ratio is a strong indicator of amyloid pathology, with AD patients showing appr. 38% decrease in AB42 / AB40 ratio in CSF when compared to the age-matched controls (Fukuyama et al. 2000. European Neurology 43: 155 - 160). The CSF A42 / A40 ratio also improves differentiation of AD patients from VaD, DLB and non-AD dementia patients (Spies et al. 2010. Current Alzheimer research 75: 470-6). While both biomarkers Ap42 concentration and the Ap42 / Ap40 ratio at the baseline in CSF were significantly decreased in the MCI patients who developed AD as compared to cognitively stable MCI patients and MCI patients who developed other forms of dementia,

[0047] • the Aβ42 / Aβ40 ratio was superior to Aβ42 concentration with regard to identifying incipient AD in MCI (p < 0.05) (Hansson et al. 2007. Dementia and Geriatric Cognitive Disorders 23: 316 - 320).

[0048] • Total Aβ correlates with increased disease severity, reflecting the extent of neurodegeneration, e. g. in AD (McLean et al. 1999. Annals of Neurology 46). Additionally, the mean Aβ load in the hippocampus correlates with the duration and severity of dementia in Alzheimer's disease (Bartoo et al. 1997. Journal of Neuropathology & Experimental Neurology 56 (5): 531-540). Other biomarkers, which are closely related to amyloidogenic pathway and are used for diagnosing and monitoring neurodegenerative diseases are:

[0049] • APP-alpha and APP-beta: Several studies have demonstrated that the levels of APP isoforms, particularly sAPPα and sAPPβ, which are produced from the cleavage of APP by α-secretase and β-secretase, respectively, are altered in neurodegenerative diseases such as AD and MCI- AD. Both sAPPα and sAPPβ are highly correlated with p-tau and total tau in CSF, suggesting that sAPPs possibly reflect neuropathological changes in the brain. Levels of sAPPα are significantly higher in MCI-AD cases compared with non-AD and FTD, whereas levels of sAPPβ are significantly increased in AD and MCI-AD and are significantly higher when compared to FTD. Additionally, sAPPα and sAPPβ have good discriminative power for the diagnosis of MCI-AD from other groups (reviewed in Araki et al. 2017. Biomarker Research 5; Araki et al. 2018. Neurology and Clinical Neuroscience 6).

[0050] Tau Protein

[0051] Tau proteins play a crucial role in maintaining the stability of microtubules in neurons, which are essential for maintaining cell structure and facilitating intracellular transport. In the context of neurodegenerative diseases, tau proteins undergo abnormal hyperphosphorylation, leading to their detachment from microtubules and subsequent self-assembly into neurofibrillary tangles (NFTs). Furthermore, hyperphosphorylation of tau can convert it into a prion-like protein, contributing to its propagation and the spread of neurodegeneration (Alonso et al. 2016. Alzheimer's & Dementia 12: 1090-1097). These tangles are a hallmark of AD and other tauopathies, such as FTD, progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD) (reviewed in Avila et al. 2004. Physiological reviews 842: 361-84).

[0052] Tau pathology typically begins in the entorhinal cortex and hippocampus, spreading to other brain regions as the disease progresses. Hyperphosphorylated tau aggregates interfere with neuronal function and are strongly associated with synaptic loss and neuronal death, contributing to cognitive decline and other clinical symptoms of AD (Braak & Braak 1991. Acta Neuropathologica, 82(4): 239-259). Several studies have shown that the severity of cognitive impairment is more strongly correlated with the burden of neurofibrillary tangles (Nelson et al. 2012. J Neuropathol Exp Neurol. 71(5): 362-81). Furthermore, the density of neurofibrillary tangles in the brain is more closely associated with cognitive decline than the presence of amyloid plaques (Nagy et al. 1995. Dementia 6 (1): 21-31).

[0053] In clinical practice today, following biomarkers are used to monitor the pathological tau forms for diagnosing and monitoring neurodegenerative diseases:

[0054] • Total tau (t-tau): Elevated levels of total tau in CSF are indicative of neuronal damage and the intensity of degeneration. Elevated t-tau levels is observed in several neurodegenerative conditions with acute and severe neuronal injury, such as traumatic brain injury, stroke and Creutzfeldt-Jakob disease (reviewed in Zetterberg 2017. Neuropathology and Applied Neurobiology 43(3), 194–199). CSF T-tau is likely increased very early in the disease progression since increased levels can be seen already in amyloid-positive cognitively unimpaired individuals. In AD, higher T-tau values predict a more rapid cognitive decline and relate to more rapid hippocampal atrophy (reviewed in Scholl et al. 2019. Molecular and Cellular Neuroscience 97:18-33)

[0055] • T-tau / AB42 ratio improves diagnostic accuracy, particularly in distinguishing Alzheimer's disease from other forms of neurodegenerative disorders, such as FTD and semantic dementia, and is significantly elevated in AD patients when compared to the healthy controls (Paterson et al. 2018. Alz Res Therapy 10, 32).

[0056] • Phosphorylated tau (p-tau): The measurement of specific phosphorylated tau epitopes, such as p-tau181, p-tau217, p-tau231, p-tau199, p-tau396 and p-tau404 in the CSF is used to assess the extent of tau pathology, with p-tau181, p-tau217, p-tau231 demonstrating the highest potential (reviewed in Scholl et al. 2019. Molecular and Cellular Neuroscience 97:18-33). Clinical research consistently demonstrate that CSF p-tau is highly increased in AD compared to healthy controls and may differentiate AD from its most relevant differential diagnoses.

[0057] o p-tau181. Several studies have shown that elevated levels of p-Tau 181 (phosphorylation at threonine 181) in blood plasma are associated with progressive neurodegeneration in AD, especially in individuals with elevated brain amyloid-β (Moscoso 2021). The rates of change in CSF levels of total tau and p-Tau 181 vary across the course of AD, with different patterns of association with brain atrophy, providing a better understanding of the dynamics of the disease progression (Llibre-Guerra et al. 2019. JAMA Netw Open. 2(12): e1917126). Moreover, p-tau181 enhances accurate differentiation between AD and dementia with Lewy bodies, as well as shows excellent discrimination between AD and non-AD dementias including FTD, exceeding the differential diagnostic and prognostic accuracy of t-tau (reviewed in Hampel and Teipel 2004. Dementia and Geriatric Cognitive Disorders 17: 350 – 354).

[0058] o p-tau217. Soluble p-Tau217 (phosphorylation at threonine 217) has been identified as a potential biomarker for AD, reflecting both amyloid and tau pathology. It has also been suggested, that p- tau217 is a more potent diagnostic biomarker for AD, when compared to p-tau 181. P-tau217 levels increase more than p-tau181 and correlate better with [18F]flortaucipir uptake. P-tau217 also shows stronger correlations with CSF and PET measures of neocortical amyloid-β burden and more accurately differentiates AD dementia from non-AD neurodegenerative disorders (Janelidze et al. 2020. Nature Communications 11: article 1683). P-tau217 concentrations correlated with β-amyloid PET in early disease stages and with both β-amyloid and tau PET in late disease stages. Also, p-tau217 mediated the association between β-amyloid and tau outside of the medial temporal lobe, indicating that p-tau is involved in β-amyloid-dependent formation of neocortical tau tangles (Mattsson-Carlgren et al. 2021. EMBO Molecular Medicine 13(6):e14022). p-tau217 showed significant amyloid-dependent changes in both preclinical and symptomatic stages of the disease, unlike p-tau231, p-tau181, amyloid-β42 / 40, glial fibrillary acidic protein, or neurofilament light. Longitudinal increases in p-tau217 were also linked to clinical deterioration and brain atrophy in preclinical AD (Ashton et al. 2022. Nature Medicine 28: 2555 - 2562).

[0059] o p-tau231. Overall levels of p-tau231 (phosphorylation at threonine 231) are significantly elevated in AD patients. Moreover, p-tau231 compared to t-tau raised sensitivity levels in the discrimination of AD and FTD from 58 to 90 %. The level of p-tau 231 declined with disease progression, correlating with cognitive performance at baseline (Hampel and Teipel 2004. Dementia and Geriatric Cognitive Disorders 17: 350–354). The analysis of the intra-vitam CSF as well as post-mortem neuropathological data in AD cohort showed significant correlations between CSF p-tau231 concentrations and NFT scores and t-tau load in several neocortical regions, as well as between amyloid beta plaques score and CSF p-tau231 concentration in frontal cortex. There is a significant correlation between p-tau231 in CSF and brain homogenates observed (Buerger et al. 2006. Brain 129(Pt 11): 3035-41).

[0060] Amyloid-beta is a peptide produced from the cleavage of amyloid precursor protein (APP). It aggregates to form insoluble plaques in the brain, which are a hallmark of Alzheimer's disease (AD). Elevated amyloid-beta levels lead to plaque formation, disrupting neuronal communication, promoting inflammation, and contributing to synaptic dysfunction and cell death. These processes are linked to the cognitive decline observed in AD. Measuring amyloid-beta load, often through techniques like PET imaging or cerebrospinal fluid (CSF) analysis, serves as a critical biomarker for diagnosing AD and assessing disease progression.

[0061] Tau is a microtubule-associated protein that stabilizes neuronal structure. When tau becomes hyperphosphorylated, it can misfold and aggregate into neurofibrillary tangles. An increased load of phospho-Tau indicates a higher level of these tangles, which disrupts cellular function, impairs axonal transport, and leads to neurodegeneration. This accumulation is closely associated with neuroinflammation and is a key feature of Alzheimer’s and other tauopathies.

[0062] Phospho-Tau levels play a role in disease mechanisms and can be utilized alongside amyloid-beta measurements to provide a comprehensive picture of neurodegenerative processes.

[0063] Certain embodiments of the present invention relate to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need, wherein administration of said PAM or fragments to said subject thereof leads to a reduction of the amyloid-beta and / or phospho-Tau protein load.

[0064] Certain embodiments of the present invention relate to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof, wherein administration of said PAM or fragments to said subject thereof leads to a reduction of the neuroinflammation.

[0065] Current clinical methodologies for assessing neurodegeneration encompass a variety of approaches, including neuroimaging techniques, cerebrospinal fluid (CSF) and blood-based biomarker analysis, and neurophysiological testing. Below is a concise summary of these methodologies frequently employed in clinical and also preclinical settings.

[0066] Neuroimaging techniques, such as PET and MRI, are non-invasive and extensively used in living patients to assess brain structure, function, and pathology, proving valuable information for the diagnosis and management of neurodegenerative disease (reviewed in Young et al. 2020. Alzheimers Res Ther.

[0067] 12(1):49)

[0068] Positron emission tomography (PET)

[0069] • [18F]-2-Fluoro-2 -deoxy-D-glucose (FDG) PET is used to diagnose dementia by assessing regional glucose metabolism in the brain. FDG is an artificial glucose analog that gets trapped in tissue after phosphorylation, indicating areas of metabolic activity. Decreased FDG uptake (hypometabolism) in specific brain regions can suggest synaptic and neuronal damage, which is a characteristic hallmark for many neurodegenerative diseases.

[0070] • Amyloid-β PET imaging detects the direct accumulation of amyloid plaques. Tracers like [11C]PiB and FDA-approved F-18 labelled compounds (flutemetamol, florbetapir, and florbetaben) bind to amyloid-β plaques, allowing their visualization in vivo. This technique provides an early and quantifiable measure of amyloid pathology, facilitating the diagnosis and tracking of the disease progression.

[0071] • Tau PET imaging utilizes tracers, such as [18F]flortaucipir, that bind to tau protein tangles, which follows specific brain region patterns correlating with cognitive decline and neurodegeneration stages. Tau PET is valuable for differential diagnosis, prognosis, and assessing treatment responses in clinical trials.

[0072] • SV2A PET imaging targets the synaptic vesicle glycoprotein 2A, a marker of synaptic density.

[0073] This method is crucial for detecting synaptic loss, which occurs early in AD and other neurodegenerative conditions. Tracers like [11C]UCB-J and [18F]UCB-H allow visualization of synaptic changes in vivo, providing insights into disease mechanisms and potential therapeutic targets.

[0074] Magnetic resonance imaging (MRI)

[0075] • Structural MRI (sMRI) is the primary tool for diagnosing and researching neurodegeneration due to its high-resolution imaging of brain structures. It helps exclude brain lesions, identify atrophy patterns, and assess vascular burden. High-resolution volumetry, particularly of the hippocampus, is crucial for early diagnosis and monitoring progression in AD. Techniques like quantitative susceptibility mapping (QSM) and T2* transverse relaxation time are used to assess iron deposition, correlating with cognitive and motor decline.

[0076] • Functional MRI (fMRI) measures brain activity by detecting changes in blood flow, linked to neuronal activity. It is essential for understanding functional connectivity and neuronal dysfunction in neurodegenerative diseases. Resting-state fMRI reveals altered connectivity in networks like the default mode network in AD, while task-based fMRI shows compensatory mechanisms and progression markers.

[0077] • Arterial Spin Labelling (ASL) measures cerebral blood flow non-invasively, serving as an alternative to FDG PET for detecting hypometabolic patterns in neurodegenerative diseases. It offers diagnostic quality similar to FDG PET while reducing radiation exposure.

[0078] • Diffusion Tensor Imaging (DTI) measures the diffusion of water molecules in tissue, providing insights into the microstructural properties of white matter tracts. It identifies microstructural alterations in tracts associated with neurodegenerative diseases like AD, ALS, and frontotemporal dementia. Common measures like fractional anisotropy are influenced by various disease-relevant properties, limiting anatomical specificity.

[0079] • Graph Theory in neuroimaging studies brain networks by analyzing nodes (brain regions) and edges (connections). It is used to understand network alterations in neurodegenerative diseases, providing insights into disease mechanisms and potential biomarkers.

[0080] CSF- and blood-based biomarker analysis. CSF analysis involves the extraction of CSF through a lumbar puncture to measure biomarkers like amyloid-β, t-tau and p-tau, NfL and others. This method is minimally invasive and is widely used in clinical settings to diagnose and monitor neurodegenerative diseases; Blood tests are increasingly used to measure biomarkers for neurodegenerative diseases due to their less invasive nature. The most common methods to assess CSF- and blood-based biomarkers are immunoassays and mass spectrometry.

[0081] Immunoassays

[0082] • Enzyme-Linked Immunosorbent Assay (ELISA) is widely used in clinical settings to quantify specific proteins, including biomarkers in blood and CSF. It employs specific antibodies to detect and measure concentrations of target proteins such as amyloid-β, tau, Nf, C-reactive protein (CRP) and many other. Known for its high sensitivity and specificity, ELISA remains a gold standard for biomarker measurement in both clinical and research environments.

[0083] • Single Molecule Array (Simoa) is an ultra-sensitive digital immunoassay technology that has gained traction in clinical settings for detecting low-abundance biomarkers in blood and CSF. It excels in measuring biomarkers like NfL and GFAP at very low concentrations, making it an invaluable tool for early diagnosis and continuous monitoring of neurodegenerative diseases due to its exceptional sensitivity and precision.

[0084] • Electrochemiluminescence Immunoassay (ECLIA) is used in clinical environments for the sensitive and specific quantification of biomarkers in blood and CSF. This technique utilizes electrochemiluminescent labels for detection, providing high sensitivity and a broad dynamic range. It is particularly useful for diagnosing and monitoring various diseases through precise biomarker measurement.

[0085] Multiplex assay are often immunoassay-based, which measure multiple neurodegenerative biomarkers simultaneously in a single sample, enhancing diagnostic accuracy and efficiency. Examples include the Luminex xMAP and Meso Scale Discovery (MSD) platforms. Below is the list of some currently available immunoassay for selected biomarkers detecting neurodegeneration:

[0086] Amyloid-β immunoassay-based quantification

[0087] • Elecsys® beta-Amyloid (1-42) CSF II Assay (Roche): Measures Aβ42 in CSF, providing high sensitivity and specificity mostly for AD research and diagnosis.

[0088] • Human Amyloid beta 42 ELISA Kit (Invitrogen, Thermo Fisher Scientific): Designed to quantify Aβ42 in human tissue culture medium, tissue homogenate, and CSF.

[0089] • INNOTEST® β-AMYLOID(1-42) (Fujirebio): Measures Aβ42 in human CSF.

[0090] • Meso Scale Discovery (MSD) V-PLEX Aβ42 Kit: Quantifies Aβ42 in human plasma and CSF.

[0091] Tau immunoassay-based quantification

[0092] • Elecsys® Phospho-Tau (181P) CSF Assay (Roche): Measures p-Tau181 in CSF, often used with Aβ assays to evaluate the pTau181 / Aβ42 ratio for AD diagnostics.

[0093] • Elecsys® Total-Tau CSF Assay (Roche): Measures total Tau protein levels in CSF.

[0094] • INNOTEST® hTAU Ag CSF (Fujirebio): Determines total tau in CSF, often used in combination with β-amyloid assays.

[0095] GFAP immunoassay-based quantification

[0096] • Quanterix Simoa GFAP Discovery Kit: Measures GFAP levels in human serum and plasma, used for research in neurodegenerative diseases and traumatic brain injury.

[0097] Mass Spectrometry (MS) is utilized in clinical laboratories for the precise measurement of neurodegenerative biomarkers in blood and CSF. Its ability to identify and quantify multiple proteins simultaneously with high sensitivity and specificity makes it suitable for detailed protein analysis in a clinical context.

[0098] • LC-MS / MS (Liquid Chromatography-Tandem Mass Spectrometry): Widely used in clinical labs for accurately quantifying biomarkers in blood and CSF. It combines liquid chromatography's separation capabilities with tandem mass spectrometry's detection power, offering high sensitivity and specificity.

[0099] MALDI-TOF (Matrix-Assisted Laser Desorption / Ionization-Time of Flight): Used in preclinical research for identifying and quantifying proteins, peptides, and other biomolecules. This technique is effective for rapid analysis of complex samples, aiding in biomarker discovery and validation.

[0100] • Immunoprecipitation-Mass Spectrometry (IP-MS): Applied in both clinical and preclinical settings to study protein-protein interactions and quantify specific proteins in complex mixtures. It combines immunoprecipitation for isolating target proteins with mass spectrometry for precise quantification, useful for detailed biomarker analysis and validation.

[0101] Protein and Tissue Analysis

[0102] • Immunohistochemistry (IHC): Widely used in clinical pathology for the localization and visualization of biomarkers within tissue sections. This technique employs antibodies to detect specific proteins in situ, providing spatial and contextual information about biomarker expression. IHC is a standard method in diagnostic histopathology and research.

[0103] • Western Blot: Primarily used in preclinical research to detect and quantify specific proteins in a sample. This technique involves the separation of proteins by gel electrophoresis, followed by transfer to a membrane and detection using antibodies. Western blot is valuable for validating the presence and abundance of biomarkers.

[0104] Cell-Based Assays

[0105] • Flow Cytometry is utilized in both clinical and preclinical settings for the quantitative analysis of cell populations and the detection of biomarkers on or within cells, allowing for the simultaneous measurement of multiple parameters at the single-cell level, making it highly useful for immunophenotyping and biomarker discovery.

[0106] Current therapeutic approaches available today usually address only one aspect of neurodegeneration. This limitation fails to account for the complex and highly diverse nature of neurodegenerative diseases, resulting in low efficacy.

[0107] Due to the highly complex and diverse nature of neurodegenerative disorders, with interrelated mechanisms that contribute to their progression, there is critical need for comprehensive and multifaceted approaches in research and treatment. Addressing these conditions holistically is essential for developing effective therapies, improving patient outcomes. An integrated approach targeting Aβ deposition, tau pathology, and neuroinflammation may offer a more effective means of slowing disease progression and improving patient outcomes. The present invention aims to fulfill this unmet need by providing a method for reducing neurodegenerative pathology. This method involves administering a peptidylglycine-alpha amidating monooxygenase (PAM) that demonstrably reduces Ap deposition, tau pathology, and activated astrocytes, thereby offering a comprehensive approach to treating neurodegenerative disorders.

[0108] Certain embodiments of the present invention relate to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof, wherein said reduction of amyloid-beta protein and or phospho-Tau load is assessed with an immunoassay, particularly an assay including but not limited to Enzyme-Linked Immunosorbent Assay (ELISA), Multiplex Assays, Singleplex Assays, Electrochemiluminescence Immunoassay (ECLIA); Positron Emission Tomography (PET) Scans including Amyloid PET and Tau PET; Mass Spectrometry, including but not limited to LC-MS / MS (Liquid Chromatography-Tandem Mass Spectrometry), MALDI-TOF (Matrix-Assisted Laser Desorption / Ionization-Time of Flight), Immunoprecipitation-Mass Spectrometry (IP-MS); Immunohistochemistry (IHC); Western Blot; Flow Cytometry.

[0109] Certain embodiments of the present invention relate to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof, wherein said subject is suffering from or is having a risk of suffering from a disease or medical condition selected from the group comprising Alzheimer's Disease, Mild cognitive impairment (MCI), Frontotemporal Dementia (FTD), Lewy Body Dementia (LBD), Parkinson's Disease with Dementia (PDD), Huntington's Disease, Amyotrophic Lateral Sclerosis (ALS) with cognitive impairment, Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD), Multiple System Atrophy (MSA), prion diseases (e.g., Creutzfeldt-Jakob Disease), Cerebral Amyloid Angiopathy (CAA), Down Syndrome (DS) with Alzheimer-like dementia, Chronic Traumatic Encephalopathy (CTE), and Neurofibrillary Tangle Dementia (NTD) Postencephalitic Parkinsonism.

[0110] Neuroinflammation is in particular defined as a pathological immune response in the central nervous system (CNS), characterized by activation of glial cells (e.g. astrocytes, microglia).

[0111] In certain embodiments neuroinflammation is detected using specific neuroimaging techniques (such as PET or MRI) and / or measuring the level of a biomarker of neuroinflammation in a bodily fluid of the subject.

[0112] In certain embodiments the level of said biomarker of neuroinflammation detected in a bodily fluid of said subject is selected from the group comprising sTREM2 (soluble Triggering Receptor Expressed on Myeloid Cells 2), YKL-40 (Chitinase-3 -like protein 1, CHI3L1), Glial Fibrillary Acidic Protein (GFAP), S100B, and Neopterin, each of which represents a direct molecular indicator of neuroinflammation within the central nervous system. In another embodiment, the biomarker of neuroinflammation is further determined in combination with one or more biomarkers in a sample of bodily fluid from the subject that reflect supportive or complementary aspects of neuroinflammation, selected from Nfl, soluble adhesion molecules (sICAM-1, sVCAM-1), proinflammatory cytokines (e.g. IL-6, IL- ip, TNF-a, IL-10, IL-12, IL-18, IFN-y, IL-15, IL-16), chemokines (e.g. MCP-1 (CCL2), IL-8 (CXCL8)), sTNFR1 / sTNFR2, C-reactive protein (CRP), complement components (Clq and / or C3), sCD14, sCD163.

[0113] Glial Fibrillary Acidic Protein (GFAP) is the primary cytoskeletal protein in astrocytes, crucial for maintaining the integrity of the blood-brain barrier. GFAP serves as a marker for reactive astrocytes, which involves an increase in the number and size of astrocyte cell bodies (McKeon et al., 2018, 90 (20): 925-930).

[0114] Soluble triggering receptor expressed on myeloid cells 2 (sTREM2) is a CSF biomarker of neuroinflammation indicative of reactive astrogliosis and microglia (Qin et al. 2024, Brain, Vol.147 (1): 163-176).

[0115] Inside the brain, YKL-40 is primarily expressed in reactive astrocytes, contributing to microglial activation induced by AB plaques (Querol-Vilaseca, M. et al., 2017. J Neuroinflammation 14, 118). Neopterin is produced by activated microglia and macrophages (Huber et. al 1984, J Exp Med, 160 (1): 310-316).

[0116] SIOOB is a calcium-binding protein predominantly expressed by astrocytes and perivascular glial cells. Increased levels of SIOOB in cerebrospinal fluid or plasma reflect astroglial activation and blood-brain barrier involvement associated with neuroinflammatory processes (Michetti et al. 2019, Vol. 148 (2): 168-187).

[0117] Neurofilament Light Chain (NfL) is a structural axonal protein that increases in CSF and plasma in response to neuronal injury that often accompanies inflammatory activation (Gaetani L, et al. J Neurol Neurosurg Psychiatry 2019 (0) :1–12)

[0118] Adhesion molecules (sICAM-1 and sVCAM-1) are soluble adhesion molecules shed from activated endothelial cells that participate in immune-cell trafficking and blood-brain barrier modulation (Uzawa et al. 2011, Arch Neurol. 68 (7): 913-917).

[0119] sCD14 and sCD163 are soluble receptors derived from activated microglia and macrophages that complement sTREM2 by capturing different stages of neuroinflammation (sCD14 reflects early activation, while sCD163 is linked to anti-inflammatory or healing phases) (Buechler et al. 2013, Vol.

[0120] 12 (6): 391 - 402). Cytokines and chemokines (e.g., IL-6, IL-1β, TNF-a, CCL2) are multifunctional mediators that reflect inflammatory signaling pathways and immune-cell communication within the CNS and periphery (Ramesh et al. 2013; Mediators of Inflammation, 1-20).

[0121] In more particular embodiments the level of said biomarker of neuroinflammation detected in a bodily fluid sample of said subject is selected from the group comprising GFAP, sTREM2, YKL-40, neopterin, S100B, sICAM-1, and sVCAM-1, IL-1β, IL-6, TNF-a, IL-8, IL-10, IL-12, IL-18, IFN-y, CCL2 (MCP-1), CCL3 (MIP-la), CCL4 (MIP-1β), CCL5 (RANTES), CXCL10 (IP- 10), NfL, sCD14, and sCD163. In certain more particular embodiments, said biomarker is selected from the group comprising GFAP, sTREM2, YKL-40, neopterin, S100B, NfL, sICAM-1, and sVCAM-1.

[0122] In a still more particular embodiment, said biomarker is selected from the group comprising GFAP, sTREM2, and YKL-40.

[0123] In even more particular embodiments, said biomarker is GFAP.

[0124] Measurement Platforms

[0125] In particular embodiments Quantification of neuroinflammatory biomarkers in CSF or plasma is performed using validated analytical methods.

[0126] Protein biomarkers such as GFAP, sTREM2, YKL-40, NfL, and adhesion molecules are measured by immunoassay-based technologies, including enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence immunoassay (ECLIA), and multiplex bead-based systems (e.g., Luminex, Olink).

[0127] For ultra-low-abundance proteins, Single Molecule Array (Simoa) may be used; this offers femtomolar sensitivity and enables detection of plasma biomarkers such as GFAP, NfL, and YKL-40, which are particular markers to be measured with this method, that were previously measurable only in CSF. In certain specific embodiments, small-molecule metabolites such as neopterin, kynurenine, and quinolinic acid are quantified by liquid chromatography coupled to tandem mass spectrometry (LC-MS / MS); this allows for high analytical specificity and accuracy. In specific embodiments, quantifying neuroinflammatory biomarkers in CSF or plasma is performed with ELISA, Simoa, ECLIA, Luminex, Olink, or LC-MS / MS; more particularly, ELISA, Simoa, ECLIA, or Luminex; even more particularly Simoa or ELISA, even more particularly Simoa.

[0128] In particular embodiments, the thresholds defining neuroinflammation refer to quantitative values that distinguish normal physiological states from active or pathological neuroinflammation. In particular embodiments, such thresholds are established from reference populations, validated clinical datasets, or assay-specific reference ranges; they typically reflect values that exceed normal biological variability. In particular embodiments, for biomarkers determined in bodily fluid samples, neuroinflammation is considered present when the measured concentration is not within the range defined for a healthy reference population and / or exceeds the neuroinflammatory threshold established for the respective biomarker in the respective bodily fluid (e.g. cerebrospinal fluid, serum, or plasma).

[0129] For the following biomarkers, the healthy range and the corresponding neuroinflammatory threshold are in particular embodiments as described below (for all biomarkers, “0” denotes the lower limit of detection (LLOQ) of the respective validated analytical method.):

[0130] GFAP (Glial Fibrillary Acidic Protein)

[0131] • CSF: healthy range 0 - 715 pg / mL; neuroinflammatory threshold > 715 pg / L.

[0132] • Serum / Plasma: healthy range 0 - 85 pg / mL; neuroinflammatory threshold > 90 pg / mL.

[0133] YKL-40 (Chitinase-3 -like protein 1, CHI3L1)

[0134] • CSF: healthy range 160-300 pg / mL; neuroinflammatory threshold > 300 pg / mL.

[0135] • Serum / Plasma: healthy range 14-155 pg / mL; neuroinflammatory threshold > 155 pg / mL. sTREM2 (soluble Triggering Receptor Expressed on Myeloid Cells 2

[0136] • CSF: healthy range 250 - 280 pg / mL; neuroinflammatory threshold > 400 pg / mL

[0137] Neopterin

[0138] • CSF: healthy range 3 - 10 pmol / mL; neuroinflammatory threshold > 30 pmol / mL.

[0139] • Serum / Plasma: healthy range 6.3-12.3 pmol / mL; neuroinflammatory threshold > 12.3 pmol / mL. S100B

[0140] • Serum / Plasma: healthy range 25-1000 pg / mL; neuroinflammatory threshold > 1000 pg / mL.

[0141] • CSF if applicable: healthy range 300 - 800 pg / mL; neuroinflammatory threshold > 800 pg / mL. sCD163

[0142] • CSF: healthy range below detection; neuroinflammatory threshold > 10 ng / mL.

[0143] • Serum / Plasma: healthy range 0.7-1 ng / mL; neuroinflammatory threshold > 1 ng / mL.

[0144] In specific embodiments said bodily fluid is selected from the group comprising whole blood, serum, plasma and cerebrospinal fluid.

[0145] In other specific embodiments said bodily fluid is CSF.

[0146] In specific embodiments the level of said biomarker of neuroinflammation is detected with an assay, in particular an immunoassay in a bodily fluid sample of said subject. A variety of immunoassays are known and are used in particular embodiments of the assays and methods of the present invention. In specific embodiments of the present invention, the immunoassay is selected from the group comprising luminescence immunoassay (LIA), immunoluminometric assay (ILMA), radioimmunoassays ("RIA"), homogeneous enzyme-multiplied immunoassays ("EMIT"), enzyme linked immunoadsorbent assays ("ELISA"), apoenzyme reactivation immunoassay ("ARIS"), chemiluminescence- (“CLIA”), electrochemiluminescence- (“ECLIA”) and fluorescenceimmunoassays, luminescence-based bead arrays, magnetic beads based arrays, protein microarray assays, rapid test formats such as for instance dipstick immunoassays, immuno-chromatographic strip tests, rare cryptate assay, automated systems / analyzers and multiplex assays.

[0147] In certain embodiments said biomarker of neuroinflammation is detected in a bodily fluid sample of said subject using an ultra- sensitive immunoassay (e.g. Simoa).

[0148] Single Molecule Array (Simoa) is an ultra-sensitive digital immunoassay technology that can detect extremely low-abundance biomarkers by isolating individual immunocomplexes on beads. Simoa has proven especially useful for measuring plasma levels of biomarkers like NfL and GFAP that were previously difficult to reliably quantify in blood. The heightened sensitivity (femtomolar range) allows early detection of subtle changes, making it invaluable for tracking disease progression or treatment effects in neurodegenerative disorders.

[0149] Neuroimaging Techniques for In Vivo Neuroinflammation Assessment

[0150] Modern neuroimaging provides powerful, non-invasive tools to monitor neuroinflammatory changes in living patients, which are used in certain embodiments of the present invention and which are described in the following.

[0151] Positron Emission Tomography (PET)

[0152] PET imaging uses radiotracers to visualize specific metabolic or molecular processes related to neuroinflammation in the brain. PET molecular imaging is the most widely used clinical method to image microglial / astrocyte activation and other inflammatory targets (including TSPO, MAO-B, P2X7, CSF1R, etc.), wherein in more particular embodiments TSPO or MAO-B are used.

[0153] In more particular embodiments, radioligands for the 18kDa translocator protein (TSPO), which is expressed on activated microglia, are used to image neuroinflammation. More particular TSPO tracers are [A11C]PK11195, [A11C]PBR28,A18F-labeled tracers (e.g. [A18F] DPA-714, [A18F]PBR111), [A11C]DAA11O6, or [A11C]ER176; they can reveal spatial and temporal patterns of microglial activation in vivo. These inflammation-targeted PET scans have been applied in conditions such as AD, multiple sclerosis, and traumatic brain injury to measure the degree of glial activation. In other more particular embodiments, L-deprenyl is used; this isan irreversible monoamine oxidase B (MAO-B) inhibitor. The enzyme MAO-B exists on the outer mitochondrial membrane, occurring predominantly in astrocytes. Even more particularly, the PET tracer11C-deuterium-L-deprenyl (11C-DED) is used; this has high affinity and specificity for MAO-B. These MAO-B / astrocyte tracers can be used to detect reactive astrocytosis, a common feature in neuroinflammation (Carter et al. 2012. Nucl Med 2012; 53:37-46).

[0154] In other more particular embodiments, the purinergic receptor type-2X7 (P2X7R) is detected; this is expressed by virtually all cells of the innate and adaptive immune system and is mainly expressed in microglia as part of the immune cells of the central nervous system (CNS). P2X7 receptor is an inflammatory ATP-gated ion channel involved in cytokine release (IL-1β). In other more particular embodiments, P2X7R tracers for imaging, e.g. [18F]JNJ-64413739, are used; these have been described e.g. in (Schmidt et al. 2023. Int. J. Mol. Sci. 24: 1374).

[0155] In other more particular embodiments, Colony-stimulating factor- 1 receptor (CSF1R) is detected; this is specifically expressed in microglia in the brain and is up-regulated in activated microglia in many neurological diseases. Therefore, CSF1R is a potential biomarker and hence an attractive imaging target of neuroinflammation. In other more particular embodiments, PET tracers (e.g.,A11C-CPPC;A11C-GW2580) are used; these have been developed and validated in preclinical models and early translational work. These tracers aim for greater microglia specificity than TSPO (Horti et al. 2019. PNAS 116(5): 1686-1691 ).

[0156] In other more particular embodiments, Imidazoline type-2 binding sites (I2BS) are detected; these are located on the mitochondrial outer membranes and co-expressed with the monoamine oxidase B (MAO-B) in astrocytes and brain imaging with I2BS PET is thought to represent a valuable marker of astrogliosis in vivo. In other more particular embodiments, the tracer [11C]BU99008 for I2BS PET is used; this is described e.g. in (Livingston et al. 2022. Mol Psychiatry 27(4): 2019-2029).

[0157] Magnetic Resonance Imaging (MRI)

[0158] MRI is used in particular embodiments; this provides high-resolution structural and functional information without radiation. MRI modalities are used to access secondary consequences of neuroinflammation. In more particular embodiments, advanced techniques are used, which are detailed in the following; these enable more direct detection of glial activation-related processes:

[0159] • Quantitative Susceptibility Mapping (QSM): QSM quantifies tissue magnetic susceptibility and enables in vivo assessment of iron accumulation, which is closely associated with microglial activation and chronic oxidative stress. Elevated iron content in deep gray matter regions has been observed in Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis, marking QSM as one of the most specific MRI-based indicators of neuroinflammation. • Magnetic resonance spectroscopy [MRS]: Myo-Inositol is a metabolite primarily produced by glial cells in the brain and its levels can indicate glial activation and inflammation measured by MRS. MRS non-invasively measures brain metabolites such as myo-inositol and choline, which are primarily derived from astrocytes and increase during glial activation. Decreased N- acetylaspartate (NAA) reflects neuronal dysfunction. MRS is therefore regarded as the most specific metabolic MRI technique for detecting neuroinflammatory changes

[0160] In certain embodiments said neuroimaging technique is selected from TSPO-PET, MAO-B-PET, P2X7-PET, CSF1R-PET, I2BS-PET, SV2APET, MRS and QSM-MRI. In more particular embodiments, said neuroimaging technique is selected from the group comprising TSPO-PET, MAO-B-PET, P2X7-PET, CSF1R-PET, and I2BS-PET.

[0161] In even more particular embodiments, said neuroimaging technique is selected from the group comprising TSPO-PET and MAO-B-PET.

[0162] In yet even more particular embodiments, said neuroimaging technique is TSPO-PET using [18F]DPA-714 tracer.

[0163] In certain embodiments, neuroinflammation can be determined by quantitative imaging metrics deviating from reference values established in healthy control populations, which are detailed further in the following.

[0164] For PET, outcome measures include in certain embodiments the standardized uptake value ratio (SUVR), reflecting tracer uptake normalized to a reference region; binding potential (BPND), indicating receptor or target binding density; distribution volume (VT), representing the equilibrium volume of distribution of the tracer; and the kinetic constant (λk3), describing the tracer transfer rate to the bound compartment.

[0165] In more particular embodiments reference ranges and indicative deviations are as follows:

[0166] • Healthy range: SUVR 0.9-1.1, BPND 0.5-1.0, VT 2.0–4.0 mL / cm3, λk30.005–0.015 min-1, or within ±10 % of the control mean. The term control mean refers to the average value obtained from a representative healthy reference population, which defines the normal physiological range for the respective parameter or biomarker and may be adjusted for demographic factors such as age or sex as appropriate.* Indicative of neuroinflammation: increase > 20 % above the healthy reference mean in relevant brain regions (e.g., cortical, subcortical, or white matter regions expressing TSPO or other neuroinflammatory targets). Biomarkers may also be referenced against an internal reference in the same subject. This may for instance be the biomarker level in a reference region of the body or tissue wherein the level of the biomarker is not affected by the pathophysiological condition (e.g. neuroinflammation). The biomarker level in said reference region of the body or tissue therefore can serve as a reference value against which the biomarker level in the region of the body or tissue (target) that is affected by the pathophysiological condition is compared. The skilled person knows which region of the body or tissue wherein the level of the biomarker is not affected by the pathophysiological condition for a given pathophysiological condition.

[0167] More particularly, for SUVR and BPND, the biomarker is normalized within the same subject, and comparison to the control mean may serve for interpretation of normal versus pathological values. For MRI / MRS, parameters sensitive to neuroinflammatory changes include the myo-inositol / creatine (ml / Cr) ratio, reflecting glial proliferation; fractional anisotropy (FA), indicative of white-matter microstructural integrity; quantitative susceptibility mapping (QSM), representing iron deposition and microglial activation; and cerebral blood flow (CBF), assessing perfusion alterations. Typical reference ranges and indicative deviations are as follows:

[0168] • Healthy range: ml / Cr 0.35-0.55, FA 0.45-0.70, QSM 20-40 ppb, CBF 45-65 mL / 100 g / min, or within ±10 % of the control mean.

[0169] • Indicative of neuroinflammation: deviation > 20 % from the healthy reference mean in the corresponding brain region.

[0170] Thresholds within these defined ranges typically represent reproducible and clinically meaningful deviations from physiological baseline values and are interpreted as indicative of neuroinflammation.

[0171] In certain embodiments, said quantitative imaging parameter is selected from the group comprising the standardized uptake value ratio (SUVR), binding potential (BPND), distribution volume (VT), and kinetic constant (λk3) when obtained from PET imaging, and from the myo-inositol / creatine ratio (ml / Cr), fractional anisotropy (FA), quantitative susceptibility mapping (QSM), and cerebral blood flow (CBF) when obtained from MRI or magnetic resonance spectroscopy (MRS).

[0172] In certain more particular embodiments, said quantitative imaging parameter is selected from the group comprising SUVR, BPND, ml / Cr ratio, and QSM, each representing reproducible and clinically validated outcome measures of neuroinflammatory activity.

[0173] In even more particular embodiments, said quantitative imaging parameter is selected from the group comprising SUVR, BPND, and ml / Cr ratio. In yet even more particular embodiments, said quantitative imaging parameter is SUVR, representing the standardized uptake value ratio derived from PET imaging using a reference region.

[0174] The skilled person is aware which reference region is suitable for the respective tracer and imaging method. The reference region refers to a brain region that remains unaffected by neuroinflammatory processes and thus provides a stable baseline for normalization of tracer uptake.

[0175] Improvement of Neuroinflammation

[0176] In certain embodiments, improvement of neuroinflammation is defined as a) a decrease in the level of at least one biomarker selected from the group comprising sTREM2, YKL-40 and GFAP by at least 10 %, particularly by at least 20 %, more particularly by at least 30 %, and even more particularly by at least 50 % relative to the baseline concentration measured in said subject prior to treatment and / or b) normalization of the biomarker level to a value that is within the range observed for healthy individuals, in particular a GFAP level of 0–715 pg / mL in a CSF sample of said subject or 0–90 pg / mL in a plasma sample of said subject; and / or a YKL-40 level of 160–300 pg / mL in a CSF sample of said subject or 14–155 pg / mL in a plasma sample of said subject; and / or a sTREM2 level or 250–280 pg / mL in a CSF sample of said subject.

[0177] In this context, “prior to treatment” in the present invention refers in particular to a time span within which a medical professional would expect that such parameter or biomarker level is essentially unchanged from when they were determined, more particularly a span of several days, weeks or months, such as 1-6 days, or 1-4 weeks, or 1-3 months, or 1-6 months, prior to the administration of PAM or fragments thereof according to the present invention. Healthy individuals may be from a control population of healthy individuals, which may be age-adjusted, as required.

[0178] In certain embodiments, improvement of neuroinflammation is considered achieved when at least one, particularly two or more, of said biomarkers exhibit such reduction or normalization, in more particular embodiments, the improvement is determined primarily by a decrease or normalization of GFAP. In other embodiments, improvement of neuroinflammation is determined by imaging techniques selected from the group comprising PET and / or MRI / MRS.

[0179] In particular embodiments, improvement of neuroinflammation is defined as a reduction of imaging signal indicative of neuroinflammation, wherein the PET-derived outcome measure is selected from the group comprising standardized uptake value ratio (SUVR), binding potential (BPND), distribution volume (VT), or kinetic constant (λk3), and the MRI / MRS-derived parameter is selected from myo-inositol / creatine ratio, fractional anisotropy, quantitative susceptibility mapping (QSM) iron load. More particularly, said improvement is achieved when the PET- and / or MRI / MRS-derived parameter a) is reduced by at least 10 %, particularly by at least 15 %, more particularly by at least 20 %, and even more particularly by at least 30 % relative to the baseline value measured in the same subject prior to treatment and / or b) normalized to values within the ranges observed in healthy controls upon follow-up imaging, wherein the imaging parameter is selected from SUVR and / or BPND and / or QSM iron load and / or ml / Cr ratio.

[0180] In certain embodiments, normalization of imaging biomarkers leading to the improvement of neuroinflammation is achieved when the SUVR falls within the range of 0.9- 1.1, particularly between 0.9 and 1.0, more particularly between 0.9 and 0.95; and / or the BPND falls within the range of 0.5-1.0, particularly between 0.5 and 0.8, more particularly between 0.5 and 0.7; and / or the QSM iron load falls within the range of 20-40 ppb, particularly between 20 and 30 ppb, more particularly between 20 and 25 ppb; and / or the ml / Cr ratio falls within the range of 0.35-0.55, particularly between 0.35 and 0.45, more particularly between 0.35 and 0.40.

[0181] The terms SUVR, BPND, VT, and λk3refer to quantitative outcome measures commonly used in molecular PET imaging. These parameters describe the relative or absolute radiotracer binding in a target brain region and are calculated according to established kinetic modeling approaches known to the person skilled in the art. The skilled person is capable of selecting an appropriate reference region, image quantification method, and modeling algorithm for the tracer used to ensure reproducible measurement of neuroinflammatory activity.

[0182] Subject matter of the present invention is modified or unmodified Peptidylglycine a-amidating monooxygenase (PAM) or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function.

[0183] Cognitive function according to the present invention refers to the mental processes that allow individuals to receive, select, store, transform, develop, and recover information from external stimuli. Cognition encompasses several well-recognized but often overlapping domains including attention, executive function, memory, language, visuospatial and constructional abilities, sensory-perceptual-motor skills and social cognition (Zhang 2019. ArXiv, 1907.02863). In the present invention, cognition and cognitive function are used interchangeably.

[0184] In certain embodiments of the present invention cognitive function comprises mental processes that allow individuals to receive, select, store, transform, develop, and recover information from external stimuli. Cognition encompasses several well-recognized but often overlapping domains including attention, executive function, memory, language, visuospatial and constructional abilities, sensory-perceptual-motor skills and social cognition. In particular embodiments cognitive function comprises memory function including spatial memory and anxiety -like behavior. Certain embodiments of the present invention relate to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of memory, wherein said subject has a reduction in cognitive function.

[0185] Methods to assess cognitive function / impairment

[0186] Cognitive impairment is a multifaceted condition characterized by deficits in cognitive processes, including memory, attention, executive function, and language. Defined broadly, it refers to a noticeable decline from an individual's prior level of cognitive performance that can interfere with daily living and quality of life. This impairment can manifest in various forms, ranging from mild cognitive impairment (MCI), which may precede dementia, to more severe conditions like Alzheimer’s disease and other neurodegenerative disorders. The assessment of cognitive impairment employs a variety of methodologies, integrating both neuropsychological testing and neuroimaging techniques.

[0187] A range of clinical methods currently used to assess cognitive function in individuals include:

[0188] Neuropsychological tests are key diagnostic tools for assessing individuals with dementia and mild cognitive impairment, but they are also used for other neurological conditions. Below are the most commonly applied clinical tests, a brief description of each, and their limitations. More comprehensive reviews of currently available neuropsychological tests are reviewed in Cullen etal, J Neurol Neurosurg Psychiatry. 2007 Aug;78(8):790-9; Zucchella et al. Neuropsychological testing, Pract Neurol., 2018;0:1-11.

[0189] • Mini-Mental State Examination (MMSE): Commonly used 30-point questionnaire that screens for cognitive impairment, addressing the domains of orientation, registration, attention and calculation, recall, and language. Widely recognized and used with extensive normative data available. Influenced by the patient’s educational and cultural background; less sensitive to mild cognitive impairment and non-Alzheimer's dementias.

[0190] • Montreal Cognitive Assessment (MoCA): Screening tool for detecting mild cognitive dysfunction, covering a broader range of cognitive domains than MMSE such as executive functions, memory, attention, language, visuoconstructional skills, conceptual thinking, calculations, and orientation. Sensitive to mild cognitive impairment but more complex to administer and requires specific training.

[0191] o 3MS (Modified Mini -Mental State): Extends the MMSE, covering a broader range of cognitive abilities with added focus on memory, language, and visuospatial skills. It provides a more comprehensive assessment but remains influenced by educational and cultural backgrounds. • ADAS-Cog (11 / 13 / 14): Multi-domain performance scale (memory, language, praxis) widely used in AD drug trials. Scoring: version-dependent total 0-70+), higher = worse. Gradation: no universal diagnostic cutoffs; severity and treatment effects judged by absolute level and change from baseline (greater increase = greater decline).

[0192] • Trail Making Test (TMT): Two-part test involving connecting numbered and lettered dots in sequence, assessing attention, processing speed, and mental flexibility. Influenced by physical and visual impairments.

[0193] • Clock Drawing Test: Involves a patient drawing a clock and setting a specific time, assessing executive function and visuospatial abilities. Quick to administer but scoring can be subjective and affected by the patient’s educational and cultural background.

[0194] • Boston Naming Test (BNT): Measures lexical retrieval and language function by asking participants to name depicted objects. Widely used, well-standardized, sensitive to aphasia and other language disorders.

[0195] • Controlled Oral Word Association Test (COWAT): Measures verbal fluency by asking individuals to rapidly generate words beginning with a given letter. Assesses executive function and language, sensitive to frontal lobe dysfunction.

[0196] • Rey Complex Figure Test and Recognition Trial (RCFT): Assesses visuospatial ability and memory through the drawing and recall of a complex figure. Provides insights into the person's organizational strategy and memory recall abilities.

[0197] • Addenbrooke’s Cognitive Examination (ACE): Comprehensive test assessing a wide range of cognitive domains such as attention, memory, verbal fluency, language, and visuospatial abilities, useful for differentiating among types of dementia. Detailed but requires more time and training to administer effectively.

[0198] • Addenbrooke’s Cognitive Examination-Revised (ACE-R): An extension of the ACE, enhancing detail in the cognitive assessment. Highly sensitive and specific for diagnosing dementia and monitoring disease progression. Longer to administer; requires trained personnel.

[0199] • Mini-Cog: Combines a 3-item recall test with a simple clock-drawing test to screen for cognitive impairment. Quick and simple, requires no special equipment or advanced training but has a limited scope and may not detect all types of cognitive impairment.

[0200] • Abbreviated Mental Test Score (AMTS): A brief 10-item scale assessing general cognitive functions such as orientation, memory, and attention. Quick to administer without the need for writing or drawing, making it suitable for patients with physical disabilities. However, some items may be culturally biased; more suitable for excluding rather than diagnosing dementia.

[0201] • General Practitioner Assessment of Cognition (GPCOG): Screening tool specifically designed for use in primary care to detect early cognitive impairment, including an informant interview component to enhance sensitivity. Less well-known and less frequently used compared to tests like MMSE and MoCA.

[0202] • Stroop Test: Assesses cognitive flexibility, processing speed, and attention by inhibiting cognitive interference. Highly sensitive to brain dysfunction and cognitive decline but requires specific materials for testing and may be affected by education and cultural background.

[0203] • Six Item Cognitive Impairment Test (6CIT): Very brief test including orientation to time and memory recall. Simple and quick with minimal cultural bias, suitable for initial screening but less detailed.

[0204] • Geriatric Depression Scale (GDS): Identifies depression in the elderly, important as depressive symptoms can overlap with cognitive impairment. Specifically designed for the elderly and sensitive to the affective component of cognitive decline but focuses on mood rather than cognitive impairment.

[0205] • Neuropsychiatric Inventory (NPI): Assesses psychiatric and problematic behavioral symptoms in dementia patients, providing comprehensive coverage of neuropsychiatric symptoms. Requires an informant and is time-consuming to administer.

[0206] • Global Deterioration Scale (GDS): Stages dementia from no cognitive decline to very severe cognitive decline, useful for assessing progression. Requires clinical judgment and may be subjective.

[0207] • Consortium to Establish a Registry for Alzheimer's Disease (CERAD) Word List: Part of the CERAD battery, used to assess memory function by testing immediate and delayed recall. Well- validated, specific for Alzheimer’s type memory deficits, but focuses primarily on memory. • CERAD battery or CERAD total score: The CERAD total score is a summary score from the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) neuropsychological battery, typically calculated by adding the raw scores of specific subtests, such as the Modified Boston Naming Test, Word List Learning, Word List Delayed Recall, and Figure Copy. This total score provides a single measure of global cognitive function, used to differentiate between cognitively normal individuals and those with conditions like Alzheimer's disease.

[0208] • Phonetic Fluency and Semantic Fluency (Animals): Tests verbal fluency by asking individuals to generate words from a category (semantic) or that begin with a specific letter (phonetic) within a limited time. Quick and effective but influenced by education, language, and cultural factors.

[0209] • MCI Screen: is a brief neuropsychological test checking for mild cognitive impairment (MCI).

[0210] The protocol consists of an immediate recall task, a triadic comparison task, a judgment task, a delayed free recall task, a cued-recall task, and a rehearsed recall task. It is scored using correspondence analysis and statistical methods for differentiating normal cognitive function from Mild cognitive impairment. It was derived from the protocol of the CERAD 10-word recall test.

[0211] • Wechsler Batteries:

[0212] o Digit Span Tests: Assess attention and working memory by having subjects repeat or sequence numbers. Widely used to evaluate attentional capacities, potentially influenced by cognitive load and distractibility.

[0213] o Wechsler Memory Scale III Logical Memory Subtest: Assesses immediate and delayed recall of stories, providing a detailed assessment of memory function, including complex information processing. Requires more time and specialized training to administer.

[0214] o Wechsler Adult Intelligence Scale III (WAIS-III) Digits Test: Tests working memory and attention by having patients recall sequences of numbers, assessing attentional capacity and mental manipulation. Can be impacted by anxiety, fatigue, or educational background.

[0215] o Wechsler Memory Scale-IV, Logical Memory II (WMS-IV LM-II): Delayed storyrecall measure of episodic memory. Scoring: age-adjusted scaled scores / percentiles (higher = better). Gradation: impairment relative to norms (≈ ≤ −1 SD suggests MCI-level deficit; ≤ −1.5 / −2 SD often seen in mild AD).

[0216] Computerized neuropsychological testing is modern clinical neuropsychological assessment, offering standardized testing environments and objective data collection for cognitive performance. The most commonly applied tests are listed below:

[0217] • CANTAB (Cambridge Neuropsychological Test Automated Battery): assessment of various aspects of cognitive function including memory, attention, executive function, and decisionmaking (Robbins et al. (1994); Dementia, 5(5), 266-281).

[0218] • CNS Vital Signs: assessment of memory, processing speed, and executive function (Gualtieri, C. T, & Johnson, L. G. (2006). Archives of Clinical Neuropsychology, 21(7), 623-643);

[0219] • Automated Neuropsychological Assessment Metrics (ANAM): assessment of cognitive functioning post-injury and to monitor cognitive readiness (Vincent et al. (2012), Archives of Clinical Neuropsychology, 27(8), 813-821);

[0220] • ImPACT (Immediate Post-Concussion Assessment and Cognitive Testing): assessment of cognitive recovery following concussion (Iverson et al. (2003) Journal of Clinical and Experimental Neuropsychology, 25(6), 683-689);

[0221] • Mindstreams: assessments of cognitive functioning in areas such as memory, attention, and problem-solving (Dwolatzky et al. (2003). BMC Geriatrics, 3, 4). Daily Living Skill Assessment is a wide-spread tool, which helps to evaluate cognitive and functional abilities in individuals with suspected cognitive impairments (reviewed in Cullen Bet al., J Neurol Neurosurg Psychiatry. 2007 Aug;78(8):790-9; Zucchella et al. Neuropsychological testing, Pract Neurol., 2018;0:1-11):

[0222] • Activities of Daily Living (ADLs): basic self-care tasks that an individual performs every day, such as bathing, dressing, eating, toileting, and transferring (moving from one place to another, like from bed to chair). The ability to perform these tasks can reflect one's physical and sometimes cognitive functioning. Common tools for assessing ADLs include the Katz ADL scale, which helps determine the level of assistance an individual might need.

[0223] • Instrumental Activities of Daily Living (IADLS): include activities such as handling finances, managing medications, cooking, shopping, using the telephone, and housekeeping. These tasks require cognitive abilities like planning, problem solving, and memory, along with physical capability. The Lawton IADL Scale is a popular instrument used to evaluate these skills and is particularly insightful for detecting early signs of cognitive decline and assessing the impact of cognitive changes on one's independence.

[0224] • Functional Activities Questionnaire (FAQ): measures the ability to perform IADLs, assessing how cognitive impairments affect day-to-day functioning. It is often completed by a caregiver or family member who provides insight into the individual’s capability to manage their everyday activities.

[0225] • ADCS-ADL (and ADCS-iADL): Informant-rated functional ability in basic and instrumental activities. Scoring: instrument-specific totals (e.g., ADCS-ADL commonly 0-78), higher = better. Gradation: no fixed diagnostic thresholds; longitudinal decline maps progression from mild (early iADL loss) to moderate / severe (broader ADL dependence).

[0226] • DAD (Disability Assessment for Dementia): Informant-based measure of independence across basic / iADL / leisure domains. Scoring: 0-100% independence, higher = better. Gradation: lower percentages reflect mild → moderate → severe disability; change over time preferred for staging Neuroimaging techniques are applied for accessing the cognition, by precisely monitoring the changes in the brain areas responsible for language, memory etc. also known as brain mapping. The most common techniques are listed below (more comprehensive review in Stufflebeam SM, Rosen BR. Neuroimaging Clin N Am. 2007 Nov; 17(4):469-84, viii-ix):

[0227] • Functional MRI (fMRI): is used to examine the brain's functional anatomy related to cognitive processes by detecting areas of increased blood flow during cognitive tasks. This helps identify specific brain regions involved in memory, language, problem-solving, and decision-making in various neurological conditions like dementia, stroke, and traumatic brain injuries. • Positron Emission Tomography (PET): imaging of metabolic processes and neurotransmitter activity in the brain, which are directly linked to cognitive function. PET scans are used to assess cerebral glucose metabolism, which declines in specific patterns in Alzheimer’s disease and other dementias, providing insights into cognitive decline. Additionally, PET can image the distribution of neurotransmitter receptors, helping to understand the neurochemical basis of cognitive functions and their disorders.

[0228] • Electroencephalography (EEG): accesses the temporal dynamics of cognitive processes by measures the brain's electrical activity, which can be analyzed to understand cognitive function across various tasks like attention, learning, and memory. EEG is also used to investigate how brain activity is linked to different cognitive states and to diagnose cognitive impairment related to diseases affecting brain function.

[0229] • Magnetoencephalography (MEG): provides detailed information about the timing and source of brain activity during cognitive tasks with better spatial resolution when compared to EEG. It is used to map cognitive brain functions such as language comprehension and visual processing. MEG is particularly useful in research for understanding the rapid changes in neural activity that accompany cognitive tasks and for mapping critical areas before brain surgery to avoid cognitive deficits.

[0230] • Diffusion Tensor Imaging (DTI): helps visualize and characterize the integrity of white matter tracts that connect different brain regions involved in cognition. By imaging these pathways, DTI provides insights into the structural connectivity that underlies cognitive functions such as language processing, spatial reasoning, and executive functions. It is particularly useful for identifying disruptions in neural pathways that can lead to cognitive impairments, as seen in conditions like multiple sclerosis and traumatic brain injuries.

[0231] Clinical global ratings are structured clinical assessments used to determine the overall stage or severity of cognitive and functional impairment, rather than to measure specific cognitive domains. They rely on a clinician’s comprehensive evaluation based on patient interviews, informant reports, and observations of daily functioning. These scales provide a global impression of disease severity and progression, integrating cognitive, behavioral, and functional aspects into a single score. Clinical global ratings are widely used in both research and clinical settings to monitor disease course, evaluate treatment response, and classify patients in clinical trials.

[0232] • Clinical Dementia Rating - Global (CDR-Global): Clinician-rated global stage from semistructured patient / informant interview. Scoring: 0 (normal), 0.5 (very mild), 1 (mild), 2 (moderate), 3 (severe); higher = worse. Gradation: CDR 0.5 ~MCI / prodromal AD; CDR > 1 indicates dementia. • Clinical Dementia Rating - Sum of Boxes (CDR-SB): Granular sum across six domains. Scoring: 0-18, higher = worse. Gradation (typical bands): ~0.5–4 very mild / MCI; ~4.5–9 mild AD; ~9.5–15 moderate; >15 severe (bands may vary by protocol).

[0233] Trial composites are multidomain cognitive and functional assessment tools developed primarily for use in clinical research to sensitively detect subtle changes in disease progression or treatment effects. Rather than relying on a single test, they integrate results from several validated instruments into a unified composite score, providing a more comprehensive measure of overall cognitive and functional performance. These composites are particularly valuable in early or preclinical stages of neurodegenerative disease, where changes in individual domains may be too small to detect reliably.

[0234] • iADRS (Integrated Alzheimer’s Disease Rating Scale): Trial composite integrating ADAS- Cogl3 (cognition) and ADCS-iADL (function) for sensitivity to change. Scoring: ≈ 0–144, higher = better. Gradation: no diagnostic cutoffs; efficacy readout is slower decline (change from baseline).

[0235] • ADCOMS (Alzheimer’s Disease Composite Score): Weighted composite from ADAS-Cog, MMSE, and CDR elements optimized for early-stage sensitivity. Scoring: unitless composite, higher = worse. Gradation: no fixed diagnostic thresholds; interpret change over time (less increase = benefit).

[0236] • NTB (Neuropsychological Test Battery — composite z-score): Protocol-defined set of neuropsych tests summarized as a composite. Scoring: z-score (mean 0, SD 1), higher = better. Gradation: baseline z < 0 indicates impairment; Az overtime tracks progression / response. • PACC (Preclinical Alzheimer’s Cognitive Composite): Composite z-score tuned for very early / preclinical decline (episodic memory plus supportive domains). Scoring: z-score, higher = better. Gradation: no diagnostic cutoffs; small longitudinal changes are informative.

[0237] In a more particular embodiment a range of clinical methods currently used to assess cognitive function in individuals include:

[0238] Neuropsychological tests are key diagnostic tools for assessing individuals with dementia and mild cognitive impairment, but they are also used for other neurological conditions. Below are the most commonly applied clinical tests, a brief description of each, and their limitations. More comprehensive reviews of currently available neuropsychological tests are reviewed in Cullen et al. 2007 Aug;78(8):790-9; Zucchella et al., Pract Neurol., 2018;0: 1-11.

[0239] Mini-Mental State Examination (MMSE): Commonly used 30-point questionnaire that screens for cognitive impairment, addressing the domains of orientation, registration, attention and calculation, recall, and language. Widely recognized and used with extensive normative data available. Influenced by the patient’s educational and cultural background; less sensitive to mild cognitive impairment and non-Alzheimer's dementias.

[0240] • Montreal Cognitive Assessment (MoCA): Screening tool for detecting mild cognitive dysfunction, covering a broader range of cognitive domains than MMSE such as executive functions, memory, attention, language, visuoconstructional skills, conceptual thinking, calculations, and orientation. Sensitive to mild cognitive impairment but more complex to administer and requires specific training.

[0241] o 3MS (Modified Mini -Mental State): Extends the MMSE, covering a broader range of cognitive abilities with added focus on memory, language, and visuospatial skills. It provides a more comprehensive assessment but remains influenced by educational and cultural backgrounds.

[0242] • Trail Making Test (TMT): Two-part test involving connecting numbered and lettered dots in sequence, assessing attention, processing speed, and mental flexibility. Influenced by physical and visual impairments.

[0243] • Clock Drawing Test: Involves a patient drawing a clock and setting a specific time, assessing executive function and visuospatial abilities. Quick to administer but scoring can be subjective and affected by the patient’s educational and cultural background.

[0244] • Boston Naming Test (BNT): Measures lexical retrieval and language function by asking participants to name depicted objects. Widely used, well-standardized, sensitive to aphasia and other language disorders.

[0245] • Controlled Oral Word Association Test (COWAT): Measures verbal fluency by asking individuals to rapidly generate words beginning with a given letter. Assesses executive function and language, sensitive to frontal lobe dysfunction.

[0246] • Rey Complex Figure Test and Recognition Trial (RCFT): Assesses visuospatial ability and memory through the drawing and recall of a complex figure. Provides insights into the person's organizational strategy and memory recall abilities.

[0247] • Addenbrooke’s Cognitive Examination (ACE): Comprehensive test assessing a wide range of cognitive domains such as attention, memory, verbal fluency, language, and visuospatial abilities, useful for differentiating among types of dementia. Detailed but requires more time and training to administer effectively.

[0248] • Addenbrooke’s Cognitive Examination-Revised (ACE-R): An extension of the ACE, enhancing detail in the cognitive assessment. Highly sensitive and specific for diagnosing dementia and monitoring disease progression. Longer to administer; requires trained personnel. • Mini-Cog: Combines a 3-item recall test with a simple clock-drawing test to screen for cognitive impairment. Quick and simple, requires no special equipment or advanced training but has a limited scope and may not detect all types of cognitive impairment.

[0249] • Abbreviated Mental Test Score (AMTS): A brief 10-item scale assessing general cognitive functions such as orientation, memory, and attention. Quick to administer without the need for writing or drawing, making it suitable for patients with physical disabilities. However, some items may be culturally biased; more suitable for excluding rather than diagnosing dementia.

[0250] • General Practitioner Assessment of Cognition (GPCOG): Screening tool specifically designed for use in primary care to detect early cognitive impairment, including an informant interview component to enhance sensitivity. Less well-known and less frequently used compared to tests like MMSE and MoCA.

[0251] • Stroop Test: Assesses cognitive flexibility, processing speed, and attention by inhibiting cognitive interference. Highly sensitive to brain dysfunction and cognitive decline but requires specific materials for testing and may be affected by education and cultural background.

[0252] • Six Item Cognitive Impairment Test (6CIT): Very brief test including orientation to time and memory recall. Simple and quick with minimal cultural bias, suitable for initial screening but less detailed.

[0253] • Geriatric Depression Scale (GDS): Identifies depression in the elderly, important as depressive symptoms can overlap with cognitive impairment. Specifically designed for the elderly and sensitive to the affective component of cognitive decline but focuses on mood rather than cognitive impairment.

[0254] • Neuropsychiatric Inventory (NPI): Assesses psychiatric and problematic behavioral symptoms in dementia patients, providing comprehensive coverage of neuropsychiatric symptoms. Requires an informant and is time-consuming to administer.

[0255] • Global Deterioration Scale (GDS): Stages dementia from no cognitive decline to very severe cognitive decline, useful for assessing progression. Requires clinical judgment and may be subjective.

[0256] • Consortium to Establish a Registry for Alzheimer's Disease (CERAD) Word List: Part of the CERAD battery, used to assess memory function by testing immediate and delayed recall. Well- validated, specific for Alzheimer’s type memory deficits, but focuses primarily on memory. • Phonetic Fluency and Semantic Fluency (Animals): Tests verbal fluency by asking individuals to generate words from a category (semantic) or that begin with a specific letter (phonetic) within a limited time. Quick and effective but influenced by education, language, and cultural factors.

[0257] • Wechsler Batteries: o Digit Span Tests: Assess attention and working memory by having subjects repeat or sequence numbers. Widely used to evaluate attentional capacities, potentially influenced by cognitive load and distractibility.

[0258] o Wechsler Memory Scale III Logical Memory Subtest: Assesses immediate and delayed recall of stories, providing a detailed assessment of memory function, including complex information processing. Requires more time and specialized training to administer.

[0259] Wechsler Adult Intelligence Scale III (WAIS-III) Digits Test: Tests working memory and attention by having patients recall sequences of numbers, assessing attentional capacity and mental manipulation. Can be impacted by anxiety, fatigue, or educational background.

[0260] Computerized neuropsychological testing is modern clinical neuropsychological assessment, offering standardized testing environments and objective data collection for cognitive performance. The most commonly applied tests are listed below:

[0261] • CANTAB (Cambridge Neuropsychological Test Automated Battery): assessment of various aspects of cognitive function including memory, attention, executive function, and decisionmaking (Robbins, et al. (1994); Dementia, 5(5), 266-281).

[0262] • CNS Vital Signs: assessment of memory, processing speed, and executive function (Gualtieri, C. T, & Johnson, L. G. (2006). Archives of Clinical Neuropsychology, 21(7), 623-643);

[0263] • Automated Neuropsychological Assessment Metrics (ANAM): assessment of cognitive functioning post-injury and to monitor cognitive readiness (Vincent, et al. (2012). Archives of Clinical Neuropsychology, 27(8), 813-821);

[0264] • ImPACT (Immediate Post-Concussion Assessment and Cognitive Testing): assessment of cognitive recovery following concussion (Iversonet al. (2003). Journal of Clinical and Experimental Neuropsychology, 25(6), 683-689);

[0265] • Mindstreams: assessments of cognitive functioning in areas such as memory, attention, and problem-solving (Dwolatzky, et al. (2003). BMC Geriatrics, 3, 4).

[0266] Daily Living Skill Assessment is a wide-spread tool, which helps to evaluate cognitive and functional abilities in individuals with suspected cognitive impairments (reviewed in et al. J Neurol Neurosurg Psychiatry. 2007 Aug;78(8):790-9; Zucchella et al. Pract Neurol., 2018;0:1–11):

[0267] Activities of Daily Living (ADLs): basic self-care tasks that an individual performs every day, such as bathing, dressing, eating, toileting, and transferring (moving from one place to another, like from bed to chair). The ability to perform these tasks can reflect one's physical and sometimes cognitive functioning. Common tools for assessing ADLs include the Katz ADL scale, which helps determine the level of assistance an individual might need.

[0268] • Instrumental Activities of Daily Living (IADLS): include activities such as handling finances, managing medications, cooking, shopping, using the telephone, and housekeeping. These tasks require cognitive abilities like planning, problem solving, and memory, along with physical capability. The Lawton IADL Scale is a popular instrument used to evaluate these skills and is particularly insightful for detecting early signs of cognitive decline and assessing the impact of cognitive changes on one's independence.

[0269] Functional Activities Questionnaire (FAQ): measures the ability to perform IADLs, assessing how cognitive impairments affect day-to-day functioning. It is often completed by a caregiver or family member who provides insight into the individual’s capability to manage their everyday activities. Neuroimaging techniques are applied for accessing the cognition, by precisely monitoring the changes in the brain areas responsible for language, memory etc. also known as brain mapping. The most common techniques are listed below (more comprehensive review in Stufflebeam SM, Rosen BR. Neuroimaging Clin N Am. 2007 Nov; 17(4):469-84, viii-ix);

[0270] • Functional MRI (fMRI): is used to examine the brain's functional anatomy related to cognitive processes by detecting areas of increased blood flow during cognitive tasks. This helps identify specific brain regions involved in memory, language, problem-solving, and decision-making in various neurological conditions like dementia, stroke, and traumatic brain injuries.

[0271] • Positron Emission Tomography (PET): imaging of metabolic processes and neurotransmitter activity in the brain, which are directly linked to cognitive function. PET scans are used to assess cerebral glucose metabolism, which declines in specific patterns in Alzheimer’s disease and other dementias, providing insights into cognitive decline. Additionally, PET can image the distribution of neurotransmitter receptors, helping to understand the neurochemical basis of cognitive functions and their disorders.

[0272] • Electroencephalography (EEG): accesses the temporal dynamics of cognitive processes by measures the brain's electrical activity, which can be analyzed to understand cognitive function across various tasks like attention, learning, and memory. EEG is also used to investigate how brain activity is linked to different cognitive states and to diagnose cognitive impairment related to diseases affecting brain function.

[0273] • Magnetoencephalography (MEG): provides detailed information about the timing and source of brain activity during cognitive tasks with better spatial resolution when compared to EEG. It is used to map cognitive brain functions such as language comprehension and visual processing. MEG is particularly useful in research for understanding the rapid changes in neural activity that accompany cognitive tasks and for mapping critical areas before brain surgery to avoid cognitive deficits.

[0274] • Diffusion Tensor Imaging (DTI): helps visualize and characterize the integrity of white matter tracts that connect different brain regions involved in cognition. By imaging these pathways, DTI provides insights into the structural connectivity that underlies cognitive functions such as language processing, spatial reasoning, and executive functions. It is particularly useful for identifying disruptions in neural pathways that can lead to cognitive impairments, as seen in conditions like multiple sclerosis and traumatic brain injuries.

[0275] In certain embodiments of the invention said reduction of cognitive function is assessed with a method selected from the group comprising Mini-Mental State Examination (MMSE); Montreal Cognitive Assessment (MoCA), Mini-Cog; General Practitioner Assessment of Cognition (GPCOG); Addenbrookes Cognitive Examination revised (ACE-R); Severe Impairment Battery (SIB); National adult reading test (NART); Neuropsychiatric Inventory (NPI); Basic / Instrumental Activities of Daily Living (BADL) / (IADL); Functional Activities Questionnaire (FAQ); depression (Geriatric Depression Scale [GDS]); disability, Interview for deterioration in daily living activities [IDDD]; verbal learning and episodic memory (evaluated with the Consortium to establish a registry for Alzheimer’s disease [CERAD] word list and Wechsler Memory Scale III logical memory subtest; attention and executive function (digits test from Wechsler adult intelligence scale III [WAIS-III], trail making test B [TMT-B], phonetic fluency (p), Stroop test, constructional praxis from CERAD); visual perception (letters test from the visual object and space perception battery [VOSP]; visuo-spatial function (number localization task from VOSP); agnosia (Poppelreuter figures test; psychomotor speed (TMT-A); semantic fluency (animal categories); and language (Boston naming test [BNT-Boston], CERAD Total Score; Cambridge Neuropsychological Test Automated Battery (CANTAB), CNS Vital Signs; Automated Neuropsychological Assessment Metrics (ANAM), ImPACT (Immediate Post-Concussion Assessment and Cognitive Testing) and Mindstreams; and trial-based composite and clinical global rating scales including the Alzheimer’s Disease Assessment Scale - Cognitive Subscale (ADAS-Cogll / 13 / 14); Clinical Dementia Rating - Global (CDR-Global); Clinical Dementia Rating - Sum of Boxes (CDR-SB); Integrated Alzheimer’s Disease Rating Scale (iADRS); Alzheimer’s Disease Composite Score (ADCOMS); Neuropsychological Test Battery (NTB); and Preclinical Alzheimer’s Cognitive Composite (PACC),

[0276] more particularly selected from the group comprising Mini-Mental State Examination (MMSE); Montreal Cognitive Assessment (MoCA), Mini-Cog; General Practitioner Assessment of Cognition (GPCOG); Addenbrookes Cognitive Examination revised (ACE-R); Severe Impairment Battery (SIB); National adult reading test (NART); Neuropsychiatric Inventory (NPI); and Basic / Instrumental Activities of Daily Living (BADL) / (IADL), Functional Activities Questionnaire (FAQ) and CERAD Total Score, and particularly further comprising at least one trial-validated global or composite endpoint selected from CDR-SB, ADAS-Cog, iADRS, or ADCOMS.

[0277] In more particular embodiments of the invention said reduction of cognitive function is assessed with a method selected from the group comprising Mini-Mental State Examination (MMSE); Montreal Cognitive Assessment (MoCA), Mini-Cog; General Practitioner Assessment of Cognition (GPCOG); Addenbrookes Cognitive Examination revised (ACE-R); Severe Impairment Battery (SIB); National adult reading test (NART); Neuropsychiatric Inventory (NPI); Basic / Instrumental Activities of Daily Living (BADL) / (IADL); Functional Activities Questionnaire (FAQ); depression (Geriatric Depression Scale [GDS]); disability, Interview for deterioration in daily living activities [IDDD]; verbal learning and episodic memory (evaluated with the Consortium to establish a registry for Alzheimer’s disease [CERAD] word list and Wechsler Memory Scale III logical memory subtest; attention and executive function (digits test from Wechsler adult intelligence scale III [WAIS-III], trail making test B [TMT-B], phonetic fluency (p), Stroop test, constructional praxis from CERAD); visual perception (letters test from the visual object and space perception battery [VOSP]; visuo-spatial function (number localization task from VOSP); agnosia (Poppelreuter figures test; psychomotor speed (TMT-A); semantic fluency (animal categories); and language (Boston naming test [BNT-Boston],

[0278] more particularly a method selected from the group comprising Mini-Mental State Examination (MMSE); Montreal Cognitive Assessment (MoCA), Mini-Cog; General Practitioner Assessment of Cognition (GPCOG); Addenbrookes Cognitive Examination revised (ACE-R); Severe Impairment Battery (SIB); National adult reading test (NART); Neuropsychiatric Inventory (NPI); and Basic / Instrumental Activities of Daily Living (BADL) / (IADL).

[0279] In certain embodiments reduction of cognitive function is defined as the patient's inability to achieve scores indicative of normal cognitive function when assessed by one or more methods according to embodiments of the present invention, such as the inability to achieve a score higher than 24 points on the Mini-Mental State Examination (MMSE), and / or the inability to achieve a score higher than 25 points on the Montreal Cognitive Assessment (MoCA), and / or the inability to achieve a score higher than 2 on the Mini-Cog, and / or the inability to score within the range of 0 to 5 points, particularly within the range of 0 to 4, more particularly within the range of 0 to 3, more particularly within the range of 0 to 2, more particularly within the range of 0 to 1, most particularly 0 on the Functional Activities Questionnaire (FAQ) ); and / or a classification of 0.5 or higher on the Clinical Dementia Rating - Global (CDR-Global); and / or a score on the Clinical Dementia Rating - Sum of Boxes (CDR-SB) within 0.5 to 9.0; and / or a worsening on ADAS-Cog defined by an increase from baseline of at least 2 to 3 points; and / or a worsening on iADRS defined by a decrease from baseline of at least 5 points.

[0280] In more particular embodiments reduction of cognitive function is defined as the patient's inability to achieve scores indicative of normal cognitive function when assessed by one or more methods according to embodiments of the present invention, such as the inability to achieve a score higher than 24 points on the Mini-Mental State Examination (MMSE), and / or the inability to achieve a score higher than 25 points on the Montreal Cognitive Assessment (MoCA), and / or the inability to achieve a score higher than 2 on the Mini-Cog, and / or the inability to score within the range of 0 to 5 points, particularly within the range of 0 to 4, more particularly within the range of 0 to 3, more particularly within the range of 0 to 2, more particularly within the range of 0 to 1, most particularly 0 on the Functional Activities Questionnaire (FAQ).

[0281] For the MMSE, scores between 25 and 30 points represent normal cognition, while 21 to 24 points indicate mild dementia, 10 to 20 points suggest moderate dementia, and 9 points or lower indicate severe dementia. For the MoCA, scores between 26 and 30 points reflect normal cognition, while scores between 18 and 25 points suggest mild cognitive impairment, and below 18 points may indicate severe cognitive impairment and possible dementia. The Mini-Cog test identifies cognitive impairment with scores of 0 to 2 points, while scores of 3 to 5 points represent normal cognition. For the FAQ, a score of 0 to 5 points indicates normal functioning, 6 to 9 points suggest mild impairment, 10 to 20 points reflect moderate impairment, and scores between 21 and 30 points indicate severe impairment

[0282] More particularly, for the ADAS-Cog 11, total scores range from 0 to 70 points, scores between 0 and 10 points are consistent with normal cognition or very mild impairment, between 11 and 30 points with mild to moderate impairment, and above 30 points with more advanced impairment. For the ADAS-Cog 13, the total score ranges from 0 to 85 point; scores below 20 points correspond to very mild impairment, scores between 20 and 45 points to mild to moderate impairment, and scores above 45 points to severe impairment.

[0283] More particularly, for the ADAS-Cog 14, the total score ranges from 0 to 90 points; scores below 25 points are associated with normal or very mild impairment, scores between 25 and 55 points with mild to moderate impairment, and scores above 55 points with severe impairment.

[0284] More particularly, for the CDR-Global, a score of 0 indicates normal cognition, 0.5 represents very mild cognitive impairment, 1 corresponds to mild dementia, 2 to moderate dementia, and 3 to severe dementia. For the CDR-Sum of Boxes (CDR-SB), scores range from 0 to 18, with higher values indicating greater impairment; scores between 0.5 and 4.0 correspond to very mild impairment, between 4.5 and 9.0 to mild impairment, between 9.5 and 15.5 to moderate impairment, and between 16 and 18 to severe impairment.

[0285] More particularly: CERAD battery total Score combines six subtests (Chandler et al. 2005. Neurology 65(1):102-6): verbal fluency (max. score 24), Boston naming test (max. score 15), word list learning (3 trials 10 words each; max. score 30), word list recall (delayed recall; max score 10), word list recognition (yes / no format; max. score 10) and constructional praxis (copying geometric figures; max. score 11), which results in a maximum score of 100 and has to be adjusted for age and education. Normal cognition (healthy aging) results in a score of ≥80, subtle deficits (possible mild cognitive impairment) results in a score of 70-79, mild cognitive impairment (very mild dementia) results in a score of 60-69 and moderate to severe cognitive impairment (dementia range) results in a score of <60.

[0286] In some embodiments cognitive impairment is grouped into severe cognitive impairment, moderate cognitive impairment, mild cognitive impairment, possible mild cognitive impairment and no cognitive impairment (normal cognition).

[0287] Measuring Improvement of cognitive function

[0288] In certain embodiments improvement of cognitive function can be assessed by comparing baseline cognitive function (before treatment) and after treatment cognitive function.

[0289] In certain embodiments repeated testing of cognitive function (e.g. by the same validated test) is used before and after treatment.

[0290] In certain embodiments testing of cognitive function is performed within 3 months, particularly within 2 months, more particularly within 1 month, more particular within 21 days, even more particular within 14 days, even more particular within 7 days, even more particular within 5 days, even more particular within 3 days, even more particular within 2 days, most particular within 24 hours before treatment. The testing of cognitive function before treatment is considered as the baseline cognitive function value. In certain embodiments testing of cognitive function is performed particularly 2 months after treatment, more particularly 3 months after treatment, more particularly 6 months or more, even more particularly 12 months or more, even more particularly 18 months or more, and most particularly 24 months or more after treatment.

[0291] In certain embodiments, testing of cognitive function may be done by one or more testing methods selected from the group comprising MMSE; MoCA; CDR-Global; CDR-SB; ADAS-Cog 11 / 13 / 14; iADRS; ADCS-ADL; ADCS-iADL; FAQ; DAD; CERAD; NTB; PACC; WMS-IV Logical Memory II; FCSRT.

[0292] In particular embodiments, the testing of cognitive function is performed using a combination comprising ADAS-Cog 11 / 13 / 14, MMSE, CDR-SB, and an ADL / IADL instrument selected from ADCS-ADL and / or ADCS-iADL, together with CDR-Global and an objective episodic-memory test selected from WMS-IV LM-II, RBANS Delayed Memory Index, or FCSRT. In more particular embodiments, the testing is performed using a combination comprising ADAS-Cog 11 / 13 / 14, MMSE, CDR-SB, and an ADL / IADL instrument selected from ADCS-ADL and / or ADCS-iADL, together with CDR-Global.

[0293] In even more particular embodiments, the testing is performed using a combination comprising ADAS-Cog 11 / 13 / 14, MMSE, CDR-SB, and / or an ADL / IADL instrument selected from ADCS-ADL or ADCS-iADL.

[0294] In most particular embodiments, the testing is performed using a combination comprising ADAS-Cog 11 / 13 / 14, MMSE, CDR-SB, and / or an ADL / IADL instrument selected from ADCS-ADL or ADCS-iADL.

[0295] In particular embodiments, improvement of cognitive function is defined as (i) an improvement in the result of a cognitive testing method assessed by comparing the result of the cognitive testing method at baseline (before treatment) to the result of the cognitive testing method after treatment or (ii) the absence of any decline in the result of a cognitive testing method, as determined by comparing the result obtained at baseline (before treatment) with the result obtained after treatment, indicating that the subject remains cognitively stable, either within the range of normal cognitive function or within a state of impaired but unchanged cognitive function.

[0296] In certain embodiments an improvement of cognitive function is an improvement in the result of a cognitive testing method assessed by comparing the result of the cognitive testing method after treatment of the subject to results that would be achieved in patients within the same group of age and Amyloidbeta / phospho -Tau load in the brain.

[0297] In other embodiments an improvement of cognitive function is an improvement in the result of a cognitive testing method assessed by comparing the result of the cognitive testing method after treatment of the subject to results before treatment (i.e. baseline).

[0298] In certain embodiments said improvement of cognitive function is an improvement in the result of a cognitive testing method from reduced cognitive function to normal cognitive function.

[0299] In certain embodiments said results of cognitive testing are related to certain parameters of the subject selected from the group comprising age, level of education, sex, baseline cognitive stage, Amyloid-β load and phospho-Tau load, APOE genotype and stability of cholinesterase inhibitor (ChEI) or memantine therapy.

[0300] In certain embodiments said improvement of cognitive function is an improvement in the result of a cognitive testing method from a severe reduction of cognitive function to a moderate or mild or possible mild reduction of cognitive function or to a normal cognitive function, or from a moderate reduction of cognitive function to a mild or possible mild reduction of cognitive function or to a normal cognitive function, or

[0301] from a mild reduction of cognitive function to a possible mild reduction of cognitive function or normal cognitive function, or

[0302] slowing down the progression of cognitive decline in said subject in comparison to a matched untreated group of subjects.

[0303] In particular embodiments said improvement of cognitive function is an improvement in the result of a cognitive testing method by at least 1 point, particularly at least 2 points, more particular by at least 3 points, even more particular by at least 4 points, most particular by at least 5 points, wherein said cognitive testing method is selected from the group comprising MMSE, MoCA and FAQ.

[0304] In particular embodiments said improvement of cognitive function is an improvement in the result of a cognitive testing method by at least 1 point, particularly at least 2 points, most particular by at least 3 points, wherein said cognitive testing method is the Mini-Cog test.

[0305] In particular embodiments said improvement of cognitive function is an improvement in the result of a cognitive testing method by at least 2 points, particularly at least 5 points, more particular by at least 10 points, most particular by at least 15 points, wherein said cognitive testing method is the CERAD total score.

[0306] In particular embodiments said improvement of cognitive function is an improvement in the result of a cognitive testing method by a decrease from baseline of at least 3 points, particularly 4 points, more particular 5 points, even more particular 6 points, most particular 8 points, wherein said cognitive testing method is ADAS-Cog 11, 13 or 14.

[0307] In particular embodiments said improvement of cognitive function is an improvement in the result of a cognitive testing method by a decrease from baseline of at least 0.5 points, particularly 1 point, more particular 1.5 points, most particular 2 points, wherein said cognitive testing method is CDR-Sum of Boxes (CDR-SB).

[0308] In particular embodiments said improvement of cognitive function is an improvement in the result of a cognitive testing method by an increase from baseline of at least 2 points, particularly 3 points, more particular 4 points, most particular 5 points, wherein said cognitive testing method is ADCS-ADL. In particular embodiments said improvement of cognitive function is an improvement in the result of a cognitive testing method by an increase from baseline of at least 1 point, particularly 2 points, more particular 3 points, most particular 4 points, wherein said cognitive testing method is ADCS-iADL. The person skilled in the art knows the respective cognitive testing methods and potential results and what a change in a score (e.g. an increase or a decrease) means in consideration of the respective score. This means for example for the MMSE, MoCA, Mini-Cog test, CERAD total score, Alzheimer’s Disease Cooperative Study - Activities of Daily Living (ADCS-ADL), instrumental ADL (ADCS-iADL), and Integrated Alzheimer’s Disease Rating Scale (iADRS) the higher the score, the better the cognition of the patient, and for example for the FAQ, Clinical Dementia Rating - Global (CDR-Global), Clinical Dementia Rating - Sum of Boxes (CDR-SB), and Alzheimer’s Disease Assessment Scale - Cognitive Subscale (ADAS-Cog 11 / 13 / 14) the lower the score, the better the cognition.

[0309] In particular embodiments said improvement of cognitive function is an improvement in the result of a cognitive testing method by at least 5 percent, particularly at least 10 percent, more particular by at least 15 percent, even more particular by at least 20 percent, most particular by at least 25 percent, wherein said cognitive testing method is selected from the group comprising Mini-Mental State Examination (MMSE); Montreal Cognitive Assessment (MoCA); Mini-Cog; Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) total score; Alzheimer’s Disease Assessment Scale - Cognitive Subscale (ADAS-Cog 11 / 13 / 14); Clinical Dementia Rating - Sum of Boxes (CDR-SB); Alzheimer’s Disease Cooperative Study - Activities of Daily Living (ADCS-ADL) and / or instrumental ADL (ADCS-iADL); and Integrated Alzheimer’s Disease Rating Scale (iADRS) or any combination thereof.

[0310] The terms “reduced cognitive function”, “reduction in cognitive function” and “impairment of cognitive function” are used interchangeably.

[0311] In certain embodiments of the invention said subject is suffering from or has a risk of suffering from a disease or medical condition selected from the group comprising:

[0312] Neurodegenerative Disorders including Alzheimer's Disease, MCI patients, Frontotemporal Dementia (FTD), Lewy Body Dementia (LBD), Parkinson's Disease with Dementia (PDD), Huntington's Disease, Amyotrophic Lateral Sclerosis (ALS) with cognitive impairment, Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD), Multiple System Atrophy (MSA), Prion Diseases (e.g., Creutzfeldt-Jakob Disease);

[0313] Vascular Disorders including Vascular Dementia, Stroke, Multi-Infarct Dementia, Subcortical Ischemic Vascular Dementia, Cerebral Amyloid Angiopathy, Strategic Single-Infarct Dementia, Transient Ischemic Attack (TIA), Hypertensive Encephalopathy; Traumatic Brain Injury (TBI) Related Disorders Chronic Traumatic Encephalopathy (CTE), Post-Concussion Syndrome, Diffuse Axonal Injury, Second Impact Syndrome, Subdural Hematoma, Epidural Hematoma, Penetrating Brain Injury, Post-Traumatic Amnesia;

[0314] Inflammatory and Infectious Disorders including Multiple Sclerosis (MS), Sepsis- Associated Encephalopathy (SAE), HIV-Associated Neurocognitive Disorder (HAND),, Autoimmune Encephalitis, Meningitis and Encephalitis;

[0315] Endocrine and Metabolic Disorders including Diabetes Mellitus, Hyperglycemia, Hypoglycemia.

[0316] Neurodegenerative diseases prevented or treated according to embodiments of the present invention are selected from the group comprising Alzheimer's Disease, mild cognitive impairment (MCI), Frontotemporal Dementia (FTD), Dementia with Lewy bodies (DLB), Parkinson's Disease with Dementia (PDD), Huntington's Disease, Amyotrophic Lateral Sclerosis (ALS) with cognitive impairment, Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD), Multiple System Atrophy (MSA), Prion Diseases (e.g., Creutzfeldt-Jakob Disease);

[0317] Alzheimer's disease (AD) is the most prevalent form of dementia. Key molecular mechanisms and histopathological hallmarks in the AD brain comprise a dynamic cascade of biochemical events including the pathological amyloidogenic cleavage of the amyloid precursor protein (APP), the generation of various beta-amyloid species including the amyloid-beta peptide (Aβ1-42), dimers, trimers, oligomers and subsequent amyloid aggregation and deposition in plaques, abnormal hyperphosphorylation and aggregation of tau protein, progressive intracellular neurofibrillary degeneration, changes within the innate immune system, inflammation and a breakdown of the bloodbrain barrier.

[0318] Mild cognitive impairment (MCI) is a heterogeneous clinical condition with several underlying causes. However, the large proportion of MCI represents a transitional state between healthy aging and very mild AD (DeCarli 2003. Lancet Neurol. 2:15-21). Accordingly, studies suggest that MCI subjects tend to progress to clinically probable AD at a rate of approximately 10%–15% per year (Markesbery 2010. J Alzheimers Dis. 19:221-228).

[0319] In particular embodiments the patient group of Alzheimer's patients may be determined by risks factors such as the occurrence of MCI, the presence of the genetic risk factor ApoE4, as well as age. In more particular embodiments the risk factor age of Alzheimer' s patients is defined as an age of at least 60 years. Dementia with Lewy bodies (DLB) is a type of dementia that worsens over time. DLB is associated with BBB dysfunction and microvascular lesions (Janelidze S et al., Neurobiol Aging. 2017 Mar;51:104-112.). Additional symptoms may include fluctuations in alertness, visual hallucinations, slowness of movement, trouble walking, and rigidity.

[0320] DLB is the most common cause of dementia after Alzheimer’s disease and vascular dementia. It typically begins after the age of 50. The underlying mechanism involves the formation of Lewy bodies in neurons, consisting of alpha-synuclein protein. A diagnosis may be suspected based on symptoms, with blood tests and medical imaging done to rule out other possible causes. At present no cure for DLB exists. For review see McKeith et al. 2017. Neurology 89: 88-100.

[0321] Frontotemporal dementia (FTD) is the clinical presentation of frontotemporal lobar degeneration, which is characterized by progressive neuronal loss predominantly involving the frontal or temporal lobes, and typical loss of over 70% of spindle neurons, while other neuron types remain intact. FTD is associated with BBB dysfunction and microvascular lesions (Janelidze S, et al., Neurobiol Aging. 2017 Mar;51:104-112). and accounts for 20% of young-onset dementia cases. Signs and symptoms typically manifest in late adulthood, more commonly between the ages of 55 and 65, approximately equally affecting men and women. Common signs and symptoms include significant changes in social and personal behavior, apathy, blunting of emotions, and deficits in both expressive and receptive language. Currently, there is no cure for FTD, but there are treatments that help alleviate symptoms. For review see Bott et al. 2014. Neurodegener Dis Manag 4(6): 439-454.

[0322] Differentiating the different dementia syndromes can be challenging, due to the frequently overlapping clinical features and related underlying pathology (“mixed Alzheimer’s disease). In particular, Alzheimer’s dementia often co-occurs with vascular dementia. People with vascular dementia present with progressive cognitive impairment, acutely or sub-acutely as in mild cognitive impairment, frequently stepwise, after multiple cerebrovascular events (strokes). For review see Venkat et al. 2015. Exp Neurol 272: 97-108.

[0323] Subcortical dementias are a group of neurodegenerative disorders characterized by the predominant involvement of subcortical brain structures, including the basal ganglia, thalamus, and brainstem, leading to a distinct clinical syndrome. Unlike cortical dementias, which primarily affect higher-order cognitive functions, subcortical dementias are marked by a combination of cognitive, motor, and psychiatric symptoms. Key features include slowed cognitive processing, impaired executive functions, memory retrieval deficits, and motor abnormalities such as bradykinesia, rigidity, and gait disturbances. Prominent examples of subcortical dementias include Parkinson's disease dementia (PDD), Huntington's disease (HD), progressive supranuclear palsy (PSP), and vascular dementia with subcortical ischemic changes. The pathophysiology of subcortical dementias involves the degeneration of subcortical neurons, often associated with proteinopathies such as alpha-synuclein in PDD, huntingtin in HD, and tau in PSP. Additionally, vascular contributions to subcortical dementia involve chronic ischemia and small vessel disease, leading to white matter lesions and lacunar infarcts. Neuroimaging techniques, including MRI and PET scans, are crucial for identifying characteristic subcortical atrophy and white matter changes. Neuropsychological assessments reveal patterns of cognitive impairment distinct from cortical dementias, emphasizing executive dysfunction and psychomotor slowing.

[0324] Huntington's disease (HD) is caused by a CAG trinucleotide repeat expansion in the HTT gene, leading to the production of mutant huntingtin protein. The disease primarily affects the basal ganglia, particularly the striatum, and extends to cortical regions as the disease progresses. Cognitive decline in HD includes difficulties with executive functions, memory retrieval, and visuospatial abilities, often accompanied by psychiatric symptoms such as depression and irritability.

[0325] Amyotrophic lateral sclerosis (ALS) involves the degeneration of upper and lower motor neurons, leading to progressive muscle weakness and atrophy. A subset of ALS patients develop frontotemporal dementia (ALS-FTD), characterized by changes in personality, behavior, and executive dysfunction. Pathologically, ALS-FTD is associated with TDP-43 proteinopathy.

[0326] Parkinson’s disease (PD), the most common neurodegenerative disease of the elderly, is characterized by progressive loss of muscle control. PD is predominant at the 6th decade of life and men are 1.5 to 2 times more likely to contract the disease than women. Head trauma, illness, or exposure to environmental toxins is identified as a risk factor. This neurodegenerative disorder is characterized by tremor, rigidity, bradykinesia, and impairment in balance. PD also causes cognitive, psychiatric, autonomic, and sensory disturbances. The pathology of PD is characterized by the gradual and selective loss of dopaminergic neurons in the substantia nigra pars compacta.

[0327] Secondary causes of dementia encompass a range of conditions where cognitive decline arises as a consequence of underlying neurological or systemic disorders.

[0328] Progressive supranuclear palsy (PSP) is a neurodegenerative disorder characterized by a combination of motor, cognitive, and behavioral symptoms, primarily caused by the accumulation of tau protein in the brain. The condition features motor dysfunction, including postural instability and gaze abnormalities, particularly a hallmark "supranuclear gaze palsy," alongside cognitive impairments that resemble frontotemporal dementia. Patients often experience executive dysfunction, memory issues, and significant behavioral changes such as apathy and emotional lability. The progression of PSP is typically gradual, with a mean survival of 6 to 10 years following diagnosis. Diagnosis is primarily clinical, supplemented by neuroimaging that reveals specific brain atrophy.

[0329] Corticobasal degeneration (CBD) is a rare, progressive neurodegenerative disorder characterized by asymmetric motor symptoms, cognitive decline, and behavioral changes. The pathophysiology involves the accumulation of tau protein, leading to the degeneration of specific brain regions, particularly the cortical and basal ganglia areas. Clinically, CBD presents with a variety of motor symptoms, including rigidity, bradykinesia, and focal dystonia, often affecting one side of the body more than the other. Patients may also experience apraxia, which manifests as difficulties in performing purposeful movements, and alien limb phenomenon, where one limb appears to act independently. Cognitive and behavioral changes can include executive dysfunction, memory impairment, and alterations in personality, resembling features of frontotemporal dementia. Diagnosis is largely clinical, with imaging studies showing characteristic atrophy in relevant brain regions. The progression of CBD varies, typically leading to significant disability within a few years.

[0330] Multiple System Atrophy (MSA) is a rare neurodegenerative disorder characterized by a combination of autonomic dysfunction, parkinsonism, and ataxia. MSA is associated with the misfolding of alpha-synuclein protein, leading to the degeneration of various brain regions, including the basal ganglia, cerebellum, and autonomic centers. Clinically, MSA presents with symptoms such as orthostatic hypotension, urinary incontinence, and motor features resembling Parkinson's disease, including rigidity and bradykinesia. Unlike Parkinson’s disease, MSA often progresses more rapidly and is marked by pronounced ataxia and significant autonomic dysfunction. Diagnosis is primarily clinical, with neuroimaging revealing characteristic patterns of atrophy. The disease course is typically progressive, leading to severe disability within a few years.

[0331] Prion Diseases, such as Creutzfeldt-Jakob Disease (CJD), are a group of rare, fatal neurodegenerative disorders caused by the accumulation of misfolded prion proteins, leading to brain damage and the formation of amyloid plaques. CJD is characterized by rapidly progressive dementia, neurological deficits, and, often, myoclonus. The disease can present in various forms, including sporadic, familial, and acquired variants. Clinically, symptoms typically emerge quickly, with patients experiencing cognitive decline, personality changes, visual disturbances, and ataxia. Diagnosis relies on clinical assessment, electroencephalography, and imaging techniques, which may show characteristic changes in the brain. Biomarkers in cerebrospinal fluid, such as 14-3-3 protein and RT-QuIC, can also aid in diagnosis. The course of CJD is usually fatal within months to a few years, and there is currently no effective treatment or cure.

[0332] Creutzfeldt-Jakob disease (CJD) is caused by the misfolding of prion proteins, leading to the accumulation of abnormal prions and widespread neuronal loss. CJD presents with rapidly progressive dementia, myoclonus, ataxia, and visual disturbances. The disease progresses rapidly, often leading to death within a year of onset. Diagnostic tools for CJD include MRI showing hyperintensities in the basal ganglia and cortical ribboning, EEG with periodic sharp wave complexes, and CSF biomarkers such as 14-3-3 protein. Neuroimaging, including MRI and PET scans, alongside cerebrospinal fluid analysis and genetic testing, are pivotal in diagnosing these conditions. Comprehensive neuropsychological assessments help delineate the specific cognitive deficits associated with each condition, aiding in differential diagnosis.

[0333] Vascular Disorders prevented or treated according to embodiments of the present invention are selected from the group comprising vascular dementia, stroke, multi-infarct dementia, subcortical ischemic vascular dementia, cerebral amyloid angiopathy, strategic single-infarct dementia, transient ischemic attack (TIA), hypertensive encephalopathy;

[0334] Vascular disorders encompass a range of conditions resulting from impaired blood flow to the brain, leading to neurological deficits and cognitive decline. These disorders can significantly impact brain function, often presenting as vascular dementia or various forms of stroke.

[0335] Vascular dementia is a form of cognitive impairment resulting from insufficient blood flow to the brain, leading to brain damage and subsequent decline in cognitive functions. It is often associated with cerebrovascular diseases, such as strokes or chronic conditions that affect cerebral circulation. Pathophysiology involves a series of ischemic events that can result in both focal and diffuse brain injury, leading to cognitive deficits that vary based on the areas affected.

[0336] Clinically, vascular dementia may present with a range of symptoms, including problems with memory, attention, and executive function, often accompanied by changes in mood and behavior. Unlike Alzheimer’s disease, which typically shows a gradual onset and progression, vascular dementia can present with a more stepwise decline, correlating with the occurrence of vascular events.

[0337] Diagnosis is primarily clinical, supported by neuroimaging techniques that can reveal evidence of infarcts, white matter changes, or other vascular abnormalities. Cognitive assessments may help differentiate vascular dementia from other forms of dementia, such as Alzheimer’s.

[0338] Stroke is a neurological emergency characterized by the sudden loss of brain function due to disrupted blood flow to the brain, resulting in brain tissue damage. There are two primary types of stroke: ischemic, which accounts for approximately 87% of cases, occurs when a blood vessel supplying the brain is obstructed, often due to a blood clot; and hemorrhagic, caused by the rupture of a blood vessel leading to bleeding in or around the brain.

[0339] The pathophysiology of ischemic stroke involves the occlusion of cerebral arteries, leading to a cascade of cellular events that can result in infarction and tissue necrosis. Hemorrhagic strokes, on the other hand, lead to increased intracranial pressure and secondary brain injury due to bleeding. The clinical presentation of stroke can vary widely depending on the affected brain regions and may include sudden weakness or numbness, difficulty speaking or understanding language, visual disturbances, and loss of coordination or balance. Diagnosis is primarily clinical, supported by neuroimaging techniques such as CT or MRI to identify the type of stroke and the extent of brain injury. Timely intervention is crucial, particularly for ischemic stroke, where treatments like thrombolysis or thrombectomy can significantly improve outcomes if administered within a narrow time window.

[0340] Multi-infarct dementia (MID) is a subtype of vascular dementia characterized by cognitive decline resulting from multiple small strokes (infarcts) in various regions of the brain. These infarcts lead to cumulative damage and disruptions in cognitive function, particularly in areas responsible for memory, attention, and executive functioning. The condition typically arises from chronic vascular risk factors such as hypertension, diabetes, and hyperlipidemia, which contribute to the development of small vessel disease and increased likelihood of ischemic events.

[0341] Clinically, MID is marked by a stepwise progression of cognitive impairment, often correlating with the occurrence of new strokes. Patients may experience fluctuations in cognitive abilities, with periods of stability interspersed with sudden declines following vascular events. Common symptoms include memory loss, difficulties with problem-solving and planning, and changes in mood or behavior.

[0342] Diagnosis of multi-infarct dementia involves a thorough clinical evaluation, neuropsychological assessments, and neuroimaging techniques like MRI or CT scans to identify the presence of multiple infarcts and rule out other forms of dementia. The presence of significant white matter changes, often referred to as leukoaraiosis, can also be indicative of MID.

[0343] Subcortical ischemic vascular dementia (SIVD) is a form of cognitive decline resulting from ischemic lesions primarily located in the subcortical areas of the brain, often due to chronic small vessel disease. This condition is typically associated with vascular risk factors such as hypertension, diabetes, and hyperlipidemia, which contribute to white matter changes and lacunar infarcts. Clinically, SIVD presents with a range of symptoms, including slowed thinking, executive dysfunction, and mood disturbances, often accompanied by motor symptoms such as gait disturbances and rigidity. Diagnosis involves neuroimaging, often revealing characteristic white matter hyperintensities and lacunar strokes.

[0344] Cerebral amyloid angiopathy (CAA) is a condition characterized by the deposition of amyloid-beta protein in the walls of cerebral blood vessels, leading to increased fragility and a higher risk of hemorrhagic strokes. CAA can contribute to cognitive decline and is often associated with aging and Alzheimer’s disease. Clinically, patients may experience recurrent lobar hemorrhages and progressive cognitive impairment, particularly in memory and executive function. Diagnosis typically involves neuroimaging, such as MRI, which can reveal characteristic patterns of microbleeds or larger hemorrhages. While there is currently no specific treatment for CAA, managing vascular risk factors and monitoring for complications are essential. Strategic single-infarct dementia refers to cognitive decline resulting from a single ischemic stroke that occurs in a critical region of the brain, essential for specific cognitive functions. This type of dementia often results from strokes in regions such as the thalamus or the basal ganglia, which are involved in memory, attention, and executive functioning. Clinically, patients may present with focal cognitive deficits that correspond to the location of the infarct, such as memory impairment or language difficulties. Diagnosis involves neuroimaging to identify the infarct and assess its impact on surrounding brain structures.

[0345] A transient ischemic attack (TIA) is a temporary episode of neurological dysfunction caused by a brief interruption of blood flow to the brain, often lasting only a few minutes to a few hours. TIAs serve as important warning signs for potential future strokes and are typically characterized by sudden onset of symptoms such as weakness, numbness, or speech difficulties. Unlike a full stroke, TIAs do not result in permanent damage; however, they indicate significant vascular risk and warrant immediate medical evaluation. Diagnosis is based on clinical presentation, history, and neuroimaging to assess for underlying vascular issues.

[0346] Hypertensive encephalopathy is a neurological condition resulting from severely elevated blood pressure, leading to acute brain dysfunction. This condition is characterized by symptoms such as headache, confusion, seizures, and altered mental status, often presenting as a medical emergency. The pathophysiology involves a breakdown of the blood-brain barrier due to high blood pressure, resulting in edema and potential ischemic injury. Diagnosis is primarily clinical, supported by blood pressure measurements and neuroimaging to rule out other causes of acute neurological symptoms.

[0347] In certain embodiments cognitive impairment which is associated with vascular disorders prevented or treated according to embodiments of the present invention are selected from the group comprising Binswanger’s Disease, Post-Stroke Cognitive Impairment, Lacunar Infarcts, White Matter Hyperintensities (WMH), CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy), Mixed Dementia and Diabetic Encephalopathy.

[0348] Binswanger’s disease is a subtype of vascular dementia and arises primarily from chronic ischemia due to small vessel disease affecting the deep white matter of the brain. It is characterized by a progressive decline in cognitive function, including memory, attention, and executive functions, often accompanied by personality changes and mood disturbances, such as depression. The disease is linked to hypertension and other vascular risk factors, leading to lipohyalinosis (a form of arterial wall damage) and demyelination. MRI findings typically reveal extensive white matter hyperintensities, reflecting ischemic changes. Patients may also exhibit gait abnormalities and parkinsonian symptoms as the disease progresses. Diagnosis is often made based on clinical presentation and MRI findings. Neuropsychological testing may reveal deficits consistent with subcortical dementia. Post-stroke cognitive impairment encompasses a range of cognitive deficits that can occur after a stroke, impacting approximately 30-50% of stroke survivors. The cognitive impairments can be diverse, affecting memory, language, attention, and executive function, and are often a result of both direct neuronal damage and secondary changes such as neuroinflammation and disruptions in cerebral blood flow. The type and severity of cognitive impairment can vary based on the location and extent of the stroke. For instance, left hemispheric strokes may lead to aphasia, while right hemispheric strokes may impact visuospatial skills. Moreover, the accumulation of small vessel disease and subsequent WMH can exacerbate cognitive decline in these patients. Cognitive evaluation using standardized neuropsychological tests, alongside imaging studies (like MRI or CT), are used to delineate the specific deficits and underlying pathology.

[0349] Lacunar infarcts are small, deep infarcts resulting from the occlusion of small penetrating arteries, often due to lipohyalinosis associated with chronic hypertension, diabetes, or other vascular risk factors. These infarcts typically range from 3 to 15 mm in size and are most commonly located in regions such as the basal ganglia, thalamus, internal capsule, and brainstem. Clinically, lacunar infarcts may manifest as pure motor hemiparesis, sensory disturbances, or dysarthria, depending on their location. They are also associated with cognitive decline and can contribute to the development of vascular dementia. MRI is crucial for identifying lacunar infarcts, which appear as small, round hyperintense lesions on T2-weighted sequences.

[0350] White matter hyperintensities (WMH) are areas of increased signal intensity on T2 -weighted MRI scans and are commonly seen in aging populations and those with vascular risk factors. WMH are associated with small vessel disease and can signify underlying chronic ischemia or demyelination. The presence of WMH is linked to cognitive decline, increased risk of stroke, and other neurological conditions. They can correlate with deficits in executive function, processing speed, and overall cognitive performance. Pathologically, WMH can reflect a range of changes, including myelin loss, axonal degeneration, and gliosis. The etiology can be multifactorial, involving hypertension, diabetes, and other vascular pathologies.

[0351] CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) is a hereditary condition caused by mutations in the NOTCH3 gene, leading to smooth muscle cell dysfunction in the small penetrating arteries of the brain. It is characterized by recurrent strokes, cognitive decline, and mood disturbances, typically presenting in mid-adulthood. The disease results in thickening of the vascular wall, which leads to reduced blood flow and ischemia in the white matter. MRI typically reveals extensive WMH and subcortical infarcts. Diagnosis is often confirmed through genetic testing for NOTCH3 mutations, along with characteristic imaging findings. Patients may also present with migraine with aura and depression. Mixed dementia refers to the simultaneous presence of multiple dementia pathologies, primarily Alzheimer's disease and vascular dementia. This condition is common, especially in older adults, and reflects a complex interplay of neurodegenerative and vascular processes. Patients may exhibit a range of cognitive deficits, including memory impairment, executive dysfunction, and behavioral changes. The clinical picture may vary widely depending on the dominant underlying pathology. Mixed dementia is associated with the presence of amyloid plaques and neurofibrillary tangles characteristic of Alzheimer's, alongside vascular changes such as WMH and lacunar infarcts. Imaging studies may reveal both neurodegenerative and vascular abnormalities.

[0352] Diabetic encephalopathy describes cognitive decline linked to diabetes mellitus, which can occur through multiple mechanisms, including vascular damage, metabolic disturbances, and neuroinflammation. Chronic hyperglycemia can lead to microvascular complications affecting cerebral blood flow and contributing to neuronal injury. The accumulation of advanced glycation end-products (AGEs) may also promote oxidative stress and inflammation, further impairing cognitive function. Cognitive deficits are often seen in areas such as attention, executive function, and memory. Imaging studies may reveal increased WMH and atrophy. Traumatic Brain Injury (TBI) Related Disorders prevented or treated according to embodiments of the present invention are selected from the group comprising Chronic Traumatic Encephalopathy (CTE), Post-Concussion Syndrome, Diffuse Axonal Injury, Second Impact Syndrome, Subdural Hematoma, Epidural Hematoma, Penetrating Brain Injury, Post-Traumatic Amnesia;

[0353] Traumatic brain injury (TBI) encompasses a spectrum of injuries to the brain resulting from external mechanical forces, such as impact or acceleration-deceleration forces. TBIs can lead to a variety of neurological and psychological disorders, depending on the severity and location of the injury. Symptoms can range from mild concussions with transient cognitive disturbances to severe cases involving loss of consciousness and significant functional impairment. The consequences of TBI can manifest immediately or develop over time, affecting cognitive function, emotional regulation, and behavior. Diagnosis typically involves clinical evaluation, neuroimaging, and neuropsychological assessments to gauge the extent of the injury and its effects.

[0354] Chronic traumatic encephalopathy (CTE) is a progressive neurodegenerative condition associated with a history of repetitive head trauma, often seen in athletes involved in contact sports. CTE is characterized by the accumulation of hyperphosphorylated tau protein, leading to neuronal degeneration and cognitive decline. Clinically, CTE can manifest with symptoms such as memory loss, mood disorders, behavioral changes, and eventually, severe cognitive impairment resembling dementia. Diagnosis is currently postmortem, relying on pathological examination of brain tissue, although research is ongoing to identify potential biomarkers for in vivo diagnosis. Post-concussion syndrome (PCS) is a complex disorder characterized by persistent symptoms following a concussion, often lasting weeks to months after the initial injury. Common symptoms include headaches, dizziness, cognitive difficulties, and emotional changes, which can significantly impact daily functioning and quality of life. The pathophysiology of PCS remains poorly understood, with potential contributions from neurochemical changes, psychological factors, and individual vulnerability. Diagnosis is primarily clinical, based on the history of concussion and the presence of ongoing symptoms.

[0355] Diffuse axonal injury (DAI) is a severe form of traumatic brain injury resulting from rotational or acceleration-deceleration forces that cause widespread damage to the brain's white matter. This injury is characterized by the shearing of axons, leading to significant functional impairment and often resulting in prolonged unconsciousness or a vegetative state. Clinically, DAI presents with a range of symptoms depending on the severity and extent of the injury, including cognitive deficits, motor dysfunction, and changes in consciousness. Diagnosis is typically confirmed through neuroimaging, such as MRI, which may reveal characteristic patterns of axonal injury.

[0356] Second impact syndrome (SIS) occurs when an individual sustains a second concussion before fully recovering from a previous one, leading to rapid and severe brain swelling. This condition is particularly dangerous in young athletes and can result in catastrophic outcomes, including permanent neurological impairment or death. The pathophysiology involves a dysregulated cerebral blood flow and increased intracranial pressure following the second injury. Clinically, SIS can manifest with loss of consciousness, respiratory failure, and severe neurological deficits shortly after the second impact. Diagnosis is primarily clinical, with a focus on the history of recent head injuries and rapid deterioration of neurological function. Immediate medical intervention is critical, often requiring intensive monitoring and potentially surgical intervention to relieve intracranial pressure.

[0357] A subdural hematoma is a collection of blood between the dura mater and the arachnoid membrane, typically resulting from trauma that causes tearing of bridging veins in the brain. This type of hematoma can be acute or chronic, with acute cases often presenting with rapid onset of symptoms such as headache, confusion, and neurological deficits following an injury. Chronic subdural hematomas may develop slowly and can present with subtle cognitive changes or focal neurological deficits, especially in older adults. Diagnosis is confirmed through neuroimaging, particularly CT or MRI, which can visualize the blood collection.

[0358] An epidural hematoma is a localized collection of blood between the skull and the dura mater, often resulting from trauma, such as a skull fracture that lacerates an underlying artery. This condition typically presents with a classic clinical picture of a "lucid interval," where a patient initially loses consciousness but then briefly regains awareness before rapidly deteriorating. Symptoms may include severe headache, confusion, and focal neurological deficits as intracranial pressure rises. Diagnosis is typically confirmed through CT imaging, which reveals the characteristic biconvex shape of the hematoma.

[0359] Penetrating brain injury occurs when an object breaches the skull and directly damages brain tissue, often leading to severe neurological deficits and complications. This type of injury can result from various mechanisms, including gunshot wounds, stab wounds, or accidental trauma. Clinically, the presentation can vary widely based on the location and extent of the injury, with potential outcomes ranging from focal neurological deficits to widespread damage and altered consciousness. Diagnosis is primarily clinical, supported by neuroimaging to assess the extent of injury and associated complications, such as hemorrhage or edema.

[0360] Post-traumatic amnesia (PTA) refers to a state of confusion and memory loss that follows a traumatic brain injury, during which an individual cannot form new memories or recall events that occurred during the period of confusion. The duration of PTA is often used as an indicator of injury severity; longer periods of amnesia are associated with worse outcomes. Clinically, PTA can manifest as disorientation, inability to remember the event leading to the injury, and confusion regarding time and place. Diagnosis is primarily clinical, with assessments of cognitive function and orientation conducted regularly to track recovery.

[0361] Inflammatory and Infectious Disorders prevented or treated according to embodiments of the present invention are selected from the group comprising multiple sclerosis (MS), sepsis-associated ancephalopathy (SAE), HIV-associated neurocognitive disorder (HAND), neurosyphilis, autoimmune encephalitis, meningitis and encephalitis.

[0362] Multiple sclerosis (MS) is a chronic autoimmune disorder characterized by the demyelination of nerve fibers in the central nervous system, leading to a variety of neurological symptoms. The exact cause of MS remains unknown, though it is believed to involve a combination of genetic, environmental, and immunological factors. Clinically, MS presents with diverse symptoms, including fatigue, motor weakness, sensory disturbances, visual problems, and cognitive impairment, often occurring in relapsing-remitting or progressive forms. Diagnosis is typically based on clinical evaluation, magnetic resonance imaging (MRI) findings showing lesions, and cerebrospinal fluid analysis for oligoclonal bands.

[0363] Sepsis-associated encephalopathy (SAE) is a brain dysfunction that occurs in the context of systemic infection and sepsis, characterized by alterations in consciousness, cognitive function, and behavior. The pathophysiology of SAE involves complex interactions between systemic inflammatory responses, neuroinflammation, and metabolic derangements that affect brain function. Clinically, patients may present with confusion, delirium, agitation, or drowsiness, which can range from mild cognitive impairment to severe coma. Diagnosis is primarily clinical, requiring the identification of sepsis and ruling out other causes of altered mental status.

[0364] HIV-associated neurocognitive disorder (HAND) refers to a spectrum of cognitive impairments that occur in individuals living with HIV, ranging from asymptomatic neurocognitive impairment to more severe forms resembling dementia. Pathophysiology involves direct effects of the virus on the central nervous system, as well as inflammatory responses that can lead to neuronal injury and cognitive decline. Clinically, HAND may manifest as difficulties with memory, attention, executive function, and processing speed. Diagnosis is based on clinical evaluation, neuropsychological testing, and consideration of HIV viral load and treatment history.

[0365] Neurosyphilis is a complication of syphilis that occurs when the Treponema pallidum bacterium invades the central nervous system. This condition can present at any stage of syphilis and may lead to a variety of neurological symptoms, including headache, cognitive impairment, vision and hearing problems, and personality changes. Pathophysiology involves inflammatory processes that affect the meninges, brain, and spinal cord. Diagnosis typically involves clinical assessment, serological tests for syphilis, and lumbar puncture for cerebrospinal fluid analysis, which may reveal pleocytosis and other signs of infection. Treatment primarily involves high-dose penicillin to eradicate the infection.

[0366] Autoimmune encephalitis is a group of disorders characterized by inflammation of the brain caused by the immune system mistakenly attacking neuronal components. This condition can be associated with specific antibodies targeting neuronal receptors or proteins, such as anti-NMDA receptor encephalitis. Clinically, autoimmune encephalitis presents with a variety of symptoms, including cognitive dysfunction, seizures, psychiatric changes, and movement disorders. Diagnosis often involves a combination of clinical evaluation, neuroimaging, and laboratory tests for specific antibodies in serum or cerebrospinal fluid.

[0367] Meningitis is an inflammatory condition affecting the protective membranes (meninges) surrounding the brain and spinal cord, often caused by infections from bacteria, viruses, or fungi. The clinical presentation of meningitis typically includes symptoms such as fever, headache, neck stiffness, and altered mental status. Diagnosis is confirmed through lumbar puncture and cerebrospinal fluid analysis, which can reveal elevated white blood cell counts, protein levels, and specific pathogens.

[0368] Encephalitis is an inflammatory condition of the brain, often caused by viral infections, but can also result from autoimmune processes or bacterial infections. Clinically, encephalitis can present symptoms such as fever, headache, seizures, confusion, and neurological deficits, which may develop rapidly. The pathophysiology involves direct infection or an inflammatory response that can lead to neuronal injury. Diagnosis typically involves clinical evaluation, neuroimaging, and lumbar puncture for cerebrospinal fluid analysis to identify the causative agent. Endocrine and metabolic disorders prevented or treated according to embodiments of the present invention are selected from the group comprising diabetes mellitus, hyperglycemia, hypoglycemia. Diabetes mellitus is a chronic metabolic disorder characterized by elevated blood glucose levels due to insufficient insulin production, impaired insulin action, or a combination of both. It is classified mainly into two types: Type 1 diabetes, which results from autoimmune destruction of pancreatic beta cells leading to absolute insulin deficiency, and Type 2 diabetes, which is characterized by insulin resistance and relative insulin deficiency. Chronic hyperglycemia can lead to a range of complications, including cardiovascular disease, neuropathy, nephropathy, and retinopathy. Diagnosis is typically based on fasting blood glucose levels, HbA1c measurements, or oral glucose tolerance tests.

[0369] Hyperglycemia refers to elevated blood glucose levels, often associated with diabetes mellitus but can occur in other conditions as well. Chronic hyperglycemia can lead to significant long-term complications, including cardiovascular disease, neuropathy, and organ damage. Symptoms may include excessive thirst, frequent urination, fatigue, and blurred vision. Diagnosis is based on blood glucose measurements, typically confirmed with fasting glucose, random glucose, or HbA1c tests.

[0370] Hypoglycemia is a condition characterized by abnormally low blood glucose levels, often defined as below 70 mg / dL (3.9 mmol / L). It can occur in individuals with diabetes due to excessive insulin administration, inadequate food intake, or increased physical activity, but it can also arise in non-diabetic conditions. Symptoms of hypoglycemia may include shakiness, confusion, sweating, irritability, and in severe cases, loss of consciousness or seizures. Diagnosis involves measuring blood glucose levels during symptomatic episodes.

[0371] Metabolic and systemic diseases prevented or treated according to embodiments of the present invention are selected from the group comprising diabetes-related cognitive impairment, chronic kidney disease -related cognitive decline and metabolic syndrome.

[0372] Diabetes-related cognitive impairment encompasses a range of cognitive deficits observed in individuals with diabetes mellitus. This condition is increasingly recognized as a significant complication of diabetes, affecting both type 1 and type 2 diabetes patients. The cognitive decline in diabetic patients is thought to arise from several interrelated mechanisms:

[0373] Vascular Damage: Chronic hyperglycemia leads to microvascular complications, including cerebral small vessel disease, which can cause white matter hyperintensities and lacunar infarcts.

[0374] Metabolic Disturbances: Elevated blood glucose levels can result in the formation of advanced glycation end-products (AGEs) and oxidative stress, both of which contribute to neuronal injury and inflammation. Insulin Resistance: Insulin plays a critical role in brain function, and resistance can impair synaptic plasticity and cognitive function.

[0375] Cognitive impairment may present difficulties with attention, executive function, memory, and processing speed. Diabetic patients often exhibit an increased risk of developing dementia, particularly Alzheimer's disease and vascular dementia.

[0376] Chronic kidney disease is associated with an increased risk of cognitive decline, which can manifest at various stages of the disease. Cognitive impairment is prevalent among patients with end-stage renal disease (ESRD) but can also be present in earlier stages. Several mechanisms contribute to cognitive decline in CKD:

[0377] Uremic Toxins: Accumulation of uremic toxins due to impaired renal function can affect brain metabolism and contribute to neuroinflammation.

[0378] Vascular Factors: CKD is closely linked to cardiovascular disease, and vascular factors (e.g., hypertension, atherosclerosis) can exacerbate cognitive decline through small vessel disease and ischemic changes.

[0379] Metabolic Disturbances: Electrolyte imbalances (such as hyperphosphatemia) and metabolic acidosis may also play a role in cognitive function.

[0380] Patients may exhibit deficits in attention, memory, executive function, and processing speed. The degree of cognitive impairment often correlates with the severity of kidney disease.

[0381] Metabolic syndrome is a cluster of interrelated risk factors that significantly increase the risk of cardiovascular disease, diabetes, and stroke. It typically includes central obesity, insulin resistance, hypertension, and dyslipidemia. The metabolic and inflammatory processes involved in metabolic syndrome contribute to both vascular damage and neurodegeneration:

[0382] Insulin Resistance: Impaired insulin signaling affects not only metabolic processes but also neuroprotective pathways in the brain.

[0383] Chronic Inflammation: Elevated inflammatory markers associated with metabolic syndrome can lead to neuroinflammation and contribute to cognitive impairment.

[0384] Vascular Dysfunction: The combination of hypertension and dyslipidemia can result in endothelial dysfunction and small vessel disease, leading to white matter lesions and increased risk of stroke. Individuals with metabolic syndrome may experience cognitive deficits, particularly in executive function and memory. The risk of developing dementia is significantly higher in those with metabolic syndrome, particularly Alzheimer’s disease and vascular dementia. Autoimmune and Inflammatory Disorders prevented or treated according to embodiments of the present invention are selected from the group comprising Lupus-related cognitive dysfunction, primary CNS vasculitis.

[0385] Lupus-related cognitive dysfunction, often referred to as neuropsychiatric lupus, is a manifestation of systemic lupus erythematosus (SLE) that affects cognitive function. Cognitive impairment can occur in up to 60% of individuals with lupus and may vary widely in presentation. Several mechanisms contribute to cognitive dysfunction in lupus patients:

[0386] Autoimmune Mechanisms: The presence of autoantibodies, such as anti-phospholipid antibodies, can lead to vascular damage and promote thrombosis, resulting in ischemic changes in the brain.

[0387] Neuroinflammation: Inflammatory mediators can cross the blood-brain barrier, leading to neuroinflammation and neuronal injury.

[0388] Vascular Changes: Lupus is associated with an increased risk of cerebrovascular accidents, which can further contribute to cognitive decline.

[0389] Patients may present with a range of cognitive impairments, including difficulties with memory, attention, executive function, and processing speed. Mood disorders such as depression and anxiety are also common, complicating the cognitive profile.

[0390] Primary CNS vasculitis (PACNS) is a rare, inflammatory condition that affects the blood vessels in the central nervous system without the involvement of systemic vasculitis. It can lead to significant neurological deficits and cognitive impairment. The exact cause of PACNS is unknown, but it involves inflammation of the small and medium-sized blood vessels in the brain, leading to ischemia and potential infarcts. The immune response may be triggered by infections or other environmental factors, but it is classified as a primary condition.

[0391] Symptoms can vary widely and may include:

[0392] • Cognitive decline, memory loss, and confusion.

[0393] • Focal neurological deficits, such as weakness or sensory changes.

[0394] • Headaches, seizures, and psychiatric symptoms.

[0395] Diagnosis is challenging and requires a combination of clinical evaluation, neuroimaging (MRI or angiography), and often a brain biopsy to confirm the presence of vasculitis. MRI may show areas of infarction, edema, or leptomeningeal enhancement.

[0396] Traumatic Brain Injury (TBI) and Related Disorders prevented or treated according to embodiments of the present invention are selected from the group comprising chronic traumatic encephalopathy, postconcussion syndrome, moderate-to-severe TBI. Chronic traumatic encephalopathy is a progressive neurodegenerative disease primarily associated with repetitive head trauma, often seen in athletes involved in contact sports (e.g., football, boxing) and military veterans. CTE is characterized by the accumulation of hyperphosphorylated tau protein in a distinctive pattern, often starting in the frontal and temporal lobes and spreading to other brain regions. This tau pathology disrupts neuronal function and leads to neurodegeneration.

[0397] The underlying mechanisms likely involve:

[0398] Repetitive Brain Injury: Repeated concussive and subconcussive impacts can trigger neuroinflammatory responses and tau pathology.

[0399] Neuroinflammation: Chronic inflammation contributes to neuronal damage and the progression of tau pathology.

[0400] Symptoms typically appear years to decades after the last exposure to head trauma and may include: Cognitive decline (memory loss, executive dysfunction), mood disturbances (depression, anxiety, impulsivity), behavioral changes (aggression, emotional instability) and motor symptoms (tremors, gait disturbances). Currently, CTE can only be diagnosed postmortem through histopathological examination of brain tissue. However, clinical criteria are being developed for diagnosis in living patients, including neuropsychological assessments and imaging studies.

[0401] Post-concussion syndrome is a complex disorder that can occur after a concussion, where symptoms persist for weeks or months after the initial injury. It can affect individuals of all ages and is often seen in sports-related injuries. The exact mechanisms behind PCS are not fully understood, but several factors may contribute:

[0402] Neurometabolic changes: Following a concussion, there can be prolonged metabolic disturbances in the brain, including changes in energy metabolism and neurotransmitter levels.

[0403] Cognitive and psychological factors: Factors such as pre-existing mental health conditions, anxiety, and psychosocial stressors can exacerbate and prolong symptoms.

[0404] Symptoms of PCS can vary widely and may include: Headaches, dizziness and balance issues, cognitive difficulties (memory problems, attention deficits), sleep disturbances and emotional symptoms (irritability, anxiety, depression).

[0405] PCS is diagnosed based on clinical history and symptom evaluation, typically occurring after a confirmed concussion. The absence of structural brain abnormalities on imaging does not preclude the diagnosis. Moderate-to-severe TBI refers to brain injuries that result in significant impairment of consciousness and cognitive function. This classification is based on the Glasgow Coma Scale (GCS) score, with moderate TBI defined as a GCS of 9-12 and severe TBI as a GCS of 3-8. The injury can result from various causes, including blunt force trauma, penetrating injuries, or blast injuries. The pathophysiological processes involved include:

[0406] Primary Injury: Immediate damage to brain tissue from the impact, leading to contusions, lacerations, and axonal injury.

[0407] Secondary Injury: Cascading biological processes that occur post-injury, such as inflammation, ischemia, and excitotoxicity, which can exacerbate neuronal death and tissue loss.

[0408] Symptoms of moderate-to-severe TBI can vary widely depending on the injury's location and severity but may include: Altered consciousness or coma, cognitive deficits (memory loss, attention problems), physical impairments (motor deficits, coordination issues) and behavioral and emotional changes (agitation, mood swings).

[0409] Diagnosis typically involves clinical evaluation and neuroimaging (CT or MRI) to assess the extent of brain injury and detect structural changes.

[0410] Down syndrome (DS) is caused by trisomy 21, where individuals have three copies of chromosome 21 instead of the usual two. This genetic anomaly leads to a range of physical and intellectual disabilities, including characteristic facial features, hypotonia, and varying degrees of cognitive impairment. Individuals with Down syndrome are at an elevated risk of developing Alzheimer’s disease-like symptoms, particularly as they age. By the age of 40, nearly half of individuals with DS show signs of cognitive decline, and by age 60, this prevalence increases to over 70%. The early accumulation of amyloid-beta plaques and neurofibrillary tangles (comprised of hyperphosphorylated tau protein) mirrors the pathology seen in Alzheimer's disease. The presence of these neurodegenerative markers correlates with the progressive cognitive decline and behavioral changes observed in this population. Symptoms include memory loss, confusion, impaired judgment, and personality changes, often leading to significant challenges in daily functioning. Early identification and supportive care can help manage these symptoms.

[0411] Neurofibrillary Tangle Dementia (NTD) Postencephalitic Parkinsonism arises following viral encephalitis, often due to infections such as influenza or other neurotropic viruses, which can lead to encephalitic inflammation and damage to brain structures. Postencephalitic parkinsonism is a rare but recognized sequela of such infections. Neurofibrillary tangles, similar to those found in Alzheimer's disease, are central to NTD and contribute to the neurodegenerative process. These tangles are primarily composed of hyperphosphorylated tau protein, leading to neuronal dysfunction and death. The basal ganglia and other regions involved in movement and cognition are particularly affected. Patients may exhibit a combination of parkinsonian symptoms, such as tremor, rigidity, and bradykinesia, alongside cognitive decline characterized by memory loss, executive dysfunction, and personality changes. The clinical presentation can resemble both Parkinson's disease and Alzheimer's disease, complicating diagnosis and management.

[0412] In certain specific embodiments of the invention, said subject is suffering from or has a risk of suffering from a disease or medical condition selected from the group consisting of mild cognitive impairment (MCI), Alzheimer’s disease, mixed Alzheimer’s disease and vascular dementia. In certain specific embodiments of the invention, said subject is suffering from or has a risk of suffering from a disease or medical condition selected from the group consisting of Alzheimer’s disease and mild cognitive impairment (MCI).

[0413] In specific embodiments of the invention, said subject is suffering from or has a risk of suffering from a disease or medical condition selected from the group consisting of mild cognitive impairment (MCI), and Alzheimer’s disease.

[0414] In certain embodiments of the present invention, an increased amyloid-beta protein load in the brain is defined as a decrease in the concentration of amyloid-beta peptides, specifically amyloid-beta42 (Aβ42) and / or the ratio of Aβ42 and Aβ40 in a sample selected from the group comprising cerebrospinal fluid (CSF) or plasma, as measured by immunoassay techniques such as ELISA, Western blot, or mass spectrometry, measuring lower when compared to the baseline levels typically found in healthy individuals and wherein an increased phosphorylated tau load is defined as an elevation in the concentration of tau proteins phosphorylated at specific residues including but not limited to Thr181, Thr231 and Ser199 in a sample selected from the group comprising cerebrospinal fluid (CSF) or plasma, as measured by immunoassay techniques such as ELISA, Western blot, or mass spectrometry, exceeding the baseline levels typically found in healthy individuals.

[0415] Certain embodiments of the present invention relate to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof, wherein an increased amyloid-beta protein load in the brain is defined as a decrease in the concentration of amyloid-beta peptides, specifically amyloid-beta42 (Aβ42) and / or the ratio of Aβ42 and Aβ40 in a sample selected from the group comprising cerebrospinal fluid (CSF) or plasma, as measured by immunoassay techniques such as ELISA, Western blot, or mass spectrometry, measuring lower when compared to the baseline levels typically found in healthy individuals and wherein an increased phosphorylated tau load is defined as an elevation in the concentration of tau proteins phosphorylated at specific residues including but not limited to Thr181, Thr231 and Ser199 in a sample selected from the group comprising cerebrospinal fluid (CSF) or plasma, as measured by immunoassay techniques such as ELISA, Western blot, or mass spectrometry, exceeding the baseline levels typically found in healthy individuals.

[0416] In certain embodiments of the present invention healthy individuals typically exhibit Aβ42 levels above 200 pg / mL measured in the CSF. The person skilled in the art is aware that lower levels of Aβ42, particularly below 200 pg / mL are often associated with Alzheimer's disease and other amyloid-related pathologies. In particular embodiments the Aβ42 / Aβ40 ratio in healthy controls ranges between 0.06 to 0.11.

[0417] In certain embodiments of the present invention the concentration of amyloid-beta peptides amyloid-beta42 (Aβ42) and / or amyloid-beta ratio (Aβ42 / Aβ40) is decreased when the Aβ42 levels are below 200 pg / ml, and / or wherein the Aβ42 / Aβ40 ratio is below a ratio of 0.06 to 0.11. Decreased levels of Aβ42 and Aβ42 / Aβ40 ratio in CSF or plasma are usually indicative of Alzheimer's disease.

[0418] In certain embodiments of the present invention healthy individuals typically exhibit Tau181 levels below 18 pg / mL measured in the CSF. Elevated levels are usually indicative of tau pathologies including Alzheimer's disease.

[0419] In certain embodiments of the present invention the concentration of tau proteins phosphorylated at specific residues including Thr181 is increased when the Tau181 levels exceed 18 pg / mL.

[0420] In certain embodiments of the present invention healthy individuals typically exhibit Tau231 levels below 50 pg / mL measured in the CSF. Elevated levels are usually indicative of tau pathologies including Alzheimer's disease.

[0421] In certain embodiments of the present invention the concentration of tau proteins phosphorylated at specific residues including Thr231 is increased when the Tau181 levels exceed 18 pg / mL.

[0422] Certain embodiments of the present invention relate to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation, wherein the amyloid-beta ratio (Aβ42 / Aβ40) is below a certain threshold and / or phospho-Tau protein load in said subject is above a certain threshold.

[0423] Certain embodiments of the present invention relate to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation, wherein the AB42 concentration in the CSF of said subject is below a certain threshold and / or the concentration of phospho-Tau phosphorylated at specific residues including but not limited to Thrl81, Thr231, Serl99 load in the CSF of said subject is above a certain threshold. Certain embodiments of the present invention relate to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation, wherein the AB42 concentration in the CSF of said subject is below a certain threshold and / or phospho-Tau phosphorylated at specific residues including but not limited to Thrl81, Thr231, Serl99 load in the CSF of said subject is above a certain threshold.

[0424] Certain embodiments of the present invention relate to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation, wherein the amyloid-beta Aβ42 in said subject is below 200 pg / mL, particularly between 200 and 150 pg / mL, more particularly between 150 and 100 pg / mL, and even more particularly between 100 and 50 pg / mL, and the most particularly between 50 pg / mL and 1 pg / mL and / or wherein the amyloid-beta Aβ42 / Aβ40 ratio in said subject is below 0.11, particularly between 0.06 and 0.11, most particularly between 0.06 and 0.03, even more particularly between 0.03 and 0.01 and / or wherein the phospho-Tau231 protein load in said subject is above 50 pg / mL, particularly between the range of 50 to 250 pg / mL, even more particularly between 250 and 500 pg / mL, the most particularly between 500 and 1200 pg / mL, when measured in CSF and wherein the phospho-Tau181 protein load in said subject is above 18 pg / mL, particularly between the range of 18 to 22 pg / mL, even more particularly between 22 and 26 pg / mL, the most particularly between 26 and 50 pg / mL, when measured in CSF. In certain embodiments of the present invention the amyloid-beta ratio (Aβ42 / Aβ40) is below a certain threshold and / or phospho-Tau protein load in said subject is above a certain threshold.

[0425] In certain embodiments of the present invention the Aβ42 concentration in the CSF of said subject is below a certain threshold and / or the concentration of phospho-Tau phosphorylated at specific residues including but not limited to Thr181, Thr231, Ser199 load in the CSF of said subject is above a certain threshold.

[0426] In certain embodiments of the present invention the Aβ42 concentration in the CSF of said subject is below a certain threshold and / or phospho-Tau phosphorylated at specific residues including but not limited to Thr181, Thr231, Ser199 load in the CSF of said subject is above a certain threshold.

[0427] In certain embodiments of the present invention the amyloid-beta Aβ42 in said subject is below 200 pg / mL, particularly between 200 and 150 pg / mL, more particularly between 150 and 100 pg / mL, and even more particularly between 100 and 50 pg / mL, and the most particularly between 50 pg / mL and 1 pg / mL and / or wherein the amyloid-beta Aβ42 / Aβ40 ratio in said subject is below 0.11, particularly between 0.06 and 0.11, most particularly between 0.06 and 0.03, even more particularly between 0.03 and 0.01 and / or wherein the phospho-Tau231 protein load in said subject is above 50 pg / mL, particularly between the range of 50 to 250 pg / mL, even more particularly between 250 and 500 pg / mL, the most particularly between 500 and 1200 pg / mL, when measured in CSF and wherein the phospho-Taul81 protein load in said subject is above 18 pg / mL, particularly between the range of 18 to 22 pg / mL, even more particularly between 22 and 26 pg / mL, the most particularly between 26 and 50 pg / mL, when measured in CSF.

[0428] Certain embodiments of the present invention relate to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation, wherein the amyloid-beta ratio (Aβ42 / Aβ40) is below a certain threshold and / or phospho-Tau protein load in said subject is above a certain threshold.

[0429] Certain embodiments of the present invention relate to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation, wherein the AB42 concentration in the CSF of said subject is below a certain threshold and / or the concentration of phospho-Tau phosphorylated at specific residues including but not limited to Thrl81, Thr231, Serl99 load in the CSF of said subject is above a certain threshold.

[0430] Certain embodiments of the present invention relate to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation, wherein the AB42 concentration in the CSF of said subject is below a certain threshold and / or phospho-Tau phosphorylated at specific residues including but not limited to Thrl81, Thr231, Serl99 load in the CSF of said subject is above a certain threshold.

[0431] Certain embodiments of the present invention relate to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation, wherein the amyloid-beta Aβ42 in said subject is below 200 pg / mL, particularly between 200 and 150 pg / mL, more particularly between 150 and 100 pg / mL, and even more particularly between 100 and 50 pg / mL, and the most particularly between 50 pg / mL and 1 pg / mL and / or wherein the amyloid-beta Aβ42 / Aβ40 ratio in said subject is below 0.11, particularly between 0.06 and 0.11, most particularly between 0.06 and 0.03, even more particularly between 0.03 and 0.01 and / or wherein the phospho-Tau231 protein load in said subject is above 50 pg / mL, particularly between the range of 50 to 250 pg / mL, even more particularly between 250 and 500 pg / mL, the most particularly between 500 and 1200 pg / mL, when measured in CSF and wherein the phospho-Tau181 protein load in said subject is above 18 pg / mL, particularly between the range of 18 to 22 pg / mL, even more particularly between 22 and 26 pg / mL, the most particularly between 26 and 50 pg / mL, when measured in CSF. Certain embodiments of the present invention relate to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function, wherein a level of PAM below a certain threshold is indicative of a reduction in cognitive function.

[0432] In certain embodiments of the invention said subject is characterized by a level of PAM and / or its isoforms and / or fragments thereof below a certain threshold in a bodily fluid of said subject.

[0433] In particular embodiments, the level of PAM and / or its isoforms and / or fragments thereof is detected as total PAM concentration and / or total PAM activity.

[0434] More particularly this means the total concentration of all PAM isoforms, and / or the total alpha-amidating activity determinably in a sample obtained from said subject.

[0435] In certain embodiments, particular thresholds for total PAM concentration in a sample of bodily fluid, obtained from said subject are equal or below 75 ng / mL, particularly equal or below 65 ng / mL, more particularly equal or below 55 ng / mL, more particularly equal or below 45 ng / mL, more particularly equal or below 35 ng / mL and most particularly equal or below 30 ng / mL.

[0436] In certain embodiments, particular thresholds for PAM activity in a sample of bodily fluid,, obtained from said subject are equal or below 11.5 µg / L*h, particularly equal or below 10.5 µg / L*h, more particularly equal or below 9.5 µg / L*h, more particularly equal or below 8.5 µg / L*h, more particularly equal or below 7.5 µg / L*h and most particularly equal or below 6.5 µg / L*h, when PAM activity is measured in serum, wherein the quantification of activity of PAM in Units is as described in Example 3and the term Units / L defines the activity of PAM in Units per 1 L of sample material.

[0437] In certain embodiments, particular thresholds for PAM activity in a sample of bodily fluid,, obtained from said subject are equal or below 16.9 µg / L*h, particularly equal or below 15.4 µg / L*h, more particularly equal or below 13.9 µg / L*h, more particularly equal or below 12.3 µg / L*h, more particularly equal or below 10.9 µg / L*h and most particularly equal or below 9.4 µg / L*h, when PAM activity is measured in Li-heparin, wherein the quantification of activity of PAM in Units is as described in Example 1 and the term Units / L defines the activity of PAM in Units per 1 L of sample material. The person skilled in the art is aware that the activity may also be determined in other bodily fluids and how to determine the respective threshold.

[0438] In certain embodiments of the invention said sample of bodily fluid of said subject is selected from the group of whole blood, serum or plasma. The levels of PAM or fragments thereof of the present invention have been determined with the described assays as outlined in Kaufmann et al. 2021 and example 3 for the measuring of amidating activity and as outlined in Ilina et al. 2023 for the measuring of PAM concentration.

[0439] The level of PAM or fragments thereof may be the total PAM concentration or PAM activity, respectively. The mentioned threshold values above might be different in other assays, if these have been calibrated differently from the assay systems used in the present invention. Therefore, the mentioned cut-off values above shall apply for such differently calibrated assays accordingly, taking into account the differences in calibration. One possibility of quantifying the difference in calibration is a method comparison analysis (correlation) of the assay in question with the respective biomarker assay used in the present invention by measuring the respective biomarker (e.g., PAM or fragments thereof) in samples using both methods. Another possibility is to determine with the assay in question, given this test has sufficient analytical sensitivity, the median biomarker level of a representative normal population, compare results with the median biomarker levels as described in the literature and recalculate the calibration based on the difference obtained by this comparison.

[0440] With the calibration used in the present invention, samples from 4106 normal (healthy) subjects (Swedish single-center prospective population-based Study (MPP-RES)) have been measured: median (interquartile range (IQR)) plasma PAM concentration was 78.6 ng / ml (66.4 ng / ml - 92.5 ng / ml).

[0441] With the calibration used in the present invention, samples from 4942 normal (healthy) subjects (Swedish single-center prospective population-based Study (MPP-RES)) have been measured: median (interquartile range (IQR)) PAM activity in serum was 12.5 µg / (L*h) (10.9 – 14.5). One Unit = one µg / (L*h) as defined in Example 1.

[0442] Threshold levels can be obtained for instance from a Kaplan-Meier analysis, where the occurrence of a disease is correlated with the quartiles of the biomarker in the population. According to this analysis, subjects with biomarker levels below the 25th percentile have a significantly increased risk for getting the diseases according to the invention. This result is further supported by Cox regression analysis with full adjustment for classical risk factors: The lowest quartile versus all other subjects is highly significantly associated with increased risk for getting a disease according to the invention.

[0443] Other particular threshold values are for instance the 20th, 10th or 5th percentile of a normal population. By using a lower percentile than the 25th percentile, one reduces the number of false positive subjects identified, but one might miss identifying subjects, who are at moderate, albeit still increased risk. Thus, one might adopt the cut-off value depending on whether it is considered more appropriate to identify most of the subjects at risk at the expense of also identifying "false positives", or whether it is considered more appropriate to identify mainly the subjects at high risk at the expense of missing several subjects at moderate risk.

[0444] In certain embodiments of the invention the reduction after treatment of the subject is decreased compared to the reduction in neuroinflammation before treatment of the subject by at least 5% particularly by at least 10%, more particular by at least 15%, even more particular by at least 20%, even more particular by at least 25%, even more particular by at least 30%, most particular by at least 35%. In certain embodiments of the invention monitoring is performed after treatment every 1 to 12 months, particularly every 2 to 9 months, particularly every 3 to 6 months, most particular every 3 months. Reduction after treatment of the subject is measured 1 -12 months, particularly – 9 months, more particularly 3 – 6 months, most particular 3 months if said subject is suffering from or has a risk of suffering from a chronic disease or medical condition selected from the group comprising Alzheimer's Disease, Mild cognitive impairment (MCI), Frontotemporal Dementia (FTD), Lewy Body Dementia (LBD), Parkinson's Disease with Dementia (PDD), Huntington's Disease, Amyotrophic Lateral Sclerosis (ALS) with cognitive impairment, Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD), Multiple System Atrophy (MSA), prion diseases (e.g., Creutzfeldt-Jakob Disease), Cerebral Amyloid Angiopathy (CAA), Down Syndrome (DS) with Alzheimer-like dementia, Chronic Traumatic Encephalopathy (CTE), and Neurofibrillary Tangle Dementia (NTD) Postencephalitic Parkinsonism.

[0445] In certain embodiments of the invention the reduction in cognitive function after treatment of the subject is decreased compared to the reduction in cognitive function before treatment of the subject by at least 5% particularly by at least 10%, more particular by at least 15%, even more particular by at least 20%, even more particular by at least 25%, even more particular by at least 30%, most particular by at least 35%.

[0446] In certain embodiments of the invention the cognitive function after treatment for the prevention of a reduction in cognitive function is monitored in said subject. Said monitoring is performed every 1 to 12 months, particularly every 2 to 9 months, particularly every 3 to 6 months, most particular every 3 months

[0447] Reduction in cognitive function after treatment of the subject is measured 1 -12 months, particularly – 9 months, more particularly 3 – 6 months, most particular 3 months if said subject is suffering from or has a risk of suffering from a chronic disease or medical condition selected from the group comprising Neurodegenerative Disorders including Alzheimer's Disease, MCI patients, Frontotemporal Dementia (FTD), Lewy Body Dementia (LBD), Parkinson's Disease with Dementia (PDD), Huntington's Disease, Amyotrophic Lateral Sclerosis (ALS) with cognitive impairment, Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD), Multiple System Atrophy (MSA), Prion Diseases (e.g., Creutzfeldt-Jakob Disease);

[0448] Vascular Disorders including Vascular Dementia, Stroke, Multi-Infarct Dementia, Subcortical Ischemic Vascular Dementia, Cerebral Amyloid Angiopathy, Strategic Single-Infarct Dementia, Transient Ischemic Attack (TIA), Hypertensive Encephalopathy;

[0449] Traumatic Brain Injury (TBI) Related Disorders Chronic Traumatic Encephalopathy (CTE), Post-Concussion Syndrome, Diffuse Axonal Injury, Second Impact Syndrome, Subdural Hematoma, Epidural Hematoma, Penetrating Brain Injury, Post-Traumatic Amnesia;

[0450] Inflammatory and Infectious Disorders including Multiple Sclerosis (MS), Sepsis-Associated Encephalopathy (SAE), HIV-Associated Neurocognitive Disorder (HAND), Autoimmune Encephalitis, Meningitis and Encephalitis;

[0451] Endocrine and Metabolic Disorders including Diabetes Mellitus, Hyperglycemia, Hypoglycemia.

[0452] In certain embodiments of the invention the reduction in neuroinflammation after treatment of the subject is decreased compared to the reduction in neuroinflammation before treatment of the subject by at least 5% particularly by at least 10%, more particular by at least 15%, even more particular by at least 20%, even more particular by at least 25%, even more particular by at least 30%, most particular by at least 35%.

[0453] In certain embodiments of the invention the neuroinflammation after treatment for the prevention of a neuroinflammation is monitored in said subject. Said monitoring is performed every 1 to 12 months, particularly every 2 to 9 months, particularly every 3 to 6 months, most particular every 3 months.

[0454] Reduction in neuroinflammation after treatment of the subject is measured 1 -12 months, particularly – 9 months, more particularly 3 – 6 months, most particular 3 months if said subject is suffering from or has a risk of suffering from a chronic disease or medical condition selected from the group comprising Alzheimer's Disease, Mild cognitive impairment (MCI), Frontotemporal Dementia (FTD), Lewy Body Dementia (LBD), Parkinson's Disease with Dementia (PDD), Huntington's Disease, Amyotrophic Lateral Sclerosis (ALS) with cognitive impairment, Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD), Multiple System Atrophy (MSA), prion diseases (e.g., Creutzfeldt-Jakob Disease), Cerebral Amyloid Angiopathy (CAA), Down Syndrome (DS) with Alzheimer-like dementia, Chronic Traumatic Encephalopathy (CTE), and Neurofibrillary Tangle Dementia (NTD) Postencephalitic Parkinsonism.

[0455] In certain specific embodiments of the invention, the modified or unmodified PAM or fragments thereof are isolated from a tissue or organ or are recombinantly produced as wild-type PAM, modified PAM, or chimeric PAM. In further embodiments of the invention, the modified or unmodified PAM or fragments thereof are recombinantly expressed using a recombinant system selected from the group comprising:

[0456] • Mammalian cell lines, including but not limited to CHO-K1, CHO-S, and CHO-DG4;

[0457] • Yeast systems, including but not limited to Pichia pastoris strains X-33, GS115, and KM71; • Bacterial systems, including but not limited to Escherichia coli strains BL21(DE3), Rosetta(DE3), and Shuffle;

[0458] • Insect cell systems, including but not limited to baculovirus expression systems using cell lines such as Sf9, Sf21, and High Five;

[0459] • Plant-based systems, including but not limited to Nicotiana benthamiana, BY-2, and rice cell cultures;

[0460] • Cell-free expression systems, including but not limited to wheat germ extract, rabbit reticulocyte lysate, and E. coli S30 extract.

[0461] In specific embodiments of the invention, the modified or unmodified PAM or fragments thereof are recombinantly expressed in CHO or E. coli cells.

[0462] In certain specific embodiments of the invention, the modified or unmodified PAM or fragments thereof are purified from an organ or tissue selected from the group comprising heart, brain (specifically hypothalamus, hippocampus, or pituitary glands), blood, plasma, adrenal glands, thyroid glands, epithelial tissue (specifically endothelium and endometrium), pancreas, and kidney.

[0463] In certain specific embodiments of the invention, the modified or unmodified PAM or fragments thereof are purified from blood, plasma, or brain.

[0464] As used herein, “PAM” includes all isoforms of PAM, in particular PAM-1, PAM-2, PAM-3, PAM-4, PAM-5 and PAM-6 (SEQ ID No. 1 to SEQ ID No. 6).

[0465] It is to be understood by the skilled artisan, that the PAM isoform sequences (SEQ ID No. 30 to 35) as represented in the sequence list, contain an N-terminal signal sequence (amino acid 1 -20) and pro-region (amino acid 21 to 30). This N-terminal signal sequence and pro-region is cleaved off prior to secretion of the protein. Therefore, in particular embodiments the PAM isoform sequences (SEQ ID No. 1 to 6) and / or fragments thereof do not contain the N-terminal signal sequence. Moreover, the person skilled in the art understands that all isoforms of PAM catalyse the conversion of peptidylglycines into alpha-amidated peptides leading to a biological activation of these hormones. These isoforms only exhibit differences in tissue distribution, enzymatic activity, and regulatory properties.

[0466] In certain embodiments of the invention the modified or unmodified fragments of PAM are modified or unmodified fragments of PAM according to SEQ ID No. 7, SEQ ID No. 8 or SEQ ID No. 9.

[0467] Certain embodiments of the invention relate to the modified or unmodified fragments of PAM according to SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 9 for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function.

[0468] Certain embodiments of the invention relate to the modified or unmodified fragments of PAM according to SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 9 for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function and said subject is suffering from or has a risk of suffering from a disease or medical condition selected from the group consisting of mild cognitive impairment (MCI), Alzheimer’s disease, mixed Alzheimer’s disease, and vascular dementia.

[0469] Certain embodiments of the invention relate to the fragments of PAM according to SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 9 for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation.

[0470] Certain embodiments of the invention relate to the fragments of PAM according to SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 9 for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation and said subject is suffering from or has a risk of suffering from a disease or medical condition selected from the group consisting of mild cognitive impairment (MCI), Alzheimer’s disease, mixed Alzheimer’s disease, and vascular dementia.

[0471] In certain embodiments said PAM or fragments thereof are isolated from a cell or tissue (including bodily fluids, e.g. blood) / organ or are recombinantly produced or are chimeric PAM or fragments thereof. Said cell or tissue / organ may be selected from mammalians, bacteria, yeast, fungi etc.

[0472] PAM for use according to the present invention can be a commercial PAM enzyme, or any formulation comprising the PAM enzyme and any means capable of producing a functional PAM enzyme in the context of the current invention, such as DNA or RNA nucleic acids encoding a PAM protein. The nucleic acid encoding PAM may be embedded in suitable vectors such as plasmids, phagemids, phages, (retro)viruses, transposons, gene therapy vectors and other vectors capable of inducing or conferring production of PAM. Also native or recombinant microorganisms, such as bacteria, fungi, protozoa and yeast may be applied as a source of PAM in the context of the current disclosure.

[0473] In some embodiments, the mammalian PAM is a human, a bovine or porcine PAM.

[0474] In certain embodiments said PAM is a protein comprising an amino acid sequence selected from the group comprising SEQ ID Nos. 1 to 10, or an amino acid sequence having at least 85%, particularly at least 90%, more particularly at least 95%, even more particularly at least 99% sequence identity therewith.

[0475] In certain embodiments, the PAM is a functional fragment (i.e., PHM (SEQ ID No. 7 and / or SEQ ID No. 9) and PAL (SEQ ID No. 8), PAM conserving at least about 85%, at least about 90%, at least about 95%, or at least about 99% of the PAM activity of the corresponding functional fragment of PAM. In some embodiments, the PAM is a variant or a derivative of PAM disclosed herein.

[0476] The percentage of identity of an amino acid or nucleic acid sequence, or the term “% sequence identity”, is defined herein as the percentage of residues in a candidate amino acid or nucleic acid sequence that is identical with the residues in a reference sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity.

[0477] In particular embodiments, the calculation of said at least percentage of sequence identity is carried out without introducing gaps. Methods and computer programs for the alignment are well known in the art, for example “Align 2” or the BLAST service of the National Center for Biotechnology Information (NCBI).

[0478] In certain specific embodiments of the invention said PAM or fragment thereof is enzymatically active. As used herein, “enzymatically active” refers to a protein or modified form of such protein or a fragment of such protein or modified form thereof, thereof that retains or essentially retains the proteins enzymatic activity. In the case of PAM, the enzymatic activity of refers in particular to the alpha-amidating activity of PAM.

[0479] The term “unmodified PAM” refers to all isoforms and single subunits of PAM, in particular those based on the SEQ ID Nos. 1 to 10, and which have not undergone any deliberate or artificial modifications, including chemical conjugation, alteration of the amino acid sequence, including amino acid exchange, deletion or insertion, fusion with other proteins, or other such methods. While there can be certain variations depending on its source organism, conditions of expression and / or purification, as well as certain naturally occurring mutants and variants known in the field, in particular unmodified PAM has an amino acid sequence of naturally occurring PAM and more particularly at least essentially retains its natural three-dimensional structure, function, and properties. The term “modified PAM” refers to all isoforms and single subunits of PAM, in particular those based on the SEQ ID Nos. 1 to 10, which are modified in accordance with the present invention, in particular as detailed herein, more particularly by amino acid manipulation, by fusion to other proteins, such as albumin, e.g. serum albumin or recombinant serum albumin, by non-covalent binding to serum albumin via a conjugated fatty acid chain to PAM, via fusion with IgG Fc regions or transferrin, via post-translational modification attaching natural or synthetic polymers, wherein the natural or synthetic polymer to be used is particularly HAP, more particularly ELP, more particularly PAS, more particularly PSA, more particularly GLK, more particularly XTEN and even more particularly PEG.

[0480] Modifications of PAM may be selected from the group comprising:

[0481] • Amino acid manipulation, also referred to as site directed mutagenesis, including the insertion, deletion or alteration of one or more amino acids within the polypeptide amino acid sequence reducing immunogenicity and proteolytic instability in vivo. Thereby alterations of one or more amino acids may lead to enhanced protease resistance in vivo.

[0482] • Conjugation and / or bioconjugation of polypeptides of interest with serum proteins, such as albumins or immunoglobulins or parts of immunoglobulins to produce fusion proteins:

[0483] • Fusion of polypeptides with Albumins, e.g. serum Albumin or recombinant serum Albumin.

[0484] • Non covalent binding to serum Albumin due to a conjugated fatty acid chain to a polypeptide of interest. The binding to albumin therefore happens in vivo after application of the fatty acid conjugated substance and is mediated by the fatty acid chain.

[0485] • Fusion of polypeptides with IgG Fc regions or Transferrin.

[0486] • Post-translational modifications attaching natural or synthetic polymers to the polypeptide of interest. Example for such polymers, but not limited to, are

[0487] ■ PEG, either single -stranded or branched PEG, having varying molecular weights covalently fused to polypeptides of interest.

[0488] ■ XTEN, an unstructured polypeptide, covalently fused to polypeptides of interest. XTEN is a 864 single amino-acid sequence composed of amino-acids Ala, Glu, Gly, Pro, Ser and Thr in a randomized manner. Half-life of an XTEN-fusion protein may be tailored by shortening of the XTEN sequence.

[0489] PAS, which is a peptide polymer consisting of amino acids proline, alanine and serine wit 100- 200 PAS repeats forming the polymer. ■ ELP (Elastin-like polypeptides) consisting of Valin-Prolin-Glycine-x-Glycine repeats, naturally found in elastin, wherein x relates to any amino-acid except for Proline. ELP can be covalently attached to a polypeptide of interest.

[0490] ■ HAP, which is a repeated sequence of glycine rich (Gly4Ser)n polypeptide, wherein n is inbetween of 100-200 and is covalently attached to the protein of interest.

[0491] ■ GLK, which is a gelatin-like fusion protein. Thereby GLK is a (Gly-X-Y)n structure, wherein X and Y are any amino-acids except for Cysteine, with n = 60 to 1500. GLK can be covalently attached to a polypeptide of interest.

[0492] ■ Carbohydrates and polysaccharides. Carbohydrates, either branched or linear can be attached to the polypeptide of interest e.g. through in vivo N-Glycosylation. Conjugation with Dextrans, Hydroxyethyls (HES), Heparosan (HEP), Hyaluronic acid (HA) represents the attachment of polysaccharides.

[0493] ■ PSA (Polysialic acid), wherein PSA polymers are covalently attached to the polypeptide of interest.

[0494] In certain embodiments of the invention said PAM or fragments thereof are modified by

[0495] a. attaching one or more natural or synthetic polymers units, wherein said polymers are selected from the group comprising

[0496] i. PEG, particularly having an average molecular weight in the range of 0.2-100 kDa, particularly 1-90 kDa, more particularly 2-80 kDa, more particularly 3-70 kDa, more particularly 4-60 kDa and even more particularly 5 to 50kDa; ii. XTEN;

[0497] iii. a peptide polymer consisting of repeats of PAS (the amino acids proline, alanine and serine, particularly comprising 100-200, particularly 120-180, more particularly 130-170, even more particularly about 150 PAS repeats;

[0498] iv. elastin-like polypeptides consisting of Valin-Prolin-Glycine-x-Glycine repeats, wherein x is any amino acid other than Proline, particularly consisting of from 150, 200, 250, 300, 400, or 500 to 550, 600, 750, 850, 950 or 1,000 amino acids; v. HAP polypeptide, having the sequence (Gly4Ser)n, wherein n is 100-200; vi. gelatin-like fusion protein, having the sequence (Gly-X-Y)n, wherein X and Y individually are any amino acid except for Cysteine, and wherein n = 60 to 1500;

[0499] vii. polysaccharides, in particular those chosen from the group comprising:

[0500] 1. dextrans;

[0501] 2. hydroxyethyls;

[0502] 3. heparosan;

[0503] 4. hyaluronic acid;

[0504] viii. poly sialic acid.

[0505] b. conjugation with a serum protein, such as albumin or an immunoglobulin or parts of an immunoglobulin;

[0506] c. site-directed mutagenesis by insertion, deletion and / or exchange of one or more amino acids within the amino acid sequence of PAM or fragments thereof.

[0507] In certain more specific embodiments of the invention said PAM or fragments thereof are modified by attaching one or more natural or synthetic polymers units, wherein said polymers are selected from the group consisting of

[0508] i. PEG, particularly having an average molecular weight in the range of 0.2-100 kDa, particularly 1-90 kDa, more particularly 2-80 kDa, more particularly 3-70 kDa, more particularly 4-60 kDa and even more particularly 5 to 50 kDa; or ii. XTEN; or

[0509] iii. HAP polypeptide, having the sequence (Gly4Ser)n, wherein n is 100-200.

[0510] In certain very specific embodiments of the invention said PAM or fragments thereof are modified by attaching one or more polymer units, wherein said polymer is PEG.

[0511] In certain very specific embodiments of the invention said polymer is PEG having an average molecular weight of 5 to 50 kDa.

[0512] In other more particular embodiments, modification of PAM is modification with PEG (PEGylation), particularly PEG with an average molecular weight of 5-10 kDa (PEG 5000 to PEG 10.000) or, more particularly with an average molecular weight of about 5 kDa (PEG 5000). In further embodiments, modification of PAM with PEG (PEGylation) shall mean the attachment of PEG 5000 or 10000 as detailed herein to serine or lysine side chains of PAM, more particularly the attachment of PEG 10000 as detailed herein to lysine side chains of PAM, or more particularly the attachment of PEG 5000 as detailed herein to lysine or serine, even more particularly to serine side chains of PAM.

[0513] In other more particular embodiments, modification of PAM is modification with XTEN as defined herein, particularly with XTEN having a sequence of SEQ ID No: 36, more particularly to lysine or serine side chains of PAM, more particularly to lysine side chains of PAM, or alternatively via a Cys amino acid C-terminally added to PAM.

[0514] In certain embodiments, modification of PAM with PEG (PEGylation) shall mean the attachment of PEG as detailed herein to lysine side chains, in particular in alkaline aqueous solution, e.g. in a buffered solution, particularly at a pH from 7.75 to 9.25, more particularly from 8.25 to 8.75, more particularly about 8.5; in particular with a molar excess of PEG polymer vs. PAM of 70- to 140-fold, more particularly 80- to 130-fold, more particularly 90- to 120-fold; in particular embodiments with a method analogous to the method described in Example 5, which can be adapted to other PAM types and / or other PEG polymers by routine methods. The PEGylation rate and molecular weight of the resulting PEGylated PAM can be analyzed by various well-known methods, such as gel filtration or SDS-PAGE. Modifications of PAM may also include modifications, which allow PAM to act as a prodrug. The term "prodrug" denotes a form or derivative of a compound which is metabolized in vivo, e.g., by biological fluids or enzymes by a subject after administration, into a pharmacologically active form of the compound in order to produce the desired pharmacological effect.

[0515] Prodrugs can thus be viewed as drugs containing specialized non-toxic protective groups used in a transient manner to alter or to eliminate undesirable properties in the parent molecule. The group of the afore mentioned prodrugs may comprise Carrier-linked prodrugs (Carrier prodrugs), Cascade prodrugs and PEG-based carrier prodrugs. A carrier-linked prodrug may be a prodrug that contains a temporary linkage of a given active substance with a transient carrier group that produces improved physicochemical or pharmacokinetic properties and that can be easily removed in vivo, usually by a hydrolytic cleavage, wherein a cascade prodrug is a prodrug for which the cleavage of the carrier group becomes effective only after unmasking an activating group and wherein several examples of PEG-based carrier prodrugs exist, most of them with the need for enzymatic activation of the linker between the active drug and the carrier, mostly initiated by enzymatic hydrolysis. Since esters are cleaved very readily and unpredictably in vivo, direct ester linkers for carrier pro drug have limitations to their usability (J. Rautio et al., Nature Reviews Drug discovery, 2008, 7 255-270).

[0516] Modified PAM may refer to PAM (SEQ ID No.: 1-10) modified via amino acid manipulations, particularly via fusion to Albumins, e.g. serum Albumin or recombinant serum Albumin, more particularly via non covalent binding to serum Albumin due to a conjugated fatty acid chain to PAM, more particularly via fusion with IgG Fc regions or Transferrin, most particularly via post-translational modifications attaching natural or synthetic polymers, whereas the natural or synthetic polymer to be used is HAP, particularly ELP, more particularly PAS, more particularly PSA, more particularly GLK, more particularly XTEN and most particularly PEG.

[0517] PEG-PAM may refer to PAM (SEQ ID No.: 1-10) modified with 1+n molecules of polyethylene glycol (PEG), whereas n is an integer in the range of 0 to 100 and one PEG molecule has a molecular weight in the range of 1-100 kDa and is either a linear molecule or a branched molecule with b+1 branches, whereas b is an integer in the range of 0 to 20.

[0518] PAM modified via site directed mutagenesis may refer to PAM (SEQ ID No.: 1-10) with an insertion of n+1 additional amino-acids and / or n+1 deletions of amino-acids and / or n+1 exchanged amino-acids in the given amino-acid sequence (SEQ ID No.: 1-10), wherein n is an integer in the range of 0-100. PAM modified via fusion to native serum Albumin or recombinant human serum Albumin may refer to PAM (SEQ ID No.: 1-10) fused to native human serum Albumin or recombinant human serum Albumin at the N-terminus or at the C-terminus of PAM according to (SEQ ID No.: 1-10).

[0519] PAM modified via fusion to IgG Fc regions may refer to PAM (SEQ ID No.: 1-10) fused to human IgGl Fc Region at the N-terminus or at the C-terminus of PAM according to (SEQ ID No.: 1-10).

[0520] PAM modified via XTEN may refer to PAM (SEQ ID No.: 1-10) fused to a XTEN moiety at the N-terminus or at the C-terminus of PAM according to (SEQ ID No.: 1-10) and / or to any surface exposed amino-acid of PAM according to (SEQ ID No.: 1-10).

[0521] Fusion may refer to a covalent or non-covalent linkage of two proteins or polypeptides, a protein and a polypeptide, a synthetic polymer and a polypeptide and / or a synthetic polymer and a protein to each other. In particular embodiments fusion may refer to covalent linkage.

[0522] A person skilled in the art knows how to create an expression vector for expression of N-terminal or C-terminal fusion proteins. The person skilled in the art knows that fusion of two proteins can be achieved by usage of state-of-the-art techniques to result in a covalent or non-covalent linkage of two proteins, a protein and a polypeptide or a protein and / or a polypeptide and a synthetic polymer. The person skilled in the art knows how to perform site directed mutagenesis.

[0523] In terms of this invention, the biological activity of the modified PAM shall remain present to an extent of at least 1 % (in particular 1-300%) when directly compared to the non-modified active moiety, particularly of at least 5% (in particular 5-300%), more particularly of at least 15% (in particular 15-300%), more particularly of at least 30% (in particular 30-300%), more particularly of at least 50% (in particular -300%) more particularly of at least 70% (in particular 70-300%), more particularly of at least 90% (in particular 90-300%), more particularly of at least 150% (in particular 150-300%), more particularly of at least 200% (in particular 200-300%) and most particularly of at least 250% (in particular 250 -300%). In terms of modified PAM, biological activity shall mean the capability of modified PAM to perform C-terminal amidation and may be tested using PAM activity assays with synthetic, labelled tripeptides, such as Tyr-Val-Gly as substrate. Particularly the activity is determined as described in Example 1 using wildtype ADM-Gly as substrate.

[0524] In certain embodiments said PAM or fragment thereof is administered in combination with ascorbate and / or copper and / or peptide with C-terminal Glycine amino acid or fragments thereof. More particularly, such fragments have essentially the same physiological activity as the respective full-length peptide with C-terminal Glycine amino acid.

[0525] In certain embodiments of the invention said peptide with C-terminal Glycine amino acid is selected from the group of comprising adrenomedullin (ADM), adrenomedullin-2, intermedin-short, pro-adrenomedullin N-20 terminal peptide (PAMP), amylin, gastrin-releasing peptide, neuromedin C, neuromedin B, neuromedin S, neuromedin U, calcitonin, calcitonin gene-related peptide (CGRP) 1 and 2, islet amyloid polypeptide, chromogranin A, insulin, pancreastatin, prolactin-releasing peptide (PrRP), cholecystokinin, big gastrin, gastrin, glucagon-like peptide 1 (GLP-1), pituitary adenylate cyclase-activating polypeptide (PACAP), secretin, somatoliberin, peptide histidine methionine (PHM), vasoactive intestinal peptide (VIP), gonadoliberin, kisspeptin, MIF-1, metastin, neuropeptide K, neuropeptide gamma, neuropeptide Y, substance P, neurokinin A, neurokinin B, peptide YY, pancreatic hormone, deltorphin I, orexin A and B, melanotropin alpha (alpha-MSH), melanotropin gamma, thyrotropin-releasing hormone (TRH), oxytocin, vasopressin or fragments thereof.

[0526] Certain embodiments of the invention relate to modified or unmodified alpha-amidating monooxygenase (PAM) or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function, wherein PAM is combined with Vitamin C.

[0527] Certain embodiments of the invention relate to modified or unmodified alpha-amidating monooxygenase (PAM) or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function, wherein PAM is combined with a peptide with C-terminal Glycine amino acid, in particular adrenomedullin (ADM), vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), glucagon-like peptide 1 (GLP-1) and neuropeptide Y or fragments thereof.

[0528] Certain embodiments of the invention relate to modified or unmodified alpha-amidating monooxygenase (PAM) or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function, wherein said fragments of ADM-Gly (SEQ ID No. 14) are selected from the group of ADM-Gly 2-53 (SEQ ID No.

[0529] 17), ADM-Gly 8-53 (SEQ ID No. 18), ADM-Gly 22-53 (SEQ ID No. 19), ADM-Gly 27-53 (SEQ ID No. 20) and ADM-Gly 33-53 (SEQ ID No. 21).

[0530] In certain embodiments of the invention modified or unmodified PAM or a fragment thereof is combined with Vitamin C.

[0531] In certain embodiments of the invention modified or unmodified PAM or a fragment thereof is combined with a peptide with C-terminal Glycine amino acid, in particular adrenomedullin (ADM), vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), glucagon-like peptide 1 (GLP-1) and neuropeptide Y or fragments thereof.

[0532] In certain embodiments of the invention fragments of ADM-Gly (SEQ ID No. 14) are selected from the group of ADM-Gly 2-53 (SEQ ID No. 17), ADM-Gly 8-53 (SEQ ID No. 18), ADM-Gly 22-53 (SEQ ID No. 19), ADM-Gly 27-53 (SEQ ID No. 20) and ADM-Gly 33-53 (SEQ ID No. 21).

[0533] In certain embodiments of the invention modified or unmodified PAM or a fragment is to be used in combination with ADM-Gly or fragments thereof and in addition is to be used with Vitamin C.

[0534] In certain embodiments of the invention modified or unmodified PAM or a fragment is to be used in combination with ADM-Gly or fragments thereof and in addition is to be used with Vitamin C, wherein administered ADM-Gly or fragments thereof is amidated by administered PAM in vivo. Moreover, said combination leads to a sustained elevation of bio-ADM.

[0535] The effect of said combination is the prolonged elevation of resulting bio-ADM in circulation in comparison to the short bioavailability of wildtype-bio-ADM when administered alone. Other than in the state of the art, an elevation of active bio-ADM levels in circulation is reached without modifications of ADM-Gly or fragments thereof in said combination.

[0536] Certain embodiments of the invention relate to PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function, wherein PAM is to be used in combination with ADM-Gly or fragments thereof and in addition is to be used with Vitamin C.

[0537] Certain embodiments of the invention relate to PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function, wherein PAM is to be used in combination with ADM-Gly or fragments thereof and in addition is to be used with Vitamin C, wherein administered ADM-Gly or fragments thereof is amidated by administered PAM in vivo. Moreover, said combination leads to a sustained elevation of bio-ADM. The effect of said combination is the prolonged elevation of resulting bio-ADM in circulation in comparison to the short bioavailability of wildtype-bio-ADM when administered alone. Other than in the state of the art, an elevation of active bio-ADM levels in circulation is reached without modifications of ADM-Gly or fragments thereof in said combination. Therefore, said combinations according to the present invention are suitable for treatment and / or prevention of cognitive impairment.

[0538] Certain embodiments of the invention relate to modified or unmodified alpha-amidating monooxygenase (PAM) or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation, wherein PAM is combined with Vitamin C.

[0539] Certain embodiments of the invention relate to modified or unmodified alpha-amidating monooxygenase (PAM) or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation, wherein PAM is combined with a peptide with C-terminal Glycine amino acid, in particular adrenomedullin (ADM), vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), glucagon-like peptide 1 (GLP-1) and neuropeptide Y or fragments thereof.

[0540] Certain embodiments of the invention relate to modified or unmodified alpha-amidating monooxygenase (PAM) or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation, wherein said fragments of ADM-Gly (SEQ ID No. 14) are selected from the group of ADM-Gly 2-53 (SEQ ID No. 17), ADM-Gly 8-53 (SEQ ID No. 18), ADM-Gly 22-53 (SEQ ID No. 19), ADM-Gly 27-53 (SEQ ID No. 20) and ADM-Gly 33-53 (SEQ ID No. 21).

[0541] Certain embodiments of the invention relate to PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation, wherein PAM is to be used in combination with ADM-Gly or fragments thereof and in addition is to be used with Vitamin C.

[0542] Certain embodiments of the invention relate to PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation, wherein PAM is to be used in combination with ADM-Gly or fragments thereof and in addition is to be used with Vitamin C, wherein administered ADM-Gly or fragments thereof is amidated by administered PAM in vivo. Moreover, said combination leads to a sustained elevation of bio-ADM.

[0543] The effect of said combination is the prolonged elevation of resulting bio-ADM in circulation in comparison to the short bioavailability of wildtype-bio-ADM when administered alone. Other than in the state of the art, an elevation of active bio-ADM levels in circulation is reached without modifications of ADM-Gly or fragments thereof in said combination. Therefore, said combinations according to the present invention are suitable for treatment and / or prevention of neuroinflammation.

[0544] In certain embodiments of the invention said peptide with C-terminal glycine amino acid is selected from the group comprising adrenomedullin (ADM), adrenomedullin-2, intermedin-short, pro-adrenomedullin N-20 terminal peptide (PAMP), amylin, gastrin-releasing peptide, neuromedin C, neuromedin B, neuromedin S, neuromedin U, calcitonin, calcitonin gene-related peptide (CGRP) 1 and 2, islet amyloid polypeptide, chromogranin A, insulin, pancreastatin, prolactin-releasing peptide (PrRP), cholecystokinin, big gastrin, gastrin, glucagon-like peptide 1 (GLP-1), pituitary adenylate cyclase-activating polypeptide (PACAP), secretin, somatoliberin, peptide histidine methionine (PHM), vasoactive intestinal peptide (VIP), gonadoliberin, kisspeptin, MIF-1, metastin, neuropeptide K, neuropeptide gamma, neuropeptide Y, substance P, neurokinin A, neurokinin B, peptide YY, pancreatic hormone, deltorphin I, orexin A and B, melanotropin alpha (alpha-MSH), melanotropin gamma, thyrotropin-releasing hormone (TRH), oxytocin, vasopressin or fragments thereof.

[0545] In other certain embodiments of the invention said peptide with C-terminal glycine amino acid is selected from the group consisting of adrenomedullin (ADM) vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), glucagon-like peptide 1 (GLP-1) and neuropeptide Y.

[0546] In particular embodiments of the present invention, said PAM is administered orally, epicutaneously, subcutaneously, intradermally, sublingually, intramuscularly, intraarterially, intravenously, via the central nervous system (CNS, intracerebrally, intracerebroventricularly, intrathecally) or via intraperitoneal administration, particularly epicutaneously, subcutaneously, intradermally, intramuscularly, or intraperitoneally, more particularly subcutaneously, intramuscularly, or intraperitoneally; “formulated to be administered (e.g. intramuscularly)” likewise reads on “formulated for (e.g. intramuscular) administration”.

[0547] In particular embodiments of the present invention, said PAM is administered epicutaneously, subcutaneously, intradermally, intramuscularly, intraarterially, intravenously, via the central nervous system (CNS, intracerebrally, intracerebroventricularly, intrathecally) or via intraperitoneal administration, particularly epicutaneously, subcutaneously, intradermally, intramuscularly, or intraperitoneally, more particularly subcutaneously, intramuscularly, or intraperitoneally; “formulated to be administered (e.g. intramuscularly)” likewise reads on “formulated for (e.g. intramuscular) administration.”

[0548] In certain more specific embodiments of the present invention, said PAM is administered subcutaneously, intradermally, intramuscularly, intraarterially or intravenously, or via intraperitoneal administration. In certain even more specific embodiments of the invention said PAM is administered subcutaneously or intravascular (intraarterially, intravenously).

[0549] The embodiments of the present invention relating to modified or unmodified PAM or fragments thereof for use in the treatment of a subject having an increased amyloid-beta and / or phospho-Tau-protein load in the brain and / or for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation likewise relate to the use of modified or unmodified PAM or fragments thereof in the production of a medicament for the treatment of a subject having an increased amyloid-beta and / or phospho-Tau-protein load in the brain and / or for the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation, the use of modified or unmodified PAM or fragments thereof for the treatment of a subject having an increased amyloid-beta and / or phospho-Tau-protein load in the brain and / or for the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation, as well as methods of treatment of a subject having an increased amyloid-beta and / or phospho-Tau-protein load in the brain and / or of treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation comprising the administration of modified or unmodified PAM or fragments thereof to a subject in need thereof. Mutatis mutandis, this relates to the corresponding pharmaceutical formulations.

[0550] The embodiments of the present invention relating to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function likewise relate to the use of modified or unmodified PAM or fragments thereof in the production of a medicament for the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function, the use of modified or unmodified PAM or fragments thereof for the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function, as well as methods of treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function comprising the administration of modified or unmodified PAM or fragments thereof to a subject in need thereof. Mutatis mutandis, this relates to the corresponding pharmaceutical formulations. The embodiments of the present invention relating to modified or unmodified PAM or fragments thereof for use in treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation likewise relate to the use of modified or unmodified PAM or fragments thereof in the production of a medicament for the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation, the use of modified or unmodified PAM or fragments thereof for the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation, as well as methods of treatment or prevention of neuroinflammation for the improvement of neuroinflammation comprising the administration of modified or unmodified PAM or fragments thereof to a subject in need thereof. Mutatis mutandis, this relates to the corresponding pharmaceutical formulations.

[0551] Pharmaceutical formulation

[0552] Further subject matter of the present invention is a pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment of a subject having an increased amyloidbeta and / or phospho-Tau-protein load in the brain and / or for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation.

[0553] Further subject matter of the present invention is a pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function.

[0554] In certain embodiments said subject that has a reduction in cognitive function.

[0555] Further subject matter of the present invention is a pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation.

[0556] In certain embodiments said subject that has a reduction in neuroinflammation.

[0557] In certain embodiments said PAM or fragments thereof are isolated from a cell or tissue (including bodily fluids, e.g. blood) / organ or are recombinantly produced or are chimeric PAM or fragments thereof. Said cell or tissue / organ may be selected from mammalians, bacteria, yeast, fungi etc.

[0558] In certain embodiments said PAM or a fragment thereof is a protein comprising an amino acid sequence selected from the group comprising SEQ ID Nos 1 to 10, or an amino acid sequence having at least 85%, particularly at least 90%, more particularly at least 95%, even more particularly at least 99% sequence identity therewith.

[0559] In certain embodiments said PAM or fragment thereof is enzymatically active.

[0560] In certain embodiments said PAM or a fragment thereof is modified by

[0561] a. attaching one or more natural or synthetic polymers units, wherein said polymers are selected from the group comprising

[0562] iv. PEG, particularly having an average molecular weight in the range of 0.2-100 kDa, particularly 1-90 kDa, more particularly 2-80 kDa, more particularly 3-70 kDa, more particularly 4-60 kDa and even more particularly 5 to 50 kDa; v. XTEN;

[0563] vi. a peptide polymer consisting of repeats of PAS (the amino acids proline, alanine and serine, particularly comprising 100-200, particularly 120-180, more particularly 130- 170, even more particularly about 150 PAS repeats;

[0564] vii. elastin-like polypeptides consisting of Valin-Prolin-Glycine-x-Glycine repeats, wherein x is any amino acid other than Proline, particularly consisting of from 150, 200, 250, 300, 400, or 500 to 550, 600, 750, 850, 950 or 1,000 amino acids;

[0565] viii. HAP polypeptide, having the sequence (Gly4Ser)n, wherein n is 100-200;

[0566] ix. gelatin-like fusion protein, having the sequence (Gly-X-Y)n, wherein X and Y individually are any amino acid except for Cysteine, and wherein n = 60 to 1500; x. polysaccharides, in particular those chosen from the group comprising:

[0567] 1. dextrans;

[0568] 2. hydroxyethyls;

[0569] 3. heparosan;

[0570] 4. hyaluronic acid;

[0571] xi. poly sialic acid.

[0572] b. conjugation with a serum protein, such as albumin or an immunoglobulin or parts of an immunoglobulin;

[0573] c. site-directed mutagenesis by insertion, deletion and / or exchange of one or more amino acids within the amino acid sequence of PAM.

[0574] In certain embodiments, the pharmaceutical formulation according to the invention comprises one or more items from the group comprising packaging, instructions to the subject and / or physician treating the subject and a leaflet. Such items may in particular comprise instructions regarding the administration of the pharmaceutical formulation to a subject, such as relating to the dosage, medical indications, administration route.

[0575] The embodiments of the present invention relating to the modified or unmodified PAM or pharmaceutical formulation relating to the modified or unmodified PAM for use in the treatment of a subject having an increased amyloid-beta and / or phospho-Tau-protein load in the brain and / or for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation analogously relate to the following: Certain embodiments of the present invention relate to the use of modified or unmodified PAM for the manufacture of a medicament for the treatment of cognitive impairment in a subject in need thereof for the treatment of a subject having an increased amyloid-beta and / or phospho-Tau-protein load in the brain and / or for the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation. Certain embodiments of the present invention relate to the use of modified or unmodified PAM for the treatment of a subject having an increased amyloid-beta and / or phospho-Tau-protein load in the brain and / or for the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation. Certain embodiments of the present invention relate to modified or unmodified PAM for the treatment of cognitive impairment in a subject in need thereof for the treatment of a subject having an increased amyloid-beta and / or phospho-Tau-protein load in the brain and / or for the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation.

[0576] The embodiments of the present invention relating to the modified or unmodified PAM or pharmaceutical formulation relating to the modified or unmodified PAM for use in treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function analogously relate to the following: Certain embodiments of the present invention relate to the use of modified or unmodified PAM for the manufacture of a medicament for the treatment of cognitive impairment in a subject in need thereof for the improvement of cognitive function. Certain embodiments of the present invention relate to the use of modified or unmodified PAM for the treatment of cognitive impairment in a subject in need thereof for the improvement of cognitive function. Certain embodiments of the present invention relate to modified or unmodified PAM for the treatment of cognitive impairment in a subject in need thereof for the improvement of cognitive function.

[0577] The embodiments of the present invention relating to the modified or unmodified PAM or pharmaceutical formulation relating to the modified or unmodified PAM for use in treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation analogously relate to the following: Certain embodiments of the present invention relate to the use of modified or unmodified PAM for the manufacture of a medicament for the treatment of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation. Certain embodiments of the present invention relate to the use of modified or unmodified PAM for the treatment of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation. Certain embodiments of the present invention relate to modified or unmodified PAM for the treatment of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation.

[0578] In particular embodiments of the present invention, said pharmaceutical formulation is formulated to be administered or is administered orally, epicutaneously, subcutaneously, intradermally, sublingually, intramuscularly, intraarterially, intravenously, via the central nervous system (CNS, intracerebrally, intracerebroventricularly, intrathecally) or via intraperitoneal administration, particularly epicutaneously, subcutaneously, intradermally, intramuscularly, or intraperitoneally, more particularly subcutaneously, intramuscularly, or intraperitoneally; “formulated to be administered (e.g. intramuscularly)” likewise reads on “formulated for (e.g. intramuscular) administration”.

[0579] In particular embodiments of the present invention, said pharmaceutical formulation is formulated to be administered or is administered epicutaneously, subcutaneously, intradermally, intramuscularly, intraarterially, intravenously, via the central nervous system (CNS, intracerebrally, intracerebroventricularly, intrathecally) or via intraperitoneal administration, particularly epicutaneously, subcutaneously, intradermally, intramuscularly, or intraperitoneally, more particularly subcutaneously, intramuscularly, or intraperitoneally; “formulated to be administered (e.g. intramuscularly)” likewise reads on “formulated for (e.g. intramuscular) administration.”

[0580] In the most particular embodiments of the present invention, said pharmaceutical formulation is formulated to be administered or is administered subcutaneously, intradermally, intramuscularly, intraarterially or intravenously, or via intraperitoneal administration.

[0581] In certain embodiments of the invention the pharmaceutical formulation is to be administered subcutaneously or intravascular (intraarterially, intravenously).

[0582] In certain embodiments of the invention said pharmaceutical formulation is a solution, particularly a ready-to-use solution.

[0583] In certain embodiments of the invention said pharmaceutical formulation is in a freeze-dried state. said pharmaceutical formulation is administered via infusion.

[0584] In certain embodiments of the invention, said pharmaceutical formulation is to be administered systemically.

[0585] In certain embodiments of the invention, said pharmaceutical formulation is to be administered subcutaneously.

[0586] According to the present invention the applied dosage might be a single bolus injection delivering the described amount of the compounds to be applied or a continuous infusion of the compounds delivering the desired amount of compounds over a distinct period of time taking into account the velocity of infusion. Thereby the compounds might be applied as a combined injection and / or infusion or as several parallel injections and / or infusion, wherein one or all compounds are delivered as an injection and / or infusions or a distinct compound is injected and another distinct compound is infused.

[0587] Certain embodiments of the present invention relate to the formulation comprising modified or unmodified PAM or fragments thereof and / or optionally one or more pharmaceutically acceptable ingredients. Certain embodiments of the present invention relate to the formulation comprising modified or unmodified PAM in combination with ascorbate and / or copper and / or peptide with C-terminal Glycine amino acid or fragments thereof.

[0588] In certain embodiments of the invention said peptide with C-terminal Glycine amino acid is selected from the group comprising adrenomedullin (ADM), adrenomedullin-2, intermedin-short, pro-adrenomedullin N-20 terminal peptide (PAMP), amylin, gastrin-releasing peptide, neuromedin C, neuromedin B, neuromedin S, neuromedin U, calcitonin, calcitonin gene-related peptide (CGRP) 1 and 2, islet amyloid polypeptide, chromogranin A, insulin, pancreastatin, prolactin-releasing peptide (PrRP), cholecystokinin, big gastrin, gastrin, glucagon-like peptide 1 (GLP-1), pituitary adenylate cyclase-activating polypeptide (PACAP), secretin, somatoliberin, peptide histidine methionine (PHM), vasoactive intestinal peptide (VIP), gonadoliberin, kisspeptin, MIF-1, metastin, neuropeptide K, neuropeptide Y, neuropeptide gamma, substance P, neurokinin A, neurokinin B, peptide YY, pancreatic hormone, deltorphin I, orexin A and B, melanotropin alpha (alpha-MSH), melanotropin gamma, thyrotropin-releasing hormone (TRH), oxytocin, vasopressin or fragments thereof.

[0589] In certain embodiments of the invention said peptide with C-terminal glycine amino acid is selected from the group consisting of adrenomedullin (ADM) vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), glucagon-like peptide 1 (GLP-1) and neuropeptide Y or fragments thereof.

[0590] Certain embodiments of the invention relate to a pharmaceutical formulation, wherein said fragments of ADM-Gly (SEQ ID No. 14) are selected from the group of ADM-Gly 2-53 (SEQ ID No. 17), ADM-Gly 8-53 (SEQ ID No. 18), ADM-Gly 22-53 (SEQ ID No. 19), ADM-Gly 27-53 (SEQ ID No. 20) and ADM-Gly 33-53 (SEQ ID No. 21).

[0591] Subject matter of the present invention is a kit comprising the pharmaceutical formulation and one or more items from the group comprising packaging, instructions to the subject and / or physician treating the subject and a leaflet. Such items may in particular comprise instructions regarding the administration of the pharmaceutical formulation to a subject, such as relating to the dosage, medical indications or administration route.

[0592] Dosage

[0593] In certain embodiments of the invention said modified or unmodified PAM or fragments thereof is administered in a dose of 2.0 Units / kg to 116.1 Units / kg, particularly 2.1 Units / kg to 71.5 Units / kg, more particularly 2.2 Units / kg to 54.1 Units / kg, more particularly 2.3 Units / kg to 38.1 Units / kg, more particularly 2.4 Units / kg to 24.1 Units / kg, even more particularly 2.5 Units / kg to 11.1 Units / kg. In certain embodiments of the invention said modified PAM is PEGylated PAM and is administered in a dose of 2.0 Units / kg to 116.1 Units / kg, particularly 2.1 Units / kg to 71.5 Units / kg, more particularly 2.2 Units / kg to 54.1 Units / kg, more particularly 2.3 Units / kg to 38.1 Units / kg, more particularly 2.4 Units / kg to 24.1 Units / kg, even more particularly 2.5 Units / kg to 11.1 Units / kg.

[0594] In certain embodiments of the invention said modified PAM is PEGylated PAM and is administered subcutaneously in a dose of 2.0 Units / kg to 116.1 Units / kg, particularly 2.1 Units / kg to 71.5 Units / kg, more particularly 2.2 Units / kg to 54.1 Units / kg, more particularly 2.3 Units / kg to 38.1 Units / kg, more particularly 2.4 Units / kg to 24.1 Units / kg, even more particularly 2.5 Units / kg to 11.1 Units / kg.

[0595] In certain embodiments of the present invention modified or unmodified PAM or fragments thereof are in addition to be used with Vitamin C and wherein Vitamin C is applied at a dosage of 1-10000 mg / kg, particularly 2-8000 mg / kg, more particularly 3-6000 mg / kg, more particularly 4-4000 mg / kg, more particularly 5-2000 mg / kg, more particularly 10-1000 mg / kg.

[0596] Certain embodiments of the present invention relate to modified or unmodified Peptidylglycine alpha-amidating monooxygenase (PAM) or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation, PAM is in addition to be used with Vitamin C and wherein Vitamin C is applied at a dosage of 1-10000 mg / kg, particularly 2-8000 mg / kg, more particularly 3-6000 mg / kg, more particularly 4-4000 mg / kg, more particularly 5-2000 mg / kg, more particularly 10-1000 mg / kg. Certain embodiments of the present invention relate to 2-116.1 Units / kg of PAM, particularly 2.1 Units / kg to 71.5 Units / kg, more particularly 2.2 Units / kg to 54.1 Units / kg, more particularly 2.3 Units / kg to 38.1 Units / kg, more particularly 2.4 Units / kg to 24.1 Units / kg, even more particularly 2.5 Units / kg to 11.1 Units / kg are combined with 1-10000 mg / kg, particularly 2-8000 mg / kg, more particularly 3-6000 mg / kg, more particularly 4-4000 mg / kg, more particularly 5-2000 mg / kg, more particularly 10-1000 mg / kg of Vitamin C.

[0597] In certain embodiments of the present invention relate to 2-116.1 Units / kg of PAM are combined with 10-1000 mg / kg of Vitamin C.

[0598] It is apparent to the skilled person that the above dosages of PAM and / or Vitamin C read equally on the embodiments of the present invention relating to modified or unmodified Peptidylglycine alpha-amidating monooxygenase (PAM) or fragments thereof for use in the treatment of a subject having an increased amyloid-beta and / or phospho-Tau-protein load in the brain and / or for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation as detailed herein, as well as on embodiments relating to a pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment of a subject having an increased amyloid-beta and / or phospho-Tau-protein load in the brain and / or for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation, and likewise on embodiments relating to a kit comprising such pharmaceutical formulation.

[0599] It is apparent to the skilled person that the above dosages of PAM and / or Vitamin C read equally on the embodiments of the present invention relating to modified or unmodified Peptidylglycine alpha-amidating monooxygenase (PAM) or fragments thereof for use in the treatment or prevention of cognitive impairment as detailed herein, as well as on embodiments relating to a pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment, and likewise on embodiments relating to a kit comprising such pharmaceutical formulation.

[0600] It is apparent to the skilled person that the above dosages of PAM and / or Vitamin C read equally on the embodiments of the present invention relating to modified or unmodified Peptidylglycine alpha-amidating monooxygenase (PAM) or fragments thereof for use in the treatment or prevention of neuroinflammation as detailed herein, as well as on embodiments relating to a pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation, and likewise on embodiments relating to a kit comprising such pharmaceutical formulation.

[0601] Definitions

[0602] The term “subjects” include human or non-human mammals. Non-human mammals may be for example pigs, dogs, cows, rats, mice, guinea pigs, rabbits, sheep, cats, monkeys, orangutans, or chimpanzees. Particular are human subjects.

[0603] As used herein, the terms "comprising" and "including" or grammatical variants thereof are to be taken as specifying at least the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof. This term encompasses the terms "consisting of and "consisting essentially of that are understood to specify only the stated feature, integers, steps or components to the exclusion of any additional features.

[0604] Thus, the terms "comprising / including / having" mean that any further component (or likewise features, integers, steps and the like) can / may be present.

[0605] The term "consisting” of means that no further component (or likewise features, integers, steps and the like) is present. The term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, biological and biophysical arts. Treatment or prevention in the context of the present invention includes maintaining the state of a certain condition (e.g. amyloid-beta and / or phospho-Tau-protein load in the brain, cognitive impairment, and / or neuroinflammation) said subject has at the start of such treatment, wherein more particularly the worsening of said subject's state of said condition is halted or slowed down by such treatment, even more particularly compared to the worsening of the state of said condition observed in untreated subjects. As the skilled will readily understand, this is an improvement of the subject's state of said condition over said subject remaining untreated.

[0606] The present invention relates to modified or unmodified Peptidylglycine alpha-amidating monooxygenase (PAM) or fragments thereof for use in the treatment of a subject having an increased amyloid-beta and / or phospho-Tau-protein load in the brain and / or for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation.

[0607] Treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function (i.e. with modified or unmodified PAM as detailed in the embodiments of the present invention) in particular is maintaining the cognitive function at the state determined for said subject at the start of such treatment, wherein more particularly the decline of said subject's cognitive function is halted or slowed down by such treatment, even more particularly compared to the decline of cognitive function observed in untreated subjects. As the skilled will readily understand, this is an improvement of the subject's cognitive function over said subject remaining untreated.

[0608] Treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation (i.e. with modified or unmodified PAM as detailed in the embodiments of the present invention) in particular includes maintaining the level of neuroinflammation at the state determined for said subject at the start of such treatment, wherein more particularly the worsening of said subject's neuroinflammation is halted or slowed down by such treatment, even more particularly compared to the worsening of neuroinflammation observed in untreated subjects. As the skilled will readily understand, this is an improvement of the subject's neuroinflammation over said subject remaining untreated.

[0609] The following embodiments form also part of the present invention: Modified or unmodified Peptidylglycine alpha-amidating monooxygenase (PAM) or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function.

[0610] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to item 1, wherein said subject has a reduction in cognitive function.

[0611] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to item 1 or 2, wherein said subject is suffering from or has a risk of suffering from a disease or medical condition selected from the group comprising:

[0612] Neurodegenerative Disorders including Alzheimer's Disease, MCI patients, Frontotemporal Dementia (FTD), Lewy Body Dementia (LBD), Parkinson's Disease with Dementia (PDD), Huntington's Disease, Amyotrophic Lateral Sclerosis (ALS) with cognitive impairment, Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD), Multiple System Atrophy (MSA), Prion Diseases (e.g., Creutzfeldt-Jakob Disease);

[0613] Vascular Disorders including Vascular Dementia, Stroke, Multi-Infarct Dementia, Subcortical Ischemic Vascular Dementia, Cerebral Amyloid Angiopathy, Strategic Single-Infarct Dementia, Transient Ischemic Attack (TIA), Hypertensive Encephalopathy;

[0614] Traumatic Brain Injury (TBI) Related Disorders Chronic Traumatic Encephalopathy (CTE), Post-Concussion Syndrome, Diffuse Axonal Injury, Second Impact Syndrome, Subdural Hematoma, Epidural Hematoma, Penetrating Brain Injury, Post-Traumatic Amnesia;

[0615] Inflammatory and Infectious Disorders including Multiple Sclerosis (MS), Sepsis- Associated Encephalopathy (SAE), HIV-Associated Neurocognitive Disorder (HAND),, Autoimmune Encephalitis,, Encephalitis, Meningitis;

[0616] Endocrine and Metabolic Disorders including Diabetes Mellitus, Hyperglycemia, Hypoglycemia.

[0617] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to items 1 to 3, wherein said reduction of cognitive function is assessed with a method selected from the group comprising Mini-Mental State Examination (MMSE); Montreal Cognitive Assessment (MoCA), Mini-Cog; General Practitioner Assessment of Cognition (GPCOG); Addenbrookes Cognitive Examination revised (ACE-R); Severe Impairment Battery (SIB); National adult reading test (NART); Neuropsychiatric Inventory (NPI); Basic / Instrumental Activities of Daily Living (BADL) / (IADL); Functional Activities Questionnaire (FAQ); depression (Geriatric Depression Scale [GDS]; disability, Interview for deterioration in daily living activities [IDDD]; verbal learning and episodic memory (evaluated with the Consortium to establish a registry for Alzheimer’s disease [CERAD] word list and Wechsler Memory Scale III logical memory subtest; attention and executive function (digits test from Wechsler adult intelligence scale III [WAIS-III], trail making test B [TMT-B], phonetic fluency (p), Stroop test, constructional praxis from CERAD); visual perception (letters test from the visual object and space perception battery [VOSP]; visuo-spatial function (number localization task from VOSP); agnosia (Poppelreuter figures test; psychomotor speed (TMT-A); semantic fluency (animal categories); and language (Boston naming test [BNT-Boston],

[0618] more particularly a method selected from the group comprising Mini -Mental State Examination (MMSE); Montreal Cognitive Assessment (MoCA), Mini-Cog; General Practitioner Assessment of Cognition (GPCOG); Addenbrookes Cognitive Examination revised (ACE-R); Severe Impairment Battery (SIB); National adult reading test (NART); Neuropsychiatric Inventory (NPI); and Basic / Instrumental Activities of Daily Living (BADL) / (IADL).

[0619] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to item 2, wherein the reduction of cognitive function is defined as the patient's inability to achieve scores indicative of normal cognitive function when assessed by one or more methods according to item 4, such as the inability to achieve a score higher than 24 points on the Mini -Mental State Examination (MMSE), and / or the inability to achieve a score higher than 25 points on the Montreal Cognitive Assessment (MoCA), and / or the inability to achieve a score higher than 2 on the Mini-Cog, and / or inability to score within the range of 0 to 5 points, particularly within the range of 0 to 4, more particularly within the range of 0 to 3, more particularly within the range of 0 to 2, more particularly within the range of 0 to 1, most particularly 0 on the Functional Activities Questionnaire (FAQ).

[0620] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to items 1 to 5, wherein a level of PAM below a certain threshold is indicative of a reduction in cognitive function.

[0621] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to items 1 to 6. wherein said PAM or fragment thereof is a protein comprising an amino acid sequence selected from the group comprising SEQ ID Nos 1 to 10, or an amino acid sequence having at least 85%, particularly at least 90%, more particularly at least 95%, even more particularly at least 99% sequence identity therewith.

[0622] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to items 1 to 7, wherein said PAM is enzymatically active.

[0623] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to items 1 to 8, wherein said PAM or fragments thereof are modified by

[0624] a. attaching one or more natural or synthetic polymers units, wherein said polymers are selected from the group comprising

[0625] i. PEG, particularly having an average molecular weight in the range of 0.2- 100 kDa, particularly 1-90 kDa, more particularly 2-80 kDa, more particularly 3-70 kDa, more particularly 4-60 kDa and even more particularly 5 to 50 kDa;

[0626] ii. XTEN;

[0627] iii. a peptide polymer consisting of repeats of PAS (the amino acids proline, alanine and serine, particularly comprising 100-200, particularly 120-180, more particularly 130-170, even more particularly about 150 PAS repeats;

[0628] iv. elastin-like polypeptides consisting of Valin-Prolin-Glycine-x-Glycine repeats, wherein x is any amino acid other than Proline, particularly consisting of from 150, 200, 250, 300, 400, or 500 to 550, 600, 750, 850, 950 or 1,000 amino acids;

[0629] v. HAP polypeptide, having the sequence (Gly4Ser)n, wherein n is 100-200;

[0630] vi. gelatin-like fusion protein, having the sequence (Gly-X-Y)n, wherein X and Y individually are any amino acid except for Cysteine, and wherein n = 60 to 1500;

[0631] vii. polysaccharides, in particular those chosen from the group comprising: 1. dextrans;

[0632] 2. hydroxyethyls;

[0633] 3. heparosan;

[0634] 4. hyaluronic acid;

[0635] viii. poly sialic acid.

[0636] b. conjugation with a serum protein, such as albumin or an immunoglobulin or parts of an immunoglobulin;

[0637] c. site-directed mutagenesis by insertion, deletion and / or exchange of one or more amino acids within the amino acid sequence of PAM.

[0638] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to items 1 to 9, wherein said PAM or fragment thereof is administered in combination with ascorbate and / or copper and / or peptide with C-terminal glycine amino acid or fragments thereof.

[0639] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to item 10, wherein said peptide with C-terminal glycine amino acid is selected from the group of comprising adrenomedullin (ADM), adrenomedullin-2, intermedin-short, pro-adrenomedullin N-20 terminal peptide (PAMP), amylin, gastrin-releasing peptide, neuromedin C, neuromedin B, neuromedin S, neuromedin U, calcitonin, calcitonin gene-related peptide (CGRP) 1 and 2, islet amyloid polypeptide, chromogranin A, insulin, pancreastatin, prolactinreleasing peptide (PrRP), cholecystokinin, big gastrin, gastrin, glucagon-like peptide 1 (GLP-1), pituitary adenylate cyclase-activating polypeptide (PA CAP), secretin, somatoliberin, peptide histidine methionine (PHM), vasoactive intestinal peptide (VIP), gonadoliberin, kisspeptin, MIF-1, metastin, neuropeptide K, neuropeptide gamma, substance P, neurokinin A, neurokinin B, peptide YY, pancreatic hormone, deltorphin I, orexin A and B, melanotropin alpha (alpha-MSH), melanotropin gamma, thyrotropin-releasing hormone (TRH), oxytocin, vasopressin or fragments thereof.

[0640] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to items 1 to 11, wherein said subject is characterized by a level of PAM and / or its isoforms and / or fragments thereof below a certain threshold in a bodily fluid of said subject.

[0641] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to item 12, wherein said level of PAM and / or its isoforms and / or fragments thereof is the total concentration of PAM or the total activity of PAM.

[0642] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to items 12 and 13, wherein the threshold of the total concentration of PAM is equal or below 75 ng / mL, particularly equal or below 65 ng / mL, more particularly equal or below 55 ng / mL, more particularly equal or below 45 ng / mL, more particularly equal or below 35 ng / mL and most particularly equal or below 30 ng / mL.

[0643] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to items 12 and 13, wherein the threshold of the activity of PAM is equal or below 11.5 µg / L*h, particularly equal or below 10.5 µg / L*h, more particularly equal or below 9.5 µg / L*h, more particularly equal or below 8.5 µg / L*h, more particularly equal or below 7.5 µg / L*h and most particularly equal or below 6.5 µg / L*h, when PAM activity is measured in serum or wherein the threshold of the activity of PAM equal or below 16.9 µg / L*h, particularly equal or below 15.4 µg / L*h, more particularly equal or below 13.9 µg / L*h, more particularly equal or below 12.3 µg / L*h, more particularly equal or below 10.9 µg / L*h and most particularly equal or below 9.4 µg / L*h, when PAM activity is measured in Li-heparin.

[0644] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to items 12 to 15, wherein the sample of bodily fluid of said subject is selected from the group of whole blood, serum, plasma.

[0645] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function.

[0646] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to item 17, wherein said subject has a reduction in cognitive function.

[0647] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to items 17 to 18, wherein said PAM or fragment thereof is a protein comprising an amino acid sequence selected from the group comprising SEQ ID Nos 1 to 10, or an amino acid sequence having at least 85%, particularly at least 90%, more particularly at least 95%, even more particularly at least 99% sequence identity therewith.

[0648] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to item 17 to 19 wherein said PAM or fragment thereof is enzymatically active.

[0649] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to items 17 to 20, wherein said PAM or fragment thereof is modified by

[0650] a. attaching one or more natural or synthetic polymers units, wherein said polymers are selected from the group comprising

[0651] i. PEG, particularly having an average molecular weight in the range of 0.2- 100 kDa, particularly 1-90 kDa, more particularly 2-80 kDa, more particularly 3-70 kDa, more particularly 4-60 kDa and even more particularly 5 to 50 kDa;

[0652] ii. XTEN;

[0653] iii. a peptide polymer consisting of repeats of PAS (the amino acids proline, alanine and serine, particularly comprising 100-200, particularly 120-180, more particularly 130-170, even more particularly about 150 PAS repeats;

[0654] iv. elastin-like polypeptides consisting of Valin-Prolin-Glycine-x-Glycine repeats, wherein x is any amino acid other than Proline, particularly consisting of from 150, 200, 250, 300, 400, or 500 to 550, 600, 750, 850, 950 or 1,000 amino acids; v. HAP polypeptide, having the sequence (Gly4Ser)n, wherein n is 100-200;

[0655] vi. gelatin-like fusion protein, having the sequence (Gly-X-Y)n, wherein X and Y individually are any amino acid except for Cysteine, and wherein n = 60 to 1500;

[0656] vii. polysaccharides, in particular those chosen from the group comprising:

[0657] 5. dextrans;

[0658] 6. hydroxyethyls;

[0659] 7. heparosan;

[0660] 8. hyaluronic acid;

[0661] viii. poly sialic acid.

[0662] b. conjugation with a serum protein, such as albumin or an immunoglobulin or parts of an immunoglobulin;

[0663] c. site-directed mutagenesis by insertion, deletion and / or exchange of one or more amino acids within the amino acid sequence of PAM.

[0664] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to items 17 to 21, wherein said pharmaceutical formulation is to be administered orally, epicutaneously, subcutaneously, intradermally, intramuscularly, intravascular (intraarterially, intravenously), or via the central nervous system (CNS, intracerebrally, intracerebroventricularly, intrathecally) or via intraperitoneal administration.

[0665] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of vascular cognitive impairment in a subject in need thereof for the improvement of cognitive function according to items 17 to 22, wherein said pharmaceutical formulation is a solution, particularly a ready-to-use solution.

[0666] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to items 17 to 23, wherein said pharmaceutical formulation is in a freeze-dried state. Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to items 17 to 24, wherein said pharmaceutical formulation is administered via infusion.

[0667] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to items 17 to 25, wherein said pharmaceutical formulation is to be administered systemically.

[0668] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to items 17 to 26, wherein said pharmaceutical formulation is to be administered subcutaneously.

[0669] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to items 17 to 27, further comprising one or more pharmaceutically acceptable ingredients, in particular one or more pharmaceutically acceptable excipients.

[0670] Pharmaceutical formulation comprising PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to items 17 to 28, the formulation comprising said PAM or fragments thereof in combination with ascorbate and / or copper and / or peptide having a C-terminal glycine amino acid or fragments thereof.

[0671] Pharmaceutical formulation comprising PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to item 29, wherein said peptide with C-terminal glycine amino acid is selected from the group of comprising adrenomedullin (ADM), adrenomedullin-2, intermedinshort, pro-adrenomedullin N-20 terminal peptide (PAMP), amylin, gastrin-releasing peptide, neuromedin C, neuromedin B, neuromedin S, neuromedin U, calcitonin, calcitonin gene-related peptide (CGRP) 1 and 2, islet amyloid polypeptide, chromogranin A, insulin, pancreastatin, prolactin-releasing peptide (PrRP), cholecystokinin, big gastrin, gastrin, glucagon-like peptide 1 (GLP-1), pituitary adenylate cyclase-activating polypeptide (PA CAP), secretin, somatoliberin, peptide histidine methionine (PHM), vasoactive intestinal peptide (VIP), gonadoliberin, kisspeptin, MIF-1, metastin, neuropeptide K, neuropeptide gamma, substance P, neurokinin A, neurokinin B, peptide YY, pancreatic hormone, deltorphin I, orexin A and B, melanotropin alpha (alpha-MSH), melanotropin gamma, thyrotropin-releasing hormone (TRH), oxytocin, vasopressin or fragments thereof.

[0672] 31. A kit comprising the pharmaceutical formulation according to items 17 to 30.

[0673] The following embodiments form also part of the present invention (Aspect B):

[0674] 1. Modified or unmodified Peptidylglycine a-amidating monooxygenase (PAM) or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation.

[0675] 2. Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof according to Aspect B item 1, wherein said subject has an increased amyloid-beta and / or phospho-Tau-protein load.

[0676] 3. Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof according to Aspect B items 1 or 2, wherein administration of said PAM or fragments thereof to said subject leads to a reduction of the amyloid-beta and / or phospho-Tau protein load.

[0677] 4. Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof according to Aspect B items 1 to 3, wherein administration of said PAM or fragments to said subject thereof leads to a reduction of the neuroinflammation.

[0678] 5. Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof according to Aspect B items 1 to 4, wherein said subject is suffering from or is having a risk of suffering from a disease or medical condition selected from the group comprising Alzheimer's Disease, Mild cognitive impairment (MCI), Frontotemporal Dementia (FTD), Lewy Body Dementia (LBD), Parkinson's Disease with Dementia (PDD), Huntington's Disease, Amyotrophic Lateral Sclerosis (ALS) with cognitive impairment, Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD), Multiple System Atrophy (MSA), prion diseases (e.g., Creutzfeldt-Jakob Disease), Cerebral Amyloid Angiopathy (CAA), Down Syndrome (DS) with Alzheimer-like dementia, Chronic Traumatic Encephalopathy (CTE), and Neurofibrillary Tangle Dementia (NTD) and Postencephalitic Parkinsonism. Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof according to Aspect B item 3, wherein said reduction of amyloid-beta protein and or phospho-Tau load is assessed with an immunoassay, particularly an Enzyme-Linked Immunosorbent Assay (ELISA), Multiplex Assays, Singleplex Assays, Electrochemiluminescence Immunoassay (ECLIA); Positron Emission Tomography (PET) Scans including Amyloid PET and Tau PET; Mass Spectrometry, including but not limited to LC-MS / MS (Liquid Chromatography-Tandem Mass Spectrometry), MALDI-TOF (Matrix-Assisted Laser Desorption / Ionization-Time of Flight), Immunoprecipitation-Mass Spectrometry (IP-MS); Immunohistochemistry (IHC); Western Blot; Flow Cytometry.

[0679] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof according to Aspect B items 1 to 6, wherein an increased amyloid-beta protein load in the brain is defined as a decrease in the concentration of amyloid-beta peptides, specifically amyloid-beta42 (Aβ42) and / or the ratio of Aβ42 and Aβ40 in a sample selected from the group comprising cerebrospinal fluid (CSF) or plasma, as measured by immunoassay techniques such as ELISA, Western blot, or mass spectrometry, measuring lower when compared to the baseline levels typically found in healthy individuals and

[0680] wherein an increased phosphorylated tau load is defined as an elevation in the concentration of tau proteins phosphorylated at specific residues including but not limited to Thr181, Thr231 and Ser199 in a sample selected from the group comprising cerebrospinal fluid (CSF), or plasma, as measured by immunoassay techniques such as ELISA, Western blot, or mass spectrometry, exceeding the baseline levels typically found in healthy individuals.

[0681] Certain embodiments of the present invention relate to modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation according to Aspect B items 1 to 7, wherein the amyloid-beta ratio (Aβ42 / Aβ40) is below a certain threshold and / or phospho-Tau protein load in said subject is above a certain threshold.

[0682] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation according to Aspect B item 8, wherein the amyloid-beta Aβ42 in said subject is below 200 pg / mL, particularly between 200 and 150 pg / mL, more particularly between 150 and 100 pg / mL, and even more particularly between 100 and 50 pg / mL, and the most particularly between 50 pg / mL and 1 pg / mL and / or wherein the amyloid-beta Aβ42 / Aβ40 ratio in said subject is below 0.11, particularly between 0.06 and 0.11, most particularly between 0.06 and 0.03, even more particularly between 0.03 and 0.01 and / or wherein the phospho-Tau231 protein load in said subject is above 50 pg / mL, particularly between the range of 50 to 250 pg / mL, even more particularly between 250 and 500 pg / mL, the most particularly between 500 and 1200 pg / mL, when measured in CSF and wherein the phospho-Tau181 protein load in said subject is above 18 pg / mL, particularly between the range of 18 to 22 pg / mL, even more particularly between 22 and 26 pg / mL, the most particularly between 26 and 50 pg / mL, when measured in CSF.

[0683] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof according to Aspect B items 1 to 9, wherein said PAM is a protein comprising an amino acid sequence selected from the group comprising SEQ ID Nos 1 to 10, or an amino acid sequence having at least 85%, particularly at least 90%, more particularly at least 95%, even more particularly at least 99% sequence identity therewith.

[0684] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof according to Aspect B items 1 to 10, wherein said PAM is enzymatically active.

[0685] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof according to Aspect B items 1 to 11, wherein said PAM is modified by

[0686] a. attaching one or more natural or synthetic polymers units, wherein said polymers are selected from the group comprising

[0687] i. PEG, particularly having an average molecular weight in the range of 0.2- 100 kDa, particularly 1-90 kDa, more particularly 2-80 kDa, more particularly 3-70 kDa, more particularly 4-60 kDa and even more particularly 5 to 50 kDa;

[0688] ii. XTEN; add forms

[0689] iii. a peptide polymer consisting of repeats of PAS (the amino acids proline, alanine and serine, particularly comprising 100-200, particularly 120-180, more particularly 130-170, even more particularly about 150 PAS repeats; iv. elastin-like polypeptides consisting of Valin-Prolin-Glycine-x-Glycine repeats, wherein x is any amino acid other than Proline, particularly consisting of from 150, 200, 250, 300, 400, or 500 to 550, 600, 750, 850, 950 or 1,000 amino acids;

[0690] v. HAP polypeptide, having the sequence (Gly4Ser)n, wherein n is 100-200;

[0691] vi. gelatin-like fusion protein, having the sequence (Gly-X-Y)n, wherein X and Y individually are any amino acid except for Cysteine, and wherein n = 60 to 1500;

[0692] vii. polysaccharides, in particular those chosen from the group comprising:

[0693] 9. dextrans;

[0694] 10. hydroxyethyls;

[0695] 11. heparosan;

[0696] 12. hyaluronic acid;

[0697] viii. poly sialic acid.

[0698] b. conjugation with a serum protein, such as albumin or an immunoglobulin or parts of an immunoglobulin;

[0699] c. site-directed mutagenesis by insertion, deletion and / or exchange of one or more amino acids within the amino acid sequence of PAM.

[0700] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof according to Aspect B items 1 to 12, wherein said PAM is administered in combination with ascorbate and / or copper and / or peptide with C-terminal glycine amino acid or fragments thereof.

[0701] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof according to Aspect B item 13, wherein said peptide with C-terminal Glycine amino acid is selected from the group of comprising adrenomedullin (ADM), adrenomedullin-2, intermedin-short, pro-adrenomedullin N-20 terminal peptide (PAMP), amylin, gastrin-releasing peptide, neuromedin C, neuromedin B, neuromedin S, neuromedin U, calcitonin, calcitonin gene-related peptide (CGRP) 1 and 2, islet amyloid polypeptide, chromogranin A, insulin, pancreastatin, prolactin-releasing peptide (PrRP), cholecystokinin, big gastrin, gastrin, glucagon-like peptide 1 (GLP-1), pituitary adenylate cyclase-activating polypeptide (PACAP), secretin, somatoliberin, peptide histidine methionine (PHM), vasoactive intestinal peptide (VIP), gonadoliberin, kisspeptin, MIF-1, metastin, neuropeptide K, neuropeptide gamma, substance P, neurokinin A, neurokinin B, peptide YY, pancreatic hormone, deltorphin I, orexin A and B, melanotropin alpha (alpha-MSH), melanotropin gamma, thyrotropin-releasing hormone (TRH), oxytocin, vasopressin or fragments thereof.

[0702] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation according to Aspect B items 1 to 14, wherein said subject is characterized by a level of PAM and / or its isoforms and / or fragments thereof below a certain threshold in a bodily fluid of said subject.

[0703] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation according to Aspect B item 15, wherein said level of PAM and / or its isoforms and / or fragments thereof is the total concentration of PAM or the total activity of PAM.

[0704] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation according to Aspect B items 15 and 16, wherein the threshold of the total concentration of PAM is equal or below 75 ng / mL, particularly equal or below 65 ng / mL, more particularly equal or below 55 ng / mL, more particularly equal or below 45 ng / mL, more particularly equal or below 35 ng / mL and most particularly equal or below 30 ng / mL.

[0705] Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation according to Aspect B items 15 and 16, wherein the threshold of the activity of PAM is equal or below 11.5 µg / L*h, particularly equal or below 10.5 µg / L*h, more particularly equal or below 9.5 µg / L*h, more particularly equal or below 8.5 µg / L*h, more particularly equal or below 7.5 µg / L*h and most particularly equal or below 6.5 µg / L*h, when PAM activity is measured in serum or wherein the threshold of the activity of PAM equal or below 16.9 µg / L*h, particularly equal or below 15.4 µg / L*h, more particularly equal or below 13.9 µg / L*h, more particularly equal or below 12.3 µg / L*h, more particularly equal or below 10.9 µg / L*h and most particularly equal or below 9.4 µg / L*h, when PAM activity is measured in Li-heparin. Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation according to Aspect B items 15 to 18, wherein the sample of bodily fluid of said subject is selected from the group of whole blood, serum, plasma.

[0706] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation.

[0707] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation according to Aspect B item 20, wherein said subject has an increased amyloid-beta and / or phospho-Tau protein load.

[0708] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuro inflammation according to Aspect B items 20 or 21, wherein administration of said PAM or fragments to said subject thereof leads to a reduction of the neuroinflammation.

[0709] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation according to Aspect B item 20 to 22, wherein said PAM or fragment thereof is a protein comprising an amino acid sequence selected from the group comprising SEQ ID Nos 1 to 10, or an amino acid sequence having at least 85%, particularly at least 90%, more particularly at least 95%, even more particularly at least 99% sequence identity therewith.

[0710] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation according to Aspect B items 20 to 23, wherein said PAM or fragment thereof is enzymatically active.

[0711] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation according to Aspect B items 20 to 24, wherein said PAM is modified by

[0712] a. attaching one or more natural or synthetic polymers units, wherein said polymers are selected from the group comprising

[0713] i. PEG, particularly having an average molecular weight in the range of 0.2- 100 kDa, particularly 1-90 kDa, more particularly 2-80 kDa, more particularly 3-70 kDa, more particularly 4-60 kDa and even more particularly 5 to 50 kDa;

[0714] ii. XTEN; add forms

[0715] iii. a peptide polymer consisting of repeats of PAS (the amino acids proline, alanine and serine, particularly comprising 100-200, particularly 120-180, more particularly 130-170, even more particularly about 150 PAS repeats;

[0716] iv. elastin-like polypeptides consisting of Valin-Prolin-Glycine-x-Glycine repeats, wherein x is any amino acid other than Proline, particularly consisting of from 150, 200, 250, 300, 400, or 500 to 550, 600, 750, 850, 950 or 1,000 amino acids;

[0717] v. HAP polypeptide, having the sequence (Gly4Ser)n, wherein n is 100-200;

[0718] vi. gelatin-like fusion protein, having the sequence (Gly-X-Y)n, wherein X and Y individually are any amino acid except for Cysteine, and wherein n = 60 to 1500;

[0719] vii. polysaccharides, in particular those chosen from the group comprising:

[0720] 13. dextrans;

[0721] 14. hydroxyethyls;

[0722] 15. heparosan;

[0723] 16. hyaluronic acid;

[0724] viii. poly sialic acid.

[0725] b. conjugation with a serum protein, such as albumin or an immunoglobulin or parts of an immunoglobulin;

[0726] c. site-directed mutagenesis by insertion, deletion and / or exchange of one or more amino acids within the amino acid sequence of PAM.

[0727] Pharmaceutical formulation comprising PAM or fragments thereof for use in the treatment or prevention of neuroinflammation according to Aspect B items 20 to 25, wherein said pharmaceutical formulation is to be administered orally, epicutaneously, subcutaneously, intradermally, intramuscularly, intravascular (intraarterially, intravenously), or via the central nervous system (CNS, intracerebrally, intracerebroventricularly, intrathecally) or via intraperitoneal administration.

[0728] Pharmaceutical formulation comprising PAM or fragments thereof for use in the treatment or prevention of neuroinflammation according to Aspect B items 20 to 26, wherein said pharmaceutical formulation is a solution, particularly a ready-to-use solution.

[0729] Pharmaceutical formulation comprising PAM or fragments thereof for use in the treatment or prevention of neuroinflammation according to Aspect B items 20 to 27, wherein said pharmaceutical formulation is in a freeze-dried state.

[0730] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation according to Aspect B items 20 to 28, wherein said pharmaceutical formulation is administered via infusion.

[0731] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation according to Aspect B items 20 to 29, wherein said pharmaceutical formulation is to be administered systemically.

[0732] Pharmaceutical formulation according to Aspect B items 20 to 30, the formulation comprising modified or unmodified PAM or fragments thereof and / or optionally one or more pharmaceutically acceptable ingredients.

[0733] Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment or prevention of neuroinflammation according to Aspect B items 20 to 31, the formulation comprising said PAM in combination with ascorbate and / or copper and / or peptide with C-terminal gl...

Claims

CLAIMS1. Modified or unmodified Peptidylglycine alpha-amidating monooxygenase (PAM) or fragments thereof for use in the treatment of a subject having an increased amyloid-beta and / or phospho-Tau- protein load in the brain and / or for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation.

2. Modified or unmodified Peptidylglycine alpha-amidating monooxygenase (PAM) or fragments thereof for the use according to claim 1, wherein said subject is suffering from or has a risk of suffering from a disease or medical condition selected from the group comprising:- Neurodegenerative Disorders including Alzheimer's Disease, Mild cognitive impairment (MCI), Frontotemporal Dementia (FTD), Lewy Body Dementia (LBD), Parkinson's Disease with Dementia (PDD), Huntington's Disease, Amyotrophic Lateral Sclerosis (ALS) with cognitive impairment, Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD), Multiple System Atrophy (MSA), Prion Diseases (e.g., Creutzfeldt-Jakob Disease);- Cerebral Amyloid Angiopathy (CAA), Down Syndrome (DS) with Alzheimer-like dementia, Chronic Traumatic Encephalopathy (CTE), and Neurofibrillary Tangle Dementia (NTD), Postencephalitic Parkinsonism;- Vascular Disorders including Vascular Dementia, Stroke, Multi-Infarct Dementia, Subcortical Ischemic Vascular Dementia, Cerebral Amyloid Angiopathy, Strategic Single-Infarct Dementia, Transient Ischemic Attack (TIA), Hypertensive Encephalopathy;- Traumatic Brain Injury (TBI) Related Disorders Chronic Traumatic Encephalopathy (CTE), Post-Concussion Syndrome, Diffuse Axonal Injury, Second Impact Syndrome, Subdural Hematoma, Epidural Hematoma, Penetrating Brain Injury, Post-Traumatic Amnesia;- Inflammatory and Infectious Disorders including Multiple Sclerosis (MS), Sepsis-Associated Encephalopathy (SAE), HIV-Associated Neurocognitive Disorder (HAND), Autoimmune Encephalitis, Encephalitis, Meningitis;- Endocrine and Metabolic Disorders including Diabetes Mellitus, Hyperglycemia, Hypoglycemia3. Modified or unmodified Peptidylglycine alpha-amidating monooxygenase (PAM) or fragments thereof for the use according to claims 1 or 2, wherein an increased amyloid-beta protein and / or phospho-Tau load in the brain is as assessed with an immunoassay, particularly an Enzyme-Linked Immunosorbent Assay (ELISA), Multiplex Assays, Singleplex Assays, Electrochemiluminescence Immunoassay (ECLIA); Positron Emission Tomography (PET) Scans including Amyloid PET and Tau PET; Mass Spectrometry, including but not limited to LC-MS / MS (Liquid Chromatography- Tandem Mass Spectrometry), MALDI-TOF (Matrix- Assisted Laser Desorption / Ionization-Timeof Flight), Immunoprecipitation-Mass Spectrometry (IP-MS); Immunohistochemistry (IHC); Western Blot; Flow Cytometry;and / or wherein an increased amyloid-beta protein load in the brain is defined as a decrease in the concentration of amyloid-beta peptides, specifically amyloid-beta42 (Aβ42) and / or the ratio of Aβ42 and Aβ40 in a sample selected from the group comprising cerebrospinal fluid (CSF) or plasma, as measured by immunoassay techniques such as ELISA, Western blot, or mass spectrometry, measuring lower when compared the baseline levels typically found in healthy individuals and wherein an increased phosphorylated tau load is defined as an elevation in the concentration of tau proteins phosphorylated at specific residues including but not limited to Thr181, Thr231 and Ser199 in a sample selected from the group comprising cerebrospinal fluid (CSF), or plasma, as measured by immunoassay techniques such as ELISA, Western blot, or mass spectrometry, exceeding the baseline levels typically found in healthy individuals.

4. Modified or unmodified Peptidylglycine alpha-amidating monooxygenase (PAM) or fragments thereof for the use according to claims 1 to 3, wherein the amyloid-beta ratio (Aβ42 / Aβ40) is below a certain threshold and / or phospho-Tau protein load in said subject is above a certain threshold.

5. Modified or unmodified Peptidylglycine alpha-amidating monooxygenase (PAM) or fragments thereof for the use according to claims 1 to 4, wherein, when measured in a CSF sample of the subject, the amyloid-beta Aβ42 in said sample is below 200 pg / mL, preferably between 200 and 150 pg / mL, more preferably between 150 and 100 pg / mL, and even more preferably between 100 and 50 pg / mL, and the most preferably between 50 pg / mL and 1 pg / mL and / or wherein the amyloidbeta Aβ42 / Aβ40 ratio in said sample is below 0.11, preferably between 0.06 and 0.11, most preferably between 0.06 and 0.03, even more preferably between 0.03 and 0.01 and / or wherein the phospho-Tau231 protein load in said sample is above 50 pg / mL, preferably between the range of 50 to 250 pg / mL, even more preferably between 250 and 500 pg / mL, the most preferably between 500 and 1200 pg / mL, when measured in CSF and wherein the phospho-Tau181 protein load in said sample is above 18 pg / mL, preferably between the range of 18 to 22 pg / mL, even more preferably between 22 and 26 pg / mL, the most preferably between 26 and 50 pg / mL.

6. Modified or unmodified Peptidylglycine alpha-amidating monooxygenase (PAM) or fragments thereof for the use according to claims 1 to 5, wherein said treatment is for the improvement of cognitive impairment or for the prevention of cognitive decline in said subject,wherein more particularly said subject has an impaired cognitive function.

7. Modified or unmodified PAM or fragments thereof for the use according to claims 1 to 5, wherein said modified or unmodified PAM or fragments thereof are for use in the treatment or preventionof cognitive impairment in a subject in need thereof for the improvement of cognitive function, wherein more particularly said subject has a reduction in cognitive function.

8. Modified or unmodified PAM or fragments thereof for use in the treatment or prevention of cognitive impairment in a subject in need thereof for the improvement of cognitive function according to claims 1 to 3, wherein a level of PAM below a certain threshold, in particular in a sample of bodily fluid of said subject, is indicative of a reduction in cognitive function, wherein more particularly said level of PAM and / or its isoforms and / or fragments thereof is the total concentration of PAM or the total activity of PAM,wherein more particularly the threshold of the total concentration of PAM is equal or below 75 ng / mL, preferably equal or below 65 ng / mL, more preferably equal or below 55 ng / mL, more preferably equal or below 45 ng / mL, more preferably equal or below 35 ng / mL and most preferably equal or below 30 ng / mL, and / or wherein the threshold of the activity of PAM is equal or below 11.5 µg / L*h, preferably equal or below 10.5 µg / L*h, more preferably equal or below 9.5 µg / L*h, more preferably equal or below 8.5 µg / L*h, more preferably equal or below 7.5 µg / L*h and most preferably equal or below 6.5 µg / L*h, when PAM activity is measured in serum or wherein the threshold of the activity of PAM equal or below 16.9 µg / L*h, preferably equal or below 15.4 pg / L*h, more preferably equal or below 13.9 pg / L*h, more preferably equal or below 12.3 pg / L*h, more preferably equal or below 10.9 pg / L*h and most preferably equal or below 9.4 pg / L*h, when PAM activity is measured in Li-heparin,wherein more particularly the sample of bodily fluid of said subject is selected from the group comprising whole blood, serum, and plasma.

9. Modified or unmodified PAM or fragments thereof for the use according to claims 6 to 8, wherein said cognitive function or said reduction of cognitive function or impairment of cognitive function is assessed with a method selected from the group comprising neuropsychological tests, computerized neuropsychological tests, daily living scale assessment and neuroimaging techniques;wherein more particularly said neuropsychological test is selected from the group comprising MiniMental State Examination (MMSE); Montreal Cognitive Assessment (MoCA), Mini-Cog; General Practitioner Assessment of Cognition (GPCOG); Addenbrooke’s Cognitive Examination (ACE); Addenbrooke’s Cognitive Examination revised (ACE-R); Severe Impairment Battery (SIB); National adult reading test (NART); Neuropsychiatric Inventory (NPI); depression (Geriatric Depression Scale [GDS]; ADAS-Cog (Alzheimer’s Disease Assessment Scale - Cognitive: 11 / 13 / 14); 3MS (Modified Mini-Mental State); clock drawing test, controlled oral word association test (COWAT); Rey Complex Figure test and recognition trial (RCFT), abbreviated mental test score (AMTS); Six item cognitive impairment test (6CIT); Consortium to Establish a Registry forAlzheimer's Disease (CERAD) batery; verbal learning and episodic memory (evaluated with the Consortium to establish a registry for Alzheimer’s disease [CERAD]); MCI Screen; word list and Wechsler Memory Scale III logical memory subtest; atention and executive function (digits test from Wechsler adult intelligence scale III [WAIS-III], trail making test B [TMT-B], phonetic fluency (p), Stroop test, constructional praxis from CERAD); Wechsler Bateries (Digit Span Tests, Wechsler Memory Scale IV Logical Memory II (WMS-IV LM-II), Wechsler Memory Scale III Logical Memory Subtest; Wechsler Adult Intelligence Scale III (WAIS-III) Digits Test); Free & Cued Selective Reminding Test (FCSRT); RBANS (Repeatable Batery for the Assessment of Neuropsychological Status); visual perception (leters test from the visual object and space perception batery [VOSP]; visuo-spatial function (number localization task from VOSP); agnosia (Poppelreuter figures test; psychomotor speed (TMT-A); semantic fluency (animal categories); and language (Boston naming test [BNT-Boston];more particularly selected from the group comprising Mini-Mental State Examination (MMSE); Montreal Cognitive Assessment (MoCA), Mini-Cog; General Practitioner Assessment of Cognition (GPCOG); Addenbrookes Cognitive Examination revised (ACE-R); Severe Impairment Batery (SIB); National adult reading test (NART); Neuropsychiatric Inventory (NPI);and / or wherein said daily living scale assessment is selected from the group comprising Basic / Instrumental Activities of Daily Living (BADL) / (IADL); Functional Activities Questionnaire (FAQ); disability, Interview for deterioration in daily living activities (IDDD), Disability Assessment for Dementia (DAD);and / or wherein said computerized neuropsychological test is selected from the group comprising Cambridge Neuropsychological Test Automated Battery (CANTAB), CNS Vital Signs, Automated Neuropsychological Assessment Metrics (ANAM), Immediate Post-Concussion Assessment and Cognitive Testing (ImPACT), Mindstreams;and / or wherein said neuroimaging method is selected from the group comprising Functional MRI (fMRI), Positron Emission Tomography (PET), including amyloid-PET, Tau-PET and fluorodeoxyglucose-PET (FDG-PET, Electroencephalography (EEG), Magnetoencephalography (MEG) and Diffusion Tensor Imaging (DTI).

10. Modified or unmodified PAM or fragments thereof for the use according to claim 9, wherein the reduction or impairment of cognitive function is defined as the patient's inability to achieve scores indicative of normal cognitive function when assessed by one or more methods according to claims 9, such as the inability to achieve a score higher than 24 points on the Mini-Mental State Examination (MMSE), and / or the inability to achieve a score higher than 25 points on the Montreal Cognitive Assessment (MoCA), and / or the inability to achieve a score higher than 2 on the MiniCog, and / or inability to score within the range of 0 to 5 points, particularly within the range of 0 to4, more particularly within the range of 0 to 3, more particularly within the range of 0 to 2, more particularly within the range of 0 to 1, most particularly 0 on the Functional Activities Questionnaire (FAQ) and / or a classification of 0.5 or higher on the Clinical Dementia Rating - Global (CDR- Global); and / or a score on the Clinical Dementia Rating - Sum of Boxes (CDR-SB) within 0.5 to 9.0; and / or a worsening on ADAS-Cog defined by an increase from baseline of at least 2 to 3 points; and / or a worsening on iADRS defined by a decrease from baseline of at least 5 points; wherein more particularly the reduction or impairment of cognitive function is defined as the patient's inability to achieve scores indicative of normal cognitive function when assessed by one or more methods according to claim 9, such as the inability to achieve a score higher than 24 points on the Mini-Mental State Examination (MMSE), and / or the inability to achieve a score higher than 25 points on the Montreal Cognitive Assessment (MoCA), and / or the inability to achieve a score higher than 2 on the Mini-Cog, and / or inability to score within the range of 0 to 5 points, particularly within the range of 0 to 4, more particularly within the range of 0 to 3, more particularly within the range of 0 to 2, more particularly within the range of 0 to 1, most particularly 0 on the Functional Activities Questionnaire (FAQ).

11. Modified or unmodified Peptidylglycine alpha-amidating monooxygenase (PAM) or fragments thereof for the use according to claims 1 to 10, wherein neuroinflammation is as determined by specific neuroimaging techniques (such as PET, MRI, MRS) and / or measuring the level of a biomarker of neuroinflammation in a sample of bodily fluid of said subject.

12. Modified or unmodified Peptidylglycine alpha-amidating monooxygenase (PAM) or fragments thereof for use in the treatment according to claim 11 wherein said biomarker of neuroinflammation in a sample of bodily fluid of said subject is selected from the group comprising GFAP, sTREM2, YKL-40, neopterin, and S100B;wherein optionally in addition the level of one or more of the following biomarkers of is determined in a sample of bodily fluid of said subject: sICAM-1, and sVCAM-1, IL-1β, IL-6, TNF-α, IL-8, IL-10, IL-12, IL-18, IFN-γ, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), CXCL10 (IP-10), NfL, sCD14, sCD163;more particularly wherein the concentration of said biomarker is measured by an immunoassaybased analytical method selected from the group comprising ELISA, electrochemiluminescence immunoassay (ECLIA), Single Molecule Array (Simoa), and multiplex bead-based immunoassay (Luminex);and / or wherein said neuroimaging technique is selected from the group comprising TSPO-PET, MAO-B-PET, P2X7-PET, CSF1R-PET, I2BS-PET, SV2A-PET, MRS and QSM-MRI.

13. Modified or unmodified Peptidylglycine alpha-amidating monooxygenase (PAM) or fragments thereof for the use according to claims 1 to 12, wherein improvement of neuroinflammation isdefined as a) a reduction of an imaging parameter selected from the group comprising SUVR, BPND, QSM iron load and mI / Cr ratio by at least 10 %, preferably by at least 15 %, more preferably by at least 20 %, and even more preferably by at least 30 % relative to the baseline value measured in said subject prior to treatment and / or b) normalization of said parameter to a value that is within a range observed in healthy individuals, in particular SUVR within 0.9-1.1, BPND within 0.5-1.0, mI / Cr ratio within 0.35-0.55, and QSM within 20-40 ppb;and / or wherein improvement of neuroinflammation is defined as a) a decrease in the level of at least one biomarker selected from the group comprising sTREM2, YKL-40 and GFAP by at least 10 %, preferably by at least 20 %, more preferably by at least 30 %, and even more preferably by at least 50 % relative to the baseline concentration measured in said subject prior to treatment and / or b) normalization of the biomarker level to a value that is within the range observed for healthy individuals, in particular a GFAP level of 0–715 pg / mL in a CSF sample of said subject or 0–90 pg / mL in a plasma sample of said subject; and / or a YKL-40 level of 160–300 pg / mL in a CSF sample of said subject or 14–155 pg / mL in a plasma sample of said subject; and / or a sTREM2 level or 250–280 pg / mL in a CSF sample of said subject.

14. Peptidylglycine alpha-amidating monooxygenase (PAM) or fragments thereof for use in the treatment according to claims 1 to 13 wherein administration of said PAM or fragments thereof to said subject leads to a reduction of the amyloid-beta and / or phospho-Tau protein load in the brain.

15. Modified or unmodified Peptidylglycine alpha-amidating monooxygenase (PAM) or fragments thereof for the use according to claims 1 to 14 wherein said PAM or fragment thereof is a protein comprising an amino acid sequence selected from the group comprising SEQ ID Nos 1 to 10, or an amino acid sequence having at least 85%, particularly at least 90%, more particularly at least 95%, even more particularly at least 99% sequence identity therewith.

16. Modified or unmodified Peptidylglycine alpha-amidating monooxygenase (PAM) or fragments thereof for the use according to claims 1 to 15, wherein said PAM of fragments thereof are modified bya. attaching one or more natural or synthetic polymers units, wherein said polymers are selected from the group comprisingi. PEG, particularly having an average molecular weight in the range of 0.2-100 kDa, particularly 1-90 kDa, more particularly 2-80 kDa, more particularly 3-70 kDa, more particularly 4-60 kDa and even more particularly 5 to 50 kDa; ii. XTEN;iii. a peptide polymer consisting of repeats of PAS (the amino acids proline, alanine and serine, particularly comprising 100-200, particularly 120-180, more particularly 130-170, even more particularly about 150 PAS repeats;iv. elastin-like polypeptides consisting of Valin-Prolin-Glycine-x-Glycine repeats, wherein x is any amino acid other than Proline, particularly consisting of from 150, 200, 250, 300, 400, or 500 to 550, 600, 750, 850, 950 or 1,000 amino acids; v. HAP polypeptide, having the sequence (Gly4Ser)n, wherein n is 100-200; vi. gelatin-like fusion protein, having the sequence (Gly-X-Y)n, wherein X and Y individually are any amino acid except for Cysteine, and wherein n = 60 to 1500;vii. polysaccharides, in particular those chosen from the group comprising:

1. dextrans;2. hydroxyethyls;3. heparosan;4. hyaluronic acid;viii. poly sialic acid.b. conjugation with a serum protein, such as albumin or an immunoglobulin or parts of an immunoglobulin;c. site-directed mutagenesis by insertion, deletion and / or exchange of one or more amino acids within the amino acid sequence of PAM.

17. Modified or unmodified Peptidylglycine alpha-amidating monooxygenase (PAM) or fragments thereof for the use according to claims 1 to 16, wherein said PAM or fragment thereof is administered in combination with ascorbate and / or copper and / or peptide with C-terminal glycine amino acid or fragments thereof,wherein more particularly said peptide with C-terminal glycine amino acid is selected from the group comprising adrenomedullin (ADM), adrenomedullin-2, intermedin-short, pro- adrenomedullin N-20 terminal peptide (PAMP), amylin, gastrin-releasing peptide, neuromedin C, neuromedin B, neuromedin S, neuromdin U, calcitonin, calcitonin gene-related peptide (CGRP) 1 and 2, islet amyloid polypeptide, chromogranin A, insulin, pancreastatin, prolactin-releasing peptide (PrRP), cholecystokinin, big gastrin, gastrin, glucagon-like peptide 1 (GLP-1), pituitary adenylate cyclase -activating polypeptide (PACAP), secretin, somatoliberin, peptide histidine methionine (PHM), vasoactive intestinal peptide (VIP), gonadoliberin, kisspeptin, MIF-1, metastin, neuropeptide K, neuropeptide gamma, substance P, neurokinin A, neurokinin B, peptide YY, pancreatic hormone, deltorphin I, orexin A and B, melanotropin alpha (alpha-MSH), melanotropin gamma, thyrotropin-releasing hormone (TRH), oxytocin, vasopressin or fragments thereof.

18. Modified or unmodified Peptidylglycine alpha-amidating monooxygenase (PAM) or fragments thereof for the use according to claims 1 to 17, wherein said subject is characterized by a level of PAM and / or its isoforms and / or fragments thereof below a certain threshold in a bodily fluid of said subject.

19. Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof for use in the treatment of a subject having an increased amyloid-beta and / or phospho-Tau-protein load in the brain and / or for use in the treatment or prevention of neuroinflammation in a subject in need thereof for the improvement of neuroinflammation, wherein said use is according to any of claims 1 to 18.

20. Pharmaceutical formulation comprising modified or unmodified PAM or fragments thereof the use according to claim 19, wherein said pharmaceutical formulation is to be administered orally, epicutaneously, subcutaneously, intradermally, intramuscularly, intravascular (intraarterially, intravenously), or via the central nervous system (CNS, intracerebrally, intracerebroventricularly, intrathecally) or via intraperitoneal administration.

21. Pharmaceutical formulation comprising PAM or fragments thereof the use according to claim 19 or 20, the formulation comprising said PAM or fragments thereof in combination with ascorbate and / or copper and / or peptide having a C-terminal glycine amino acid or fragments thereof, wherein in particular said peptide with C-terminal glycine amino acid is selected from the group comprising adrenomedullin (ADM), adrenomedullin-2, intermedin-short, pro-adrenomedullin N-20 terminal peptide (PAMP), amylin, gastrin-releasing peptide, neuromedin C, neuromedin B, neuromedin S, neuromdin U, calcitonin, calcitonin gene-related peptide (CGRP) 1 and 2, islet amyloid polypeptide, chromogranin A, insulin, pancreastatin, prolactin-releasing peptide (PrRP), cholecystokinin, big gastrin, gastrin, glucagon-like peptide 1 (GLP-1), pituitary adenylate cyclase- activating polypeptide (PACAP), secretin, somatoliberin, peptide histidine methionine (PHM), vasoactive intestinal peptide (VIP), gonadoliberin, kisspeptin, MIF-1, metastin, neuropeptide K, neuropeptide gamma, substance P, neurokinin A, neurokinin B, peptide YY, pancreatic hormone, deltorphin I, orexin A and B, melanotropin alpha (alpha-MSH), melanotropin gamma, thyrotropinreleasing hormone (TRH), oxytocin, vasopressin or fragments thereof.

22. A kit comprising the pharmaceutical formulation according to claims 19 to 21.

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