WO2015165980A2

WO2015165980A2 - Treatment and prevention of alzheimer's disease (ad)

Info

Publication number: WO2015165980A2
Application number: PCT/EP2015/059364
Authority: WO
Inventors: Markus Mandler; Radmila Santic; Achim Schneeberger; Frank Mattner; Walter Schmidt
Original assignee: Affiris Ag
Priority date: 2014-04-29
Filing date: 2015-04-29
Publication date: 2015-11-05
Also published as: WO2015165980A3

Abstract

The invention discloses a monocyte inducing agent and/or a monocyte activating agent and/or a monocyte recruiting agent for use in the treatment and prevention of AD.

Description

Treatment and prevention of Alzheimer's Disease (AD)

The present invention relates to means and methods for the treatment and the prevention of dementias associated with β- amyloid deposition, preferably Alzheimer's Disease (AD).

Various dementias are characterized by the aberrant accumu^¬ lation of Amyloid-β polypeptides (Αβ) resulting in β-amyloid deposition. The most prominent form of β-Amyloidoses is AD. Oth^¬ er examples include but are not limited to Dementia with Lewy bodies and Dementia in Down syndrome.

AD is the most prevalent neurodegenerative disorder current^¬ ly affecting 28 million people worldwide. It typically presents with a characteristic amnestic dysfunction associated with other cognitive-, behavioural- and neuropsychiatric changes. AD is characterized by the abnormal accumulation of intra- and extra^¬ cellular amyloid deposits - closely associated with extensive astrocytosis and microgliosis as well as dystrophic neurones and neuronal loss. These amyloid deposits mainly consist of Αβ- peptides Αβ40 and Αβ42 derived from the Amyloid Precursor Pro^¬ tein (APP; gi : 112927), which is expressed on various cell types in the nervous system. Αβ peptides are considered to be directly involved in the pathogenesis and progression of AD.

Besides amyloid deposits, neurofibrillary tangles (NFT) em^¬ body the second characteristic neuropathological hallmark of AD, first described by Alois Alzheimer. These lesions occur in the hippocampus, amygdale association cortices, and certain subcor^¬ tical nuclei. NFTs are located in the cytoplasm of neurons and are composed of hyperphosphorylated tau protein. Tau is an axon- al, microtubule binding protein that promotes microtubule assem^¬ bly and stability under normal conditions. Hyperphosphorylation of Tau results in loss of microtubule association and subsequent disassembly of microtubules, which in turn leads to an impair^¬ ment of axonal transport and subsequent axonal and neuronal de^¬ generation. It is still unclear whether tau hyperphosphorylation and tangle formation are a cause or a consequence of AD.

Besides amyloid and Tau/hyperphosphorylated Tau pathology, neuroinflammation can be considered as the third integral pillar of pathophysiologic changes causing neurodegeneration in AD. The neuroinflammatory phenotype in AD is characterized by robust and widespread activation of microglia and astrocytes in the affect- ed brain regions, resulting in endogenous expression of pro^¬ inflammatory cytokines, cell adhesion molecules, and chemokines. These changes are thought to result from glial reaction to events related to ongoing toxicity elicited by amyloid and Tau/hyperphosphorylated Tau and their mediators.

It is currently believed that one potential treatment strat^¬ egy for AD and related disorders could be based on immunotherapy to prevent or reduce the accumulation of neurotoxic agents like Αβ or Tau/hyperphosphorylated Tau.

Various active and passive treatment strategies targeting Tau/hyperphosphorylated Tau led to a reduction of Tau/hyperphosphorylated Tau deposition and associated neuropa- thological changes in animal models, however, no positive data in human AD patients are available so far. Quite in contrast, there have been a significant number of clinical trial failures in the most recent past: Results "from the Phase III clinical trials of two monoclonal antibodies — bapineuzumab and solane- zumab — that target amyloid-β indicated little clinical benefit of immunological attack on amyloid-β at the dementia stage of sporadic disease" (Aisen et al . , Nat. Rev. Drug Disc. 12 (2013), 324-325; Mullard, Nat. Rev. Drug Disc. 11 (2012), 657-660). Also other studies of hypothesis-driven candidate disease modifiers "such as anti-inflammatory drugs, secretase inhibitors and modu^¬ lators, hormonal therapies, statins and other drugs have been disappointing", including the "clinical failure of the two lead^¬ ing γ-secretase inhibitors, semagacestat [..] and avagacestat" (Aisen et al . , 2013; Mullard, 2012) . Commentators have termed this poor clinical outcome of AD clinical trials as "the culmi^¬ nation of a ^xlost decade' in Alzheimer's disease therapeutic trials, with no substantial success since the approval of meman- tine" (Aisen et al . , 2013) . In the course of this development, the US-FDA also amended the rules for approving new treatments for AD and recommended the use of AD specific biomarkers, such as radiologic biomarkers using PET (positron emission tomogra^¬ phy) scans (Kozauer et al . , N. Engl. J. Med. 368 (2013), 1170- 1171) .

WO 94/16327 Al discloses therapeutic agents that involve an "amyloid protein ion channel". However, this concept of amyloid protein ion channel of WO 94/16327 Al was not further prosecuted and was finally challenged scientifically (Sokolov et al . , J. Gen. Physiol. 128 (2006), 637-647; commentary by Eliezer, J. Gen. Physiol. 128 (2006), 631-633).

In addition, the teachings of WO 94/16327 Al imply an active interaction of Al-ions with potential Αβ—Ion channels in vivo, thereby inhibiting these channels. Thus, in order for aluminium to full fill this task, the compound has to reach the brain as the site of activity in the suggested concentrations. In the hu^¬ man brain normal levels of aluminium range from 0.25 to 0.75 mg/kg wet weight, with the grey matter (mainly responsible for regulating cognitive function affected in AD) containing about twice the concentration found in the white matter (The EFSA Journal (2008) 754, 24-88; Annex to the EFSA Journal (2008) 754, 1-34 opinion "Safety of aluminium from dietary intake") . There is evidence that with increasing age, aluminium concentrations may even increase in the human brain tissue. Similarly, several studies also indicate that brains derived from AD patients show higher Al-levels than healthy control brains (reviewed in Yokel, NeuroToxicology 21 (2000), 813-828). Thus the suggested thera^¬ peutically active Al concentration is already present in healthy and diseased brain (in the range of the intended use-formulation as described in WO 94/16327 Al, claim 12: 0.01-lOmg/kg) . In ad^¬ dition, bioavailability of Al in brain after parenteral and oral uptake is kept low relying on actively regulated, highly effi^¬ cient influx/efflux mechanisms and requires high peripheral dos^¬ es to reach suggested therapeutic cerebral concentrations. It is therefore without plausible scientific basis that an additional increase in peripheral Al would lead to additional cerebral Al levels required for exerting direct, therapeutically beneficial effects without eliciting potential toxic effects.

Furthermore, Figure 7 and 8 of this application disclose that topically applied aluminium-oxyhydroxide is able to lower cognitive decline significantly in an APP-transgenic model for Alzheimer's disease (Tg2576) without significantly changing cer^¬ ebral Αβ levels. This is implying an ΑΡΡ/Αβ independent mecha^¬ nism underlying beneficial functional effects exerted by alumin^¬ ium-oxyhydroxide in this AD model.

WO 99/27944 Al discloses AD vaccines being essentially based on the presence of an agent effective to induce an immunogenic response against Αβ . WO 2011/120924 Al refers to an Αβ vaccine, which is essentially based on Αβ1-6 peptide bound to a virus^¬ like particle. WO 2006/005707 A2, WO 2009/149486 A2 and WO 2009/149485 A2 disclose Αβ mimotope peptides for use in vaccines for the prevention and treatment of AD.

Heneka et al . (Nature, 493 (7434) (2012): 674-678) suggest the treatment of AD by inhibition of NLRP3 in order to reduce amyloid-β aggregation. Aimanianda et al . (TIPS, 30 (6) (2009): 287-295) discloses that alum activates NLRP3.

Magga et al . (J. Cell. Mol. Med. 16 (2012): 1060-1073) re^¬ port the production of monocytic cells from bone marrow stem cells and their therapeutic use in AD. Lebson et al . (Cell Transp. Cogn. Com. 17 (2008): 470/471) disclose monocyte gene therapy in AD APP+PS1 transgenic mice. WO 2012/055981 Al sug^¬ gests the use of a "TLR4 agonist free of endotoxin" for the pre^¬ vention or reduction of amyloid deposition. Malm et al . (GLIA 58 (2010) : 889-900) review the role and therapeutic potential of monocytic cells in AD.

WO 2009/105641 Al discloses the use of M-CSF for the treat^¬ ment of amyloidosis. Boissionneault et al . (Brain 132 (4) (2008) : 1078-1092) report the effects of M-CSF on amyloid depo^¬ sition and cognitive impairment in AD. Luo et al . (Neuroscience letters 367 (2) (2013) : 210-172) disclose that Colony- stimulating factor 1 receptor (CSF1R) signalling in injured neurons facilitates protection and survival.

Accordingly, so far no effective, disease modifying treat^¬ ment is available to stop the progressive neurodegeneration and associated cognitive decline in human patients. Available treat^¬ ment modalities for AD include three acetylcholinesterase in^¬ hibitors (AChEI) and one N-Methyl-D-aspartate (NMDA) antagonist. Their effects are small and only symptomatic in nature (see e.g. Corbett et al . , Nat. Rev. Drug Discov. 11 (2012), 833-846). Thus, there is a high medical need for a disease-modifying drug.

It is an object of the present invention to provide means and methods for the treatment and prevention of AD enabling a cure to AD in the meaning that the status of the diseased pa^¬ tient is not further developing or even ameliorated. Another object is to provide means and methods for preventing the develop^¬ ment of AD in persons having or being at risk of developing AD. More specifically, it is an object of the present invention to provide efficient AD treatment, as proven with respect to at least one significant biomarker, as measured by brain imaging modalities using MRI (Magnetic resonance imaging) or emission tomography based techniques.

Therefore, the present invention provides a monocyte induc^¬ ing agent and/or a monocyte activating agent and/or a monocyte recruiting agent for use in the treatment and prevention of de^¬ mentias associated with β-amyloid deposition, preferably AD.

In the course of the present invention it has surprisingly turned out that a monocyte inducing agent and/or a monocyte ac^¬ tivating agent and/or a monocyte recruiting agent have proven in clinical trials to be effective in real disease modifying ef^¬ fects in AD patients leading to clinical efficacy hitherto not seen in any of the clinical trials for AD medication so far. The present invention therefore provides a breakthrough technology for this disease. For the first time, a significant disease mod^¬ ifying effect could be detected in AD patients. Moreover, the present invention has also turned out to be effective without the significant side effects reported in other clinical trials for AD medication, especially in the field of AD immunotherapy.

More specifically, the present invention has achieved a sta^¬ tistically significant disease modifying effect in AD patients with respect to MRI scans of the volume of the (right) hippocam^¬ pus. Moreover, for the first time, the correlation of a clinical biomarker and a radiologic biomarker has been shown in the course of clinical trials performed for the present invention. Structural MRI has been highlighted as a significant biomarker, in the most recent scientific literature (Risacher et al . , Annu . Rev. Clin. Psychol. 9 (2013), 621-648; Vermuri et al . , Neurology 73 (2009), 287-293 and 294-301; Weiner et al . , Alzh. Dememt . 9 (2013), elll-94; Frisoni et al . , Nat. Rev. Neurol. 6 (2010), 67- 77; Fox et al . , Arch. Neurol. 57 (2000), 339-344).

MRI provides great power to effect cross-sectional groupwise discrimination and better correlation with general cognition and functional status cross-sectionally . MRI reflects clinically de^¬ fined disease stage even better than various CSF biomarkers tested (Vermuri et al . , Neurology 73 (2009), 287-293 and 294- 301). Numerous studies have demonstrated significantly reduced hippocampal and entorhinal cortex (EC) volume, as well as re^¬ duced cortical thickness in the medial and lateral temporal cor- tex, parietal lobe, and frontal lobes, in patients destined to convert from MCI to probable AD (MCI-converters) , up to two years prior to clinical conversion (Risacher et al . , 2013) .

Accordingly, this biomarker was investigated in the course of the clinical trials performed for the present invention in parallel with the standard clinical parameters (monitoring func^¬ tional and cognitive function of AD patients) .

With the present invention, a significant improvement in the development of AD patients compared to the usual development of AD patients (gradual cognitive, functional and behavioural de^¬ cline) can be achieved so as to satisfy the long-felt need of providing a disease-modifying treatment of AD.

The present invention provides a monocyte inducing agent and/or a monocyte activating agent and/or a monocyte recruiting agent for use in the treatment and prevention of dementias asso^¬ ciated with β-amyloid deposition, preferably Alzheimer's Disease (AD) , but also Dementia with Lewy bodies, Dementia of Down Syn^¬ drome or other β-Amyloidoses . Representative tests for safe^¬ guarding monocyte activating/inducing/recruiting properties of any agent are disclosed e.g. in examples 3.3, 3.4 and 3.5, re^¬ spectively.

According to the present invention, inducing/recruiting/activating agent (s) that are needed for the treatment are administered to a patient in an effective amount so as to obtain the disease-modifying effect.

The cells of interest in the present invention are peripher^¬ al monocytic cells entering the CSF/brain. In the mouse, at the site of action (in the brain) they are CD1 lb⁺CX₃CRl^lowCCR2⁺CXCR4^high cells, as distinct from CD1 lb+CX₃CRl^highCCR2^~CXCR4^low resident microglia. In general, murine inflammatory monocytes are defined as CCR2⁺, CX3CRl^low and GR1⁺. In contrast, resident monocytes in mice are classified as CCR2^", CX3CRl^high and GR1^".

In human monocytes, the situation seems not completely un^¬ derstood. Human monocytes that are CD14^highCDl 6^~ are considered classical monocytes. Together with the CD14^highCDl 6⁺ (i.e. interme^¬ diate monocytes) these are considered inflammatory monocytes. In contrast, CD14⁺CD16⁺ cells are non-classical monocytes (also con^¬ sidered patrolling monocytes) . According to the present inven^¬ tion, Grl+/CDllb⁺ cells can be used, as well as CD14++CD16- ; it is also preferred to use subsets of such cells. Preferred monocytes for use according to the present inven^¬ tion are Grl ⁽⁺⁾ /CD1 lb ⁽⁺⁾ cells, CD1 lb⁺CX₃CRl^lowCCR2⁺CXCR4^high cells or CD14⁺⁺CD16^" cells.

Preferred monocyte inducing agents or monocyte activating agents or monocyte recruiting agents are selected from the group consisting of M-CSF, GM-CSF, IFN-gamma, TGF-beta, TNF-alpha, IL- l, I L-2, I L-4, I L-5, I L-6, 11-10, IL-13, Fractalkine/CX3CL1 , mem^¬ bers of the Chemokine (CC-motif) ligand family (CCL) , especially CCL2, CC13, CCL4, CCL12, members of the IL-8 family, especially CXCl-8 (11-8), Toll-like receptor agonists, especially TLR4 ago^¬ nists, preferably LPS or MPLA and an aluminium salt. As also e.g. defined by the tests in examples 3.3, 3.4 and 3.5, these agents have the following properties: M-CSF, GM-CSF: induction (/proliferation) regulator, IFN-gamma: activation + induction (/proliferation) regulator, TGF-beta: induction (/proliferation) + activation regulator, TNF-alpha: activation regulator, IL-1: activation regulator, IL-2 : induction (/proliferation) + activation regulator, IL-4: induction (/proliferation) + activation regulator, IL-5: induction (/proliferation) regulator, IL-6: activation regulator, 11-10: activation regulator, IL-13: activation regulator, Fractalkine/CX3CL1 : recruitment regulator, members of the Chemokine (CC-motif) ligand family (CCL) , especially CCL2, CC13, CCL4, CCL12: activation + recruitment regulator, members of the IL-8 family, especially CXCl-8 (11-8) : recruit^¬ ment regulator, and Toll-like receptor agonists, especially TLR4 agonists, preferably LPS or MPLA and an aluminium salt: induc^¬ tion (/proliferation) + activation + recruitment regulator.

The monocytes or monocyte inducing agent or monocyte acti^¬ vating agent or monocyte recruiting agent for use according to the present invention is preferably administered subcutaneously, intra-cranially or into the bone marrow.

According to a preferred embodiment, the monocytes are human phagocytes, especially autologous phagocytes of the AD patient to whom the monocytes are administered for treating AD. These phagocytes can be obtained from an AD patient, treated ex vivo with a monocyte inducing agent or a monocyte activating agent or a monocyte recruiting agent and then administered to the AD pa^¬ tient from whom said phagocytes have been obtained (as autolo^¬ gous cells) .

According to another preferred embodiment, the monocyte in- ducing agent or monocyte activating agent or monocyte recruiting agent is an aluminium salt, especially aluminium oxyhydroxide .

Preferably, the aluminium salt has the general formula

Me_a ⁺Al_b ³⁺An^c~ ·η¾0, wherein

Me⁺ is Na⁺, K⁺, Li⁺, Rb⁺, Cs⁺ or NH₄ ⁺;

An is P0₄ ^3", SO₄ ^2", O(OH)^3", 0₂ ^~ or OH^";

a is 0, 1, 2, or 3;

b is 1 or 2 ;

c is 1, 2, 3, 4, 5, or 6; and

n is 0 to 48.

Preferably, the aluminium salt is selected from aluminium oxyhydroxide, aluminium phosphate, or aluminium sulphate.

Another preferred monocyte inducing agent or monocyte acti^¬ vating agent or monocyte recruiting agent is a cytokine or a mixture of cytokines, preferably a mixture of at least three, especially at least five, cytokines.

Preferably, the monocyte inducing agent or monocyte activat^¬ ing agent or monocyte recruiting agent is administered in an amount of at least 1.2 mg per dose, preferably at least 1.5 mg per dose, especially at least 1.8 mg per dose.

Preferably, the monocyte inducing agent or monocyte activat^¬ ing agent or monocyte recruiting agent is an aluminium salt in an amount of 1.2 to 10.0 mg per dose, preferably 1.5 to 5 mg per dose, especially 1.8 to 2.5 mg, (given as A1₂0₃ equivalent) per dose .

According to a preferred embodiment, the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is administered intracranially or into the cerebrospinal fluid.

Preferably, the monocyte inducing agent or monocyte activat^¬ ing agent or monocyte recruiting agent is free of proteasome based adjuvants.

It is also preferred to administer a monocyte inducing agent or a monocyte activating agent or a monocyte recruiting agent in an amount effective for obtaining an astrocytosis reduction and/or a blood brain barrier breakdown and/or nitric oxide oxidative stress and/or neuronal death in the AD patient.

So far all active immunotherapeutic approaches tested in hu^¬ mans have used stimulation of the adaptive immune system to ex^¬ ert its therapeutic effect mainly mediated by humoral immune re^¬ sponses specific for the target in patients. Over the past sev- eral years, stimulation of the second arm of the immune system, the innate immune system, has emerged as a potentially effective method for activating target independent defence mechanisms like phagocytosis as treatment modality for AD.

Activation of innate immune reactions is usually initiated by cells already present in all affected tissues as well as by cells recruited to the site where activation is required. These cells are mainly macrophages, monocytes, microglia and dendritic cells presenting pattern recognition receptors (PRRs) , which recognise pathogen-derived molecules distinguishable from host molecules (also referred to as pathogen-associated molecular patterns (PAMPs) ) . Upon PRR-PAMP interaction these cells undergo activation and release inflammatory mediators responsible for the clinical signs of inflammation and thereby induce the innate immune defence.

Current research mainly focused on methods to stimulate the innate immune system via activation of a special class of PRRs, the Toll-like receptors (TLRs) . TLRs are a family of innate im^¬ mune mediators that are expressed by a variety of immune and non-immune cells (Crack PJ et al . , Immunol Cell Biol 2007; 85:476-480) .

Several TLR-agonists (functioning as PAMPs) have been used as candidates for AD prevention and therapy. Along these lines WO 2009/105641 A2 is directed to a method of preventing or re^¬ ducing amyloid deposition in a subject by stimulating the innate immune system of the selected subject under conditions effective to reduce the amyloid deposits. In particular a TLR9 agonist is used in this disclosure.

Michaud et al . (Proc Natl Acad Sci U S A. 2013 Jan 29; 110 (5) : 1941-6) demonstrate that Toll-like receptor 4 stimula^¬ tion using a detoxified ligand (TLR4 agonist: monophosphoryl lipid A) improves Alzheimer's disease-related pathology in trans^¬ genic mice. Accordingly, WO 2012/055981 Al is directed to compo^¬ sitions and methods for preventing and/or reducing amyloid depo^¬ sition in a subject comprising treatment of a subject with a composition comprising a TLR4 agonist free of endotoxin. In addition TLR4 agonists free of endotoxin for preventing and/or reducing Alzheimer's disease are described. These include aminoal- kyl glucosaminide phosphate (AGP), 3D-MPL, AS01B or an AGP in combination with an oil in water emulsion. However Michaud et al . , 2013, also demonstrate that two sim^¬ ilar TLR4 agonists can exert differential effects in vivo as LPS has been demonstrated to exacerbate whereas MPL ameliorated amy^¬ loid deposition. Additionally, even though acute application of LPS was found to induce microglial and monocytic Αβ phagocytosis more effectively than MPL in this study, chronic systemic expo^¬ sure of TLR4 agonists (i.e. LPS) exacerbated the Αβ plaque load in an APP_swe/PSl transgenic AD model. It is believed that this ex^¬ acerbation is mainly caused by excessive and sustained inflamma^¬ tion induced by systemic LPS. Accordingly, MPL also induced Αβ phagocytosis, but only modest inflammation following chronic systemic application which could explain the different effects of this alternative TLR4 agonist compared to LPS.

Beside pure TLR9 and TLR4 agonists also injection of pro- teasome derived Protollin™ was tested for prevention and therapy in animal models. Protollin™ is an adjuvant comprising Proteo- somes™ non-covalently complexed with the TLR4 agonist LPS for intranasal application. Protollin™ application in APP Tg mice significantly improved cognitive function and stimulated the mi^¬ croglia activation, which correlated with a reduction of Αβ burden and no apparent toxicity (Frenkel, D. et al . JCI 115, 2423- 2433 (2005); Frenkel, D. et al . Ann Neurol 63, 591-601 (2008)).

Beside TLRs other receptors regulating the stimulation of effectors of the innate immune response, e.g. dendritic cells, macrophages, monocytes and microglia, have been described. These receptors also mainly constitute pattern recognition receptors (PRR including NOD-like receptors (NLRs) , RIG-I-like receptors (RLRs) and C-type lectin receptors (CLRs) . These receptors sig^¬ nal through different pathways and induce the production of dis^¬ tinct cytokines and chemokines that play a key role in the prim^¬ ing, expansion and polarization of the immune responses. Activa^¬ tion of the NLR family members NLRP3 and NLRC4 triggers the for^¬ mation of a protein complex, called inflammasome, which has been implicated in the induction of pro-inflammatory cytokines IL-Ιβ and IL-18.

In the course of the present invention it was shown that in^¬ duction of a mildly proinflammatory environment resulting in activation of cells of the innate immune system and thereby e.g. inducing phagocytotic activities in monocytes, macrophages and microglia cells, could help to ameliorate defects in immune cell function detectable in the aging brain and could thus be the un^¬ derlying mechanism of action for treating AD via improved clearance of deleterious protein aggregates.

In the course of the present invention it was further shown that recruitment of peripheral innate immune cells, e.g. circu^¬ lating monocytes, to the brain could help to ameliorate defects in immune cell function detectable in the degenerating nervous system in AD patients.

In the brain, a specific form of macrophages representing the endogenous brain defense and innate immune system is active, the so called microglia. Microglial cells derive from mesoder^¬ mal/ mesenchymal progenitors and have migrated into the brain during development. After invading the CNS, microglial precur^¬ sors disseminate homogeneously throughout the neural tissue. Mi^¬ croglia continuously monitor their local microenvironment and constitute the first line of defense in the CNS against invading pathogens (Nimmerjahn et al . , Science 308, 1314-1318 (2005). Da- valos, D. et al . , Nat. Neurosci. 8, 752-758 (2005). Microglial responses are dependent on changes in the local and systemic chemokine and cytokine milieu which is changing during acute and chronic insults to the brain, e.g. by stroke or chronic disease like AD. Usually such changes lead to the induction of gliosis exerting an important role in the pathogenesis of various CNS diseases including AD (Streit et al . , Prog. Neurobiol. 57,563- 581 (1999) ) .

In addition to tissue resident microglia, blood-circulating immune cells can undergo tissue/organ specific recruitment, de^¬ velop into macrophages and subsequently can take over essential microglial functions in the brain (Varvela et al . , PNAS October 30, 2012 vol. 109 no. 44, 18150-18155.

Current concepts have identified monocytes as potential pre^¬ cursors for such macrophages that continuously develop in the bone marrow, circulate in the blood and migrate into tissues

(e.g. the AD brain), where they become local macrophages or den^¬ dritic cells. Importantly, it has been shown that such monocytes commit for specific functions while still in the circulation

(Gordon et al . , Nat. Rev. Immunol. 5, 953-964 (2005)).

To date, microglia as well as newly recruited, bone marrow derived monocytes are considered to act as both, detrimental

(e.g. secretion of neurotoxic factors) and beneficial (e.g. ex- erting neuroprotection and restoration) for AD disease progression. Interestingly, recent data imply that such resident and bone marrow-derived cells (i.e. CCR2⁺ positive monocytes) have distinct neuroprotective function in AD (Naert et al . , J Mol Cell Biol (2013) 5 (5) : 284-293) . AD patients show CCR2⁺ mono^¬ cytopenia and an impaired recruitment of such CCR2⁺ monocytes. In addition, CCR2⁺ monocytes seem to be able to restrict amyloidosis in the AD brain. Furthermore, a defect in monocytopoiesis re^¬ sulting in a decrease of CX3CRl^lowLy6-C^highGrl⁺CCR2⁺ subset of monocytes was detectable in an APP/PS1 AD mouse model (Naert et al . , 2012) . In addition, bone marrow derived CDllb+ cells (mainly monocytic) were shown to have the ability to deliver therapeutic genes to the brain of amyloid-depositing transgenic mice following ex vivo transfection and reinfusion into animals (Lebson et al.; J Neurosci. 2010 Jul 21 ; 30 (29) : 9651-8 ) . Thus CCR2⁺ monocyte recruitment may constitute an important line of defense against AD associated degeneration including Αβ deposition, synaptic dysfunction, and cognitive decline (Naert et al.2012) . This is further supported by the increase in CCR2+ monocytes and corre^¬ lating partial rescue of cognitive function in the APP/PS1 AD mouse following the unspecific stimulation of CCR2+ monocyte formation using repeated systemic high dose applications of M- CSF.

Thus, one potential treatment paradigm for AD could be based on the specific induction and recruitment of peripheral mono^¬ cytes to the AD brain in order to enhance microglial activity in the brain and thereby to alleviate amyloid deposition, synaptic and neuronal dysfunction and cognitive decline in AD patients.

A large variety of organic and inorganic compounds are known to stimulate a vigorous innate immune response. These include mineral oils and different metal salts, notably aluminium com^¬ pounds (e.g. aluminium oxyhydroxide, the hydrated form of alu^¬ minium oxide (A1₂0₃) . These particulate compounds exert TLR inde^¬ pendent activity and play an important role as activators of in^¬ nate immunity in vivo. Aluminium compounds are known to induce a local TLR-independent , proinflammatory reaction at the site of administration which was shown to induce cytokine secretion, attraction of innate immune cells as well as delivering Alum- complexed antigens to dendritic cells (DCs) . It has been dis^¬ cussed controversially whether particulate aluminium compounds activates innate immune cells via the cytoplasmic NLRP3, which associates with ASC and caspase 1 to form the inflammasome .

Recently, aluminium oxyhydroxide has been observed to bind lipid moieties on APCs (e.g. dendritic cells) and promote lipid sorting in the plasma membrane, leading to signal transduction and immune response initiation as well as increased antigen up^¬ take and enhanced antigen presentation on APCs and B- lymphocytes. Aluminium oxyhydroxide has also been described to be able to activate the complement system, predominantly the al^¬ ternative pathway involving generation of the alternative C3- convertase .

Aluminium oxyhydroxide, thus, employs distinctive pathways to activate cells of the innate immune system: Within hours of exposure, aluminium oxyhydroxide induces a type 2 innate re^¬ sponse characterized by an influx of eosinophils, monocytes, neutrophils, DCs, NK cells and NKT cells. In addition, cytokines and chemokines are produced within hours of injection, including IL-Ιβ and IL-5. Optimal production of some of these depends on macrophages and mast cells, while delivery of the specific anti^¬ gen complexed to aluminium particles, to activate the adaptive arm of the immune system, is mediated via DCs.

Thus, the activation and subsequent action of proponents of the innate immune response due to exposure to particulate alu^¬ minium compounds (e.g. Alhydrogel, Alhydrogel adsorbed with bio^¬ logical macromolecules ) and their local (i.e. at the site of in^¬ jection) as well as systemic effects (i.e. activation and re^¬ cruitment of peripheral immune cells to the target organ, e.g. the AD brain) can be harnessed to treat or prevent pathophysio^¬ logic alterations in AD patients leading to an amelioration or prevention of clinical disease manifestation.

The most preferred embodiment of the present invention com^¬ prises the effective administration of aluminium oxyhydroxide (particularly as Alhydrogel) to AD patients.

Aluminium salts have a long-standing use as adjuvants in vaccines, however, during the years the pharmaceutical use of such salts has been reduced to mostly two suspension prepara^¬ tions, namely Alhydrogel (aluminium-oxyhydroxide) and AdjuPhos (aluminiumhydroxyphosphate) , onto which antigens are adsorbed for vaccine preparations (reviewed in E. B. Lindblad (2004) Vac^¬ cine 22, 3658-3668; E. B. Lindblad (2004) Immunology and Cell Biology 82, 497-505; R. K. Gupta (1998) Adv. Drug Delivery Rev. 32, 155-172) .

Despite its long use, the mode of action of Alhydrogel as an adjuvant is poorly understood. The initial hypothesis, that Alhydrogel forms a depot at the injection side has turned out to be only one part of a multi-faceted story (reviewed in C. Exley, P. Siesjo, H. Eriksson (2010) Trends Immunol. 31, 103-109; S. L. Hem, H. HogenEsch (2007) Expert Rev. Vaccines 6, 685-698; P. Marrack, A. S. McKee, M. W. Munks (2009) Nature Rev. Immunol. 9, 287-293; S. G. Reed, M. T. Orr, C. B. Fox (2013) Nat. Med. 19, 1597-1608) .

The main presentations of aluminium adjuvants used in humans are aluminium hydroxide (or aluminium oxyhydroxide) and aluminium phosphate. Both presentations are usually prepared by expos^¬ ing a soluble aluminium salt (historically potassium alum, i.e. KA1 (S0₄) 2 · 12H20, was often used) to alkaline conditions, upon which a suspension is formed. Characterisation with X-ray crystallography and IR spectroscopy revealed a boehmite-like struc^¬ ture (aluminium oxyhydroxide) for aluminium hydroxide and an amorphous structure corresponding to aluminium hydroxyphosphate for aluminium phosphate.

Aluminium oxyhydroxide preparations have a point of zero charge at a pH of ~ pH 11, while aluminium hydroxyphopsphate might have a point of zero charge as low as pH 4 (depending on the phosphate content) . Therefore aluminium oxyhydroxide and al^¬ uminium hydroxyphosphate have an opposite surface charge at neu^¬ tral pH, with the latter being negatively charged. It has to mentioned, however, that the surface charge may change depending on the exact buffer composition, especially phosphate ions have the capacity to lower the surface charge of aluminium oxyhydrox^¬ ide .

For aluminium oxyhydroxide, the preparation is devoid of an^¬ ions such as sulphates, nitrates, or chlorides and has a speci^¬ fied heavy metal content of less than 20 ppm. The suspension of aluminium oxyhydroxide has a particle size distribution between 2 ym and approximately 10 ym, which are aggregates composed of smaller fibers of ~2nm x 4.5 nm x lOnm.

According to this most preferred embodiment, the current in^¬ vention relates to the use of European Pharmacopoeial grade (Al- uminium-oxyhydroxide, monograph 1664), more specifically to the product manufactured by Brenntag Biosector (2% Alhydrogel) test^¬ ed towards EP compliance. Alhydrogel is available in three vari^¬ eties: Alhydrogel 1.3%; Alhydrogel 2% and Alhydrogel "85". Alhy^¬ drogel 2% was elected as the International Standard Preparation for aluminium hydroxide gels. The pharmaceutical preparation ac^¬ cording to the present invention is aseptically formulated into a suitable buffer, preferably an isotonic phosphate buffer (ImM to 100 mM) , preferably at a concentration of ≥ 1.0 mg/ml Alhydrogel (given as A1₂0₃ equivalent; this metric (Al as "A1₂0₃ equivalent") is used generally for the present invention; ac^¬ cordingly, all doses and amounts referred to in the present ap^¬ plication, as far they are relating to aluminum salts (especially as far as they are relating to aluminium oxyhydroxide) refer to A1₂0₃ equivalents (of aluminium oxyhydroxide (Alhydrogel) ) ) , even more preferably at a concentration of ≥ 1.5 mg/ml Alhydro^¬ gel (given as A1₂0₃ equivalent) , most preferable at a concentra^¬ tion of ≥ 2.0 mg/ml Alhydrogel (given as A1₂0₃ equivalent) . The amount of aluminium salt for Alhydrogel is given as A1₂0₃ equiva^¬ lent in line with the strength as stated by the manufacturer (i.e. 2% Alhydrogel equates to 2% A1₂0₃ , i.e. 20 mg/mL) . This concentration is directly convertible into the respective con^¬ centration of aluminium by using the respective molecular masses (20 mg/mL A1₂0₃ (Mw 101 , 96 ) _corresponds to 10.6 mg/mL aluminium (molecular mass 26, 98)) . Depending on the salt used this value cam easily be converted into the necessary amount/concentration of a different aluminium salt (it is clear that these values are based solely on the amount of aluminium (salt) , and other as^¬ pects, such as the contribution of the particulate nature of Alhydrogel is not taken into account.

Alhydrogel 2%, often also referred to as alum, is an alumin^¬ ium oxyhydroxide wet gel suspension.

In the most preferred embodiment of the present invention, the aluminium salt to be administered to the AD patient is an aluminium oxyhydroxide suspension, preferably European Pharmaco^¬ poeia grade aluminium-oxyhydroxide (monograph 1664), especially Alhydrogel. The aluminium oxyhydroxide is administered in an amount effective to achieve an AD ameliorating effect, as de^¬ fined by the EMEA Guideline on medical products for the treat^¬ ment of AD and other dementias (Document Ref. CPMP/EWP/553/95 Rev.l of 24 July 2008) . Accordingly, any administration proce- dure or dosage regimen for the aluminium salt formulation, especially aluminium-oxyhydroxide formulation, according to the pre^¬ sent invention that is suitable to achieve the AD modifying ef^¬ fect as provided by the present invention is subject to the pre^¬ sent invention. Although it is possible to deliver the prepara^¬ tion according to the present invention by way of slow infusion, the preferred strategy for administration is by administration of doses, for example by subcutaneous injection. Preferably, therefore the administration dose of aluminium oxyhydroxide is of at least 1.2 mg to an AD patient. A preferred range of amount to be administered to a patient is an amount of aluminium oxyhy^¬ droxide of 1.2 mg to 5.0 mg. The AD ameliorating effect of alu^¬ minium oxyhydroxide administration is even more pronounced at an amount of at least 1.5 mg. According to another preferred embod^¬ iment aluminium oxyhydroxide is administered in an amount of 1.5 mg to 5.0 mg, preferably 1.5 to 3.0 mg, especially 1.5 to 2.5 mg, to an AD patient. Another preferred embodiment comprises ad^¬ ministration of aluminium oxyhydroxide in an amount of 1.6 mg to 2.5 mg, preferably 1.8 to 2.2 mg, especially 1.9 to 2.0 mg, to an AD patient.

According to another preferred embodiment, the aluminium ox^¬ yhydroxide is administered in amount of 2.2 mg or higher. This amount is even higher as prescribed in the US general biological products standards (U.S.C. 21 CFR 610.15 (as of 1 April 2013)). Such preferred higher ranges of aluminium oxyhydroxide are i.a. 2.2 to 10 mg, 2.2 to 8 mg, 2.2 to 5 mg, and 2.2 to 4 mg for one administration dose.

Preferably, the aluminium salt is the single effective sub^¬ stance to be applied in the administration dose. The aluminium salt preparation according to the present invention may, however, contain various auxiliary substances that have no specific clinical effect but are useful in the dosage form to be adminis^¬ tered, be it for administration purposes, storage purposes, or other purposes. According to a preferred embodiment, the alumin^¬ ium oxyhydroxide preparation to be applied according to the pre^¬ sent invention contains a pharmaceutically acceptable carrier, diluent or excipient, for example water for injection. Prefera^¬ bly, the aluminium oxyhydroxide preparation according to the present invention additionally contains one or more stabilisa- tors, especially thiomersal, detergents, antioxidants, complex- ing agents for mono- or divalent metal ions, especially eth- ylenediaminetetraacetic acid (EDTA) , sugars, sugar alcohols, glycerol, and/or buffer substances, especially TRIS or phosphate buffer substances. This, of course, also includes mixtures of such auxiliary substances.

The dosage form to be administered to the patients can be provided in any convenient volume, preferably as injectable sus^¬ pension, e.g. with a volume of between 0.1 and 10 ml, more pre^¬ ferred of 0.2 to 5 ml, especially of 0.4 to 3 ml. Specifically preferred volumes are 0.5, 1, 1.5 and 2 ml. The pharmaceutical preparations according to the present invention are produced ac^¬ cording to pharmaceutical Good Manufacturing Practice (GMP) , as required and defined by the European and/or US Pharmacopeia.

According to a preferred embodiment, the aluminium oxyhy- droxide is administered to a patient in a suspension with a pH of 4 to 10, preferably of 5 to 9, more preferred of 6 to 8, es^¬ pecially from 7.0 to 7.5. Preferably, the suspension is an iso^¬ tonic suspension.

Preferably, the aluminium salt is administered by a route that is as convenient as possible for the AD patient but is still effective to achieve an AD modifying effect. Most effec^¬ tive treatment routes of aluminium oxyhydroxide according to the present invention are subcutaneous, intranodal, intradermal, or intramuscular administration, especially subcutaneous administration. Subcutaneous administration is performed as a bolus into the subcutis, the layer of skin directly below the dermis and epidermis, especially in the fatty tissue in the subcutis.

Administration regimes can be optimised individually for each AD patient, depending on the treatment success, as measured by various parameters, especially by cognitive and functional performances and by biomarkers, especially PET scans concerning hippocampus volume (see below) . In the course of the clinical trials conducted for the present invention, at least monthly ad^¬ ministrations of aluminium oxyhydroxide to an AD patient have proven to be successful in ameliorating AD. In order to achieve a long lasting therapeutical effect, such at least monthly ad^¬ ministrations should be continued for at least three months, es^¬ pecially at least six months.

Administration of the aluminium salt according to the present invention may also be performed at least twice a month (for example bi-weekly or weekly) ; also in such a dosage regimen, aluminium oxyhydroxide should be administered to an AD patient at least for a period of three months, preferably for at least six months, more preferred for at least twelve months, especially at least 24 months.

According to a preferred embodiment aluminium oxyhydroxide is administered to an AD patient subcutaneously in the (outer area of the) upper arm, preferably alternating in the left and in the right upper arm (i.e. administering the first dose into the right (or left) upper arm and the second dose into the left (right arm) , and so on) . Other convenient (or alternative) areas for subcutaneous administration are just above and below the waist (except the area right around the navel (a 2-inch cir^¬ cle) ) , the upper area of the buttock, preferably just behind the hip bone, the front of the thigh, midway to the outer side, 4 inches below the top of the thigh to 4 inches above the knee, etc ..

Alternatively, the dose to be administered can also be split into two (or more) split doses that are administered simultane^¬ ously (at the same physician date; at least at the same day) to the AD patient. For example, a dose of 2 mg may be split to split doses of 1.8 and 0.2 mg, 1.7 and 0.3 mg, 1.5 and 0.5 mg, 1.34 and 0.76 mg, 1.0 and 1.0 mg, 1.05 and 0.95 mg, 1.0, 0.5 and 0.5 mg, 0.6, 0.6 and 0.7 mg, 0.2, 0.5, and 1.3 mg, 0.5, 0.5, 0.5 and 0.5 mg, 0.2, 0.3, 0.5 and 1.0 mg, etc.. The split doses may be administered at different administration sites or, prefera^¬ bly, at the same site of administration. The "same site of ad^¬ ministration" is within an area of 10 cm² of the skin, preferably within an area of 5 cm² of the skin, especially within 1 cm² of the skin. Preferred split doses contain aluminium oxyhydroxide in an amount of 0.8 to 5.0 mg, preferably of 1.0 to 3.0, espe^¬ cially from 1.0 to 1.5 mg.

In order to achieve a very long lasting effect of the AD amelioration, the treatment according to the present invention is performed for longer than one year. According to a preferred embodiment of the present invention, the aluminium salt is ad^¬ ministered at least monthly for at least two years, preferably at least four years, especially at least 8 years, to an AD pa^¬ tient .

Administration of the aluminium oxyhydroxide according to the present invention may be performed by any suitable admin^¬ istration device. For convenience reasons, the aluminium oxyhy- droxide dose is administered by an injection device, especially a syringe, to an AD patient. The pharmaceutical preparations for use in the present invention can be provided in any suitable form. Preferably, they are provided in a storage stable form. Storage stability can be assured by various means, such as ster^¬ ilisation, addition of stabilisers, freezing, lyophilisation, etc. Preferably, combinations of such means are used to enhance storage stabilities of such preparations. When aluminum-salt agents, such as aluminium oxyhydroxide are frozen or lyophi- lized, an aggregation of adjuvant particles during processing may be observed. By cooling such formulations, especially alu^¬ minium oxyhydroxide (Alhydrogel) formulations, at faster rates or by the addition of sufficient amounts of a glass forming ex- cipient, such as trehalose, aggregation of Alhydrogel, can be prevented or minimized. It was proposed that freeze- concentration of buffer salts induces modifications in surface chemistry and crystallinity of such aluminium agents, which in turn favour aggregation. These modifications and the resulting aggregation of such Alhydrogel particles can be excluded or min^¬ imized through choice of buffer ions, or kinetically inhibited by rapidly forming a glassy state during freezing (see e.g. Clausi et al . , J Pharm Sci. 2008 Jun; 97 ( 6) : 2049- 61 ) .

The pharmaceutical compositions to be applied to AD patients according to the present invention are manufactured (and fin^¬ ished) into suitable containers, and sold for example in sealed vials, ampoules, cartridges, flexible bags (often constructed with multi-layered plastic) , glass or polypropylene bottles or, preferably, in syringes, especially in prefilled (ready-to-use or ready-to-reconstitute) syringes.

According to a preferred embodiment of the present inven^¬ tion, the aluminium oxyhydroxide is administered in an amount of at least 1.8 mg to an AD patient.

Preferred patients to which aluminium oxyhydroxide prepara^¬ tions according to the present invention is administered are AD patients that are early stage patients, including those patients that are often also referred to as "patients with mild cognitive impairment" (MCI) . The concept of MCI was developed in the 1990s to capture patients with early clinical signs of Alzheimer dis- ease (AD) who did not yet fulfil the criteria for dementia. The amnestic variant of MCI features the following: memory com^¬ plaints, preferably qualified by an informant; memory impairment for age, as indexed by low cognitive performance in one or more neuropsychological tests that tap into learning abilities (for example, prose recall, word list) ; preserved general cognitive function (for example, Mini-Mental State Examination score of 24 out of 30 or above) ; intact activities of daily living; and no dementia. About two-thirds of all patients with amnestic MCI harbour the pathological features of AD and develop the clinical syndrome of Alzheimer dementia within 5 years, whereas the re^¬ maining one-third have non-progressive or very slowly progres^¬ sive causes of cognitive impairment (for example, depression or age-related cognitive impairment) . Proposed new diagnostic cri^¬ teria for AD developed in 2007 (Dubois et al . , Lancet Neurol. 6 (2007), 734-746) suggested that the disease can be recognized at the MCI stage if the patient is positive for at least one of the following four markers: medial temporal atrophy on MRI; temporo^¬ parietal cortical hypometabolism on 18F-fluorodeoxyglucose PET; abnormality of cerebrospinal fluid markers (tau, amyloid^42 or phospho-tau) ; and positivity on amyloid imaging with PET. This patient population is not only included in the AD patients to be treated according to the present invention, it is a specifically preferred group of patients for which the treatment method ac^¬ cording to the present invention is specifically effective. This is in line with the revised criteria for AD clinical trials adopted by the US-FDA (Aisen et al . , 2013; Kozauer et al . , 2013) . Accordingly, it is preferred to treat patients in an ear^¬ ly state of AD, as defined by a relatively high MMSE (mini- mental state examination or Folstein test) score. Preferably the AD patient to be treated according to the present invention is a patient with an MMSE score of between 23 and 30 (30 being the maximum), preferably between 24 and 30, more preferably between 25 and 29, especially between 26 and 29. Other preferred patient groups are patients greater than or equal to 27 points (indicat^¬ ing a normal cognition) , 25 to 27 (slightly below normal cognition) or 19 to 24 (mild points cognitive impairment) .

Early stage AD patients can also be selected by other scores, preferably scores that combine cognitive and functional parameters (and numerical limits) for limiting AD population to be (effectively treated), such as ADAS-cog, etc.

The present invention provides for the first time an AD treatment that is disease modifying. The effectiveness of the treatment according to the present invention is proven by the parameters required by the drug authorisation authorities, espe^¬ cially the EMEA and the US-FDA. For example, the EMEA guideline for AD treatment requires primary endpoints reflecting the cog^¬ nitive and the functional domain. Accordingly, a combined (Com^¬ posite) score is used for the clinical assessment of the present invention. This composite score combines two established scores, one for the cognitive function (ADAS-cog (Alzheimer's Disease Assessment Scale-cognitive subscale) ) and one for the functional ability (ADCS-ADL (Alzheimer's Disease Co-operative Study - Ac^¬ tivities of Daily Living Inventory) ) . The adapted ADAS-cog com^¬ bines items that assess cognitive function. The adapted ADCS-ADL includes items that are sensitive to functional ability. Cogni^¬ tive skills are expected to decline toward the beginning of the disease and one's ability to perform basic functions are ex^¬ pected to decline later in the disease. The combined primary outcome (Composite score according to the present invention) combines both the adapted ADAS-cog and adapted ADCS-ADL to cre^¬ ate a composite that is sensitive to decline in cognitive and basic functions. The following equation is used to derive the combined primary outcome, i.e. combined composite:

Combined composite according to the present invention:

= 1.67*Word recall + 1.35*Orientation + 1.42*Word Recognition + 0.55*Recall Instructions + 0.81*Spoken Language + 1.01*Word Finding + 5.42*ONB + 0.15*VPAL + 0.19*Category Fluency + 0.28*Belongings + 0.35*Shopping + 0.23*Hobbies + 0.38*Beverage + 0.37*Meal + 0.23*Current Events + 0.26*TV + 0.33*Keeping Ap^¬ pointments + 0.37*Travel + 0.33*Alone + 0.35*Appliance + 0.49*Clothes + 0.36*Read + 0.62 telephone + 0.33*Writing

Furthermore, AD biomarkers were observed with the present invention that are characteristic for AD development. EMEA and FDA criteria recommend newer techniques, such as MRI, especially atrophy of entorhinal or (para-) hippocampal cortex. With the present invention, PET (Positron emission tomography) -MRI was applied. More specifically, volume of right hippocampus (im^¬ portant for learning and memory of material that is difficult to verbalise) is used according to the present invention as signif- icant AD biomarker for treatment success.

According to the present invention, a clinical effect in AD treatment can be observed which can be measured by a reduction in cognitive and/or functional decline (over a treatment period of about one year) by at least 30 % (calculated by the score de^¬ cline) , preferably by at least 50 %, especially by at least 70 %, compared to a normal development of decline in AD patients. Preferably, cognitive and functional parameters remain essen^¬ tially unchanged during treatment. This can be achieved by the present invention especially in patients with earliest stage pa^¬ tients (as suggested and recommended by the guidelines of EMEA and FDA) , for example AD patients with MMSE of 23 or higher, preferably of 24 or higher, more preferred of 25 or higher, es^¬ pecially of 26 or higher. For those patients, Composite score change during treatment according to the present invention was still around the initial score after 18 months. This is signifi^¬ cantly more than the minimum requirements for "disease modifying effects" as required by the EMEA ("From a regulatory point of view, a medicinal product can be considered as disease modify^¬ ing, if the progression of the disease as measured by cognitive and functional assessment tools is reduced or slowed down and if these results are linked to an effect on the underlying disease process"; "a disease modifying effect will be considered when the pharmacologic treatment delays the underlying pathological or pathophysiological disease processes and when this is accom^¬ panied by an improvement of clinical signs and symptoms of the dementing condition") .

The invention is further explained by way of the following examples and the figures, yet without being limited thereto.

Fig. 1 shows the results of the clinical trial according to the present invention with respect to the change in Composite score composed of (partial) Adapted ADL change and Adapted ADAS- cog change for all patients who have received the 2 mg and 1 mg aluminium oxyhydroxide treatment.

Fig. 2 shows a comparison of the mild population of patients (the mild population is defined by a baseline MMSE score of 24 and higher) of both groups showed that this effect is most pro^¬ nounced in the cohort of patients in earlier disease stages.

Fig. 3 shows slowing of disease progression apparent in the 2 mg and 1 mg aluminium group as evidenced by Adapted ADAS-cog (ADAS-cog items only; Least Squares Means) for the 1 mg and 2 mg aluminium oxyhydroxide group compared to the historical control

(p-values: 1 mg vs. HC-ADNI , S , HC : <0.0001; 2 mg vs. HC- ADNI , S , HC : <0.0001) .

Fig. 4 shows development of volume (in mm³) of right hippo^¬ campus for 2 mg and 1 mg aluminium oxyhydroxide treatment group of the mild population of patients (the mild population is de^¬ fined by a baseline MMSE score of 24 and higher) , showing that this effect is most pronounced in the cohort of patients in ear^¬ lier disease stages.

Fig. 5 shows the Quality of Life-Alzheimer's disease (QOL- AD) for caregivers. Caregivers completed the measure as a ques^¬ tionnaire about their patients' QOL . The measure consisted of 13 items, rated on a 4 point scale, with 1 being poor and 4 being excellent. Outcomes are shown as the change over time using a least squares means from a mixed model.

Fig. 6 shows immune response of the mice tested in the Tg2576 animal model: Tg2576-mice were injected 6x, s.c, at 4- week intervals with either conjugate-vaccine containing 30yg net peptide, KLH formulated with Alum or Alum only. Alum doses used were equivalent to 2mg/ml. Vaccination induced Abs were measured in plasma samples taken at sacrification (SeqID 1 (n=10), SeqID 2 (n=8), KLH-Alum (n=10) and Alum only (n=8)) . Samples were ana^¬ lyzed for their concentration of IgG Abs against specific pep^¬ tides. Values depicted are the titer calculated as OD max/2 (at 405nm) plus SEM. IgG response forwards the respective immunizing peptide (SeqID 1: anti SeqID 1; SeqID 2: anti SeqID 2, KLH-Alum: anti KLH, Alum: anti AD02); B) Reactivity towards human Αβΐ- 40/42 after immunization. SeqID 1 (n=10) and SeqID 2 (n=8), treated animals show anti Αβ40/42 reactivity, KLH-Alum and Alum only treated animals do not show reactivity above background. Background for this assay was set to 1/100, indicated by black lines and an asterisk in A+B .

Fig. 7 shows memory and learning of the mice tested: Groups of Tg2576 mice (n≤10/group) received 6 monthly injections of KLH/ALUM (n=9) or SeqID 1-KLH-Alum (n=10)-, SeqID 2-KLH-Alum

(n=7) -conjugate vaccines or ALUM only (n=8) . Naive wt animals

(n=20) were used as positive controls for Contextual fear condi^¬ tioning (CFC) . Contextual learning and memory was assessed by CFC-analysis using % of time freezing at the end of CFC testing. Parameter depicted is the % of time the animals are 99% immobile during a representative 2-minute period on day two of the CFC testing paradigm. *..p<0.05; **..p<0.01.

Fig. 8 shows amyloid load in the animals tested: Groups of Tg2576 mice (n≤10/group) received 6 monthly injections of KLH/ALUM (n=9) or SeqID 1 (n=10)-, SeqID 2 (n=7 ) -conj ugate vac^¬ cines or ALUM only (n=8) . Alum dose in all formulations equiva^¬ lent to 2mg/ml. Brains were isolated, 8 weeks after the 6^th im^¬ munization. Quantification of the relative total brain area covered by amyloid deposits (in % of total tissue analyzed) is based on immuno-fluorescence staining using the Αβ specific mAb 3A5. Representative subregions of the cortex (A, B) and dentate gyrus (C, D) of controls (A, C) and SeqID 1- (B, D) immunized mice are shown. E) SeqID 1-KLH Alum + SeqID 2-KLH Alum reduces the relative area covered by amyloid deposits compared to KLH- Alum controls significantly (diffuse and dense cored amyloid; *..p<0.05, **..p<0.01). A slight but insignificant reduction in Αβ deposition is detectable in Alum only treated vs. KLH-Alum treated animals, (ns) Arrowhead in C indicates unspecific fluo^¬ rescence from a cerebral vessel. Scale bar: 200μΜ; pictures tak^¬ en at lOx magnification.

Fig. 9 shows effect of aluminium oxyhydroxide containing preparations on peripheral monocytes in wt mice. Female C57BL/6 and Balb/c-mice were injected lx (s.c.) with PBS, 0.2mg/ml or 2mg/ml aluminium oxyhydroxide or left untreated (na^'l^'ve) and num^¬ ber of peripheral monocytes was assessed by FACS analysis 24h (A.. C57B1/6; B.. Balb/c) and 48h after injection (C. Balb/c) . Phagocytic activity was assessed 48h after injection in Balb/c mice (D) . *..p<0.05 compared to na^'l^'ve animals)

Fig. 10 shows effect of aluminium salt containing prepara^¬ tions on peripheral monocytes and central microglia and mono^¬ cytes in Tg2576 mice (tg and wt littermates) analysed by FACS analysis. Tg2576 mice were injected 6x (s.c.) with PBS, 2mg/ml aluminium oxyhydroxide, 2mg/ml aluminium phosphate or MPLA and wt littermates were kept untreated as control animals. The num^¬ ber of peripheral monocytes (A) , microglia (B) , infiltrating monocytes (C) , activated cells (D) , Ml (E) and M2 positive cells (F) was assessed 4 weeks after the 6^th injection. EXAMPLES :

1. Excerpt of an AD clinical trial (AFF006; Eudract : 2009- 016504-22)

Materials and Methods :

Data supporting the invention are derived from a randomized clinical trial in early AD patients. The study (AFF006; Eudract: 2009-016504-22) randomized early AD patients into 5 treatment arms. Patients of 2 study arms received either 1 mg aluminium or 2 mg aluminium. In total, 99 early AD patients were enrolled in^¬ to the 2 study arms. Participation of a given patient lasted 18 months .

Study design:

AFF006 was conducted as a randomized, placebo-controlled, parallel group, double-blind, multi-center phase II study and assessed the clinical and immunological activity as well as the safety and tolerability of repeated s.c. administrations of i.a. aluminium (different doses) in patients with early AD, as de^¬ fined in the protocol. It was performed in a total of 6 coun^¬ tries: Austria, France, Germany, Slovakia, Czech Republic and Croatia .

The clinical trial comprised 10 regular outpatient visits and 6 telephone interviews. Up to four weeks before start of treatment, a screening visit (Visit 1) was performed to ensure suitability of the patients for the clinical trial and to estab^¬ lish the patients' baseline characteristics. Following screen^¬ ing, eligible patients were randomly allocated to the treatment groups. After randomization at week 0, patients received 6 in^¬ jections with either 1 or 2 mg aluminium. Injections were applied s.c. by the investigator at weeks 0, 4, 8, 12, 40 and 65 (Visit 2, 3, 4, 5, 7 and 9) .

At Visits 2, 3, 4, 5, 6, 7 and 9 possible local and systemic reactions to the vaccine and vital signs (blood pressure, heart rate, respiratory rate and body temperature) were assessed. In addition, a physical and neurological examination was performed. Efficacy parameters were assessed at Visits 1, 2, 3, 5, 6, 7, 8, 9, 10. The final visit (Visit 10) was performed twelve weeks af- ter the last administration of study drug (Visit 9) . An early discontinuation visit (EDV) was performed when a patient discontinued from the clinical trial.

Study population

The study was done in patients with early AD. Diagnosis was defined by the following criteria:

probable Alzheimer's disease as defined by NINCDS/ADRDA cri^¬ teria (1)

- MMSE score >20 (2)

result of Free and Cued Selective Reminding Test (FCSRT) re^¬ sult of total recall ≤40 or free recall ≤17, indicating hippocampal damage impairing the patient's episodic memory (3)

the result of a centrally read MRI of a patient's brain must be compatible with the diagnosis AD, in particular, pres^¬ ence of a medial temporal lobe atrophy (Scheltens Score ≥2) (4)

Other in-/exclusion criteria applied (e.g., written informed consent; age between 50 and 80 years, treatment with immunosup^¬ pressive drugs (exclusion) ) .

Administration of study drug

During the study Visits 2, 3, 4, 5, 7 and 9 the patient re^¬ ceived study drug by the investigator, in total: six injections over a 65-week treatment period. Injections were applied to the external surface of the upper arm, approximately 8-10 cm above the elbow. Prerequisite regarding the actual site was the pres^¬ ence of an intact regional lymph node station. If the draining lymph node stations of both upper arms were not intact, injec^¬ tion was placed into the thigh close to the inguinal lymph nodes. Two alternating injection sites (e.g. left and right up^¬ per arm, left upper arm and left thigh) were used throughout the 6 injections.

Injections were applied to the subcutaneous tissue (s.c). Special care was taken to avoid intravasal application by care^¬ ful aspiration before each injection. All administrations were performed at the trial site.

Volume-based morphometry Hippocampus (left and right) , and whole lateral ventricle ROIs were delineated on an anatomical MRI template in order to generate the atlas for volumetric measures. The volumes of the hippocampus and lateral ventricles for each subject were deter^¬ mined using a fully-automated method which combines transfor^¬ mations derived from the nonlinear registration of the atlas la^¬ bels to individual subject scans and subject-specific image in^¬ formation (Collins et al . , J. Comput . Assist. Tomogr., 18: 192- 205, 1994). Lateral ventricle and hippocampal segmentations that failed post-processing QC review were manually corrected. The total intracranial volume (TIV) was estimated from the brain mask generated during pre-processing and the average TIV (TIV_avg) for each subject was determined by averaging the estimated TIV across visits. The normalization factor (TIV_tem_pi_ate/TIV_avg_subj ect ) was used to normalize the hippocampal and ventricular volumes for each subject in order to account for differences in head size .

Safety assessments:

Safety evaluations included the following:

- adverse events (AEs) and serious adverse events (SAEs) (number of patients who withdrew due to AEs; reason for withdrawal)

- Laboratory assessments: hematology, biochemistry, coagulation, serology, urinalysis, APP crossreactivity

- vital signs (blood pressure, heart rate, respiratory rate and body temperature)

- physical and neurological examination

Efficacy assessments:

The primary efficacy variables are the change from baseline (CFB) in cognition as measured by an adapted ADAS-cog, CFB in function as measured by an adapted ADCS-ADL and a combination of CFB in cognition and function as measured by a combined compo^¬ site:

1. Co-Primary: Adapted ADAS-cog;

2. Co-Primary: Adapted ADCS-ADL;

3. Combined Primary Outcome: Composite.

ADAS-cog and other items included in the adapted ADAS-cog were measured at Visits 1, 2, 3, 5, 6, 7, 8, 9 and 10 or EDV. ADCS-ADL were measured at Visits 2, 5, 6, 7, 8, 9 and 10 or EDV . Items that contributing to the combined primary outcome were measured at Visits 2, 5, 6, 7, 8, 9 and 10 or EDV.

The primary efficacy outcomes all range from 0 to 100. For each adapted scale and composite, a lower score indicates better performance. However, some items included in a scale may be op^¬ posite in direction, i.e. a higher score indicates better performance. Before a composite was calculated, contributing items that are scored in the opposite direction were reversed. An item is reversed in score by subtracting the observed value from the maximum possible value for the item. This reverses the scale of the items so that a lower score now indicates better perfor^¬ mance. The following items included in the adapted ADAS-cog and combined composite require reverse scoring: Verbal PAL, NTB Cat^¬ egory Fluency and CogState ONB.

Secondary Efficacy Outcomes:

Quality of Life (QOL) caregiver

QOL caregiver is a brief, 13-item questionnaire designed to specifically obtain a rating of the QOL of the patient from the caregiver's perspective. Questions cover relationships with friends and family, concerns about finances, physical condition, mood, and an overall assessment of life quality. All items are rated on a four-point scale, with 1 being poor and 4 being ex^¬ cellent. The total score is the sum of all items, which can range from 13 to 52. QOL caregiver values are presented here as the change from baseline. Outcomes were measured at Visits 1,6, 8, and 10.

Statistical analysis

Baseline data

Subjects were described using demographic information and baseline characteristics recorded during the screening phase (Visit 1) .

Demographic information assessed was age, gender, racial group, smoking habits, level of education, height and weight. Subject demographics was summarized by treatment for the Safety, ITT and Per Protocol populations. Primary efficacy analysis

The primary, secondary and exploratory efficacy outcomes were analyzed by comparing change over time between the groups. The efficacy analyses utilized the mixed model described below. The mixed model analysis compared the estimated change from baseline between the 3 vaccine and the 2 aluminium groups in all efficacy outcome scores at each visit. The model used separate repeated measures longitudinal models for each efficacy end- point. This analysis assessed whether or not there is a differ^¬ ence in estimated CFB values between treatment groups.

SAS · PROC MIXED was used to fit a mixed model with repeated measures (MMRM) , with CFB of each of the efficacy outcomes (e.g., Adapted ADAS-cog) as the response variable and the fol^¬ lowing covariates and fixed effects:

^• Age (covariate) ;

Level of Education (fixed effect split into categories of ≤12 years, >12 years) ;

^• Gender (fixed effect) ;

^• Baseline Test Score of Efficacy Parameter (covariate) ;

^• Center (fixed effect) ;

^• Treatment (fixed effect) ;

^• APOEe4 status (fixed effect, positive or negative) ;

Use of AChE Inhibitors (fixed effect, determined from medica^¬ tions) ;

^• Time (covariate, time will be defined in terms of visits) ;

^• Time by Treatment Interaction (Time*Treatment ) ;

The covariance structure for the model was first-order het^¬ erogeneous autoregressive (ARH[1]). Least-squares means were es^¬ timated at each visit in the study. The LS mean at a particular visit was interpreted as the expected CFB in the efficacy out^¬ come at that time point (Visit) when the specified treatment was administered. Least squares means and standard errors were esti^¬ mated from the mixed model at each visit and are shown for the various groups.

The adapted ADAS-cog combines items that assess cognitive function. The adapted ADCS-ADL includes items that are sensitive to functional ability. Cognitive skills are expected to decline toward the beginning of the disease and one's ability to perform basic functions are expected to decline later in the disease. The combined primary outcome (referred to herein as "Composite score") combines both the adapted ADAS-cog and adapted ADCS-ADL to create a Composite score that is sensitive to decline in cog^¬ nitive and basic functions. The following equation is used to derive the combined primary outcome, i.e. combined Composite score :

Combined Composite score:

The percent contribution of each item to the combined Compo^¬ site score can be found in Table 1 below:

Item Percent Contribution

ADAS-cog Word Recall 16.6

ADAS-cog Orientation 10.8

ADAS-cog Word Recognition 17.0

ADAS-cog Recall Instructions 2.8

ADAS-cog Spoken Language 4.1

ADAS-cog Word Finding 5.1

CogState One-Back Memory 8.5

NTB VPAL 8.5

NTB Category Fluency 8.5

ADCS-ADL Belongings 0.8

ADCS-ADL Shopping 1.4

ADCS-ADL Hobbies 0.7

ADCS-ADL Beverage 1.1

ADCS-ADL Meal 1.5

ADCS-ADL Current Events 0.7

ADCS-ADL TV 0.8

ADCS-ADL Keeping Appointments 1.0

ADCS-ADL Travel 1.5

ADCS-ADL Alone 1.0 ADCS-ADL Appliance 1.4

ADCS-ADL Clothes 1.5

ADCS-ADL Read 0.7

ADCS-ADL Telephone 3.1

ADCS-ADL Writing 1.0

Results

AFF006 recruited a study population reminiscent of early AD patients based on demographic data (Table 2) and data showing the baseline characteristics of the study groups (Table 3) .

Both the frequency and the intensity of the local reactions depend on the aluminium dose administered (Table 4) . Such local reactions (LR) serve as a measure of the activation of the in^¬ nate immune response.

2 mg aluminium group compares favourably even to the 1 mg aluminium group (other groups) with regard to parameters inform^¬ ing on the progression of the disease (Fig. 1) . Comparison of the mild population of patients of both groups showed that this effect is most pronounced in the cohort of patients in earlier disease stages (Fig. 2) . Slowing of disease progression over 18 months is specifically apparent in the 2 mg aluminium group, ex^¬ emplified with Adapted ADAS-cog (Fig. 3) .

Results obtained were compared to public datasets. Histori^¬ cal datasets identified were the ADNI 1 mild AD cohort (observa^¬ tional study) , the mild placebo patients from the ADCS Homocys^¬ teine trial (HC, MMSE>=20) and the placebo group from the ADCS NSAID study of Rofecoxib and Naproxen (NS, MMSE>=20) . These 3 cohorts were combined to yield the historical control (HC- ADNI,NS;HC) . Data points were available for 344 patients at month 6, 317 patients at month 12 and 226 patients at month 18. The ADNI trial only performed assessments at 6, 12 and 24 months, so the 18 month value was imputed with a straight line. The NS study was only 12 months long, so no 18 month data was available from this study.

Although the adapted ADAS-cog used some items from the ADAS- cog supplemented with items from the NTB and the CogState Bat^¬ tery, these items were not available for all of the historical studies. So, an adapted ADAS-cog 2 was created which used the same weightings as the adapted ADAS-cog for the ADAS-cog items, but did not include the NTB and CogState items (1.67*Word recall + 1.35*Orientation + 1.42*Word Recognition + 0.55*Recall In^¬ structions + 0.81*Spoken Language + 1.01*Word Finding).

The adapted ADAS-cog2 shows substantially more decline in the historical control group than the 1 and 2 mg aluminium oxyhy- droxide treated groups from the AFF006 study (Figure 3) . The p- values were: 1 mg vs. HC-ADNI, NS, HC : <0.0001; 2 mg vs. HC- ADNI, NS, HC: <0.0001. Also the MRI data show a statistically significant disease modifying effect for the 2 mg group of pa^¬ tients and a correlation of the hippocampus volume with clinical endpoints, e.g. right hippocampus with adapADAS : p=0.0006 or Composite score: p=0.0095) (Fig. 4). It has to be specifically mentioned that the present investigation has provided for the first time a parallel development of clinical data with a radio^¬ logic biomarker (MRI in the present case) ) .

Fig. 4 shows that the patients treated according to the pre^¬ sent invention showed almost no AD related reduction in hippo^¬ campus volume over a period of 18 months whereas the rate of brain atrophy per year in AD patients is in the range of 3 to 6 % per year (Risacher et al . , 2013, Table 2; the rate in healthy elderly individuals is usually in the range of 0.5 to 2.2 (see also this table 2 in Risacher et al . ) .

Fig. 5 shows that caregivers of patients treated according to the present invention rated the QOL of the patient as signif^¬ icantly improved over a period of 18 months following 2mg com^¬ pared to lmg Alum and other groups (not shown) .

Table 2: Patient Population and Disposition

lmg 2mg

Patient Disposition

(N=48) (N=51)

Number of Subjects n (%)

Completed 41 ( 85.4%) 45 ( 88.2%)

Discontinued 7 ( 14.6%) 6 ( 11.8%)

P-value¹

Reason for Discontinuation from the Study:

Death 2 ( 4.2%) 0 ( 0.0%) Adverse Event 0 ( 0.0%) 0 ( 0.0%)

Withdrawal by Subject 4 ( 8.3%) 5 ( 9.8%)

Lost to Follow-up 0 ( 0.0%) 0 ( 0.0%)

Other 1 ( 2.1%) 1 ( 2.0%)

Demographics - Race, Gender, Education, Age lmg 2mg

Demographics

(N=48) (N=51)

Race

Asian / Pacific Islan0 ( 0.0%) 1 ( 2.0%)

der

Caucasian 48 (100.0%) 50 ( 98.0%)

Gender

Male 28 ( 58.3%) 19 ( 37.3%)

Female 20 ( 41.7%) 32 ( 62.7%)

P-value¹

Education

Years

Mean (SD) 12.3 (4.03) 11.8 (3.18)

Median 12 11

(Ql, Q3) (9.0, 15.0) (10.0, 13.0)

Min, Max 8, 26 6, 22

P-value¹

Age (yrs)

n 48 51

Mean (SD) 70.3 (6.56) 68.9 (8.36)

Median 71 69

(Ql, Q3) (65.0, 75.5) (64.0, 77.0)

Min, Max 57, 80 50, 80

P-value¹

Weight (kg)

n 48 51

Mean (SD) 70.45 (10.375) 67.62 (13.700)

Median 70.5 65 (Ql, Q3) (64.00, 77.70) (57.00, 78.00)

Min, Max 47.5, 101.0 45.0, 100.0

P-value¹

BMI (kg/m²)

n 48 51

Mean (SD) 24.66 (2.903) 24.81 (3.627)

Median 24.8 24.2

(Ql, Q3) (22.95, 26.15) (22.30, 27.30)

Min, Max 17.8, 31.2 18.2, 35.4

P-value¹

Table 4 : Adverse Event Summary of Local Reactions

MedDRA System Organ Class

lmg 2mg

Preferred Term

(N=48) (N=51)

Number of subjects with re^¬

31 ( 64.6%) 42 ( 82.4%) ported adverse event

Number of unique events 96 162

General Disorders and Admin^¬

31( 64.6%), 209 42 ( 82.4%), 487 istration Site Conditions

Injection Site Erythema 26 ( 54.2%), 64 37 ( 72.5%), 143

Injection Site Swelling 13 ( 27.1%) , 27 26 ( 51.0%) , 86

Injection Site Warmth 18 ( 37.5%), 31 25 ( 49.0%), 67

Injection Site Induration 13 ( 27.1%) , 32 14 ( 27.5%) , 34

Injection Site Pain 14 ( 29.2%) , 41 31 ( 60.8%), 99

Injection Site Pruritus 4 ( 8.3%), 5 10 ( 19.6%), 17

Injection Site Nodule 4 ( 8.3%), 5 11 ( 21.6%) , 31

Injection Site Hypersensiti^¬

2 ( 4.2%), 2 4 ( 7.8%), 9 vity

Injection Site Haematoma 2 ( 4.2%), 2 1 ( 2.0%), 1

Injection Site Discolouration 0 ( 0.0%), 0 0 ( 0.0%), 0

Injection Site Inflammation 0 ( 0.0%), 0 0 ( 0.0%), 0

Injection Site Reaction 0 ( 0.0%), 0 0 ( 0.0%), 0

Fatigue 0 ( 0.0%), 0 0 ( 0.0%), 0

Feeling Hot 0 ( 0.0%), 0 0 ( 0.0%), 0 Hypothermia 0 ( 0.0%), 0 0 ( 0.0%), 0

Injection Site Urticaria 0 ( 0.0%), 0 0 ( 0.0%), 0

Pyrexia 0 ( 0.0%), 0 0 ( 0.0%), 0

Investigations: Lymph Node

0 ( 0.0%), 0 0 ( 0.0%), 0 Palpable

Investigations: Body Tempera^¬

0 ( 0.0%), 0 0 ( 0.0%), 0 ture Increased

Blood and Lymphatic System

0 ( 0.0%), 0 1 ( 2.0%), 1 Disorders: Lymphadenopathy

Gastrointestinal Disorders:

0 ( 0.0%), 0 1 ( 2.0%), 1 Glossitis

Gastrointestinal Disorders:

0 ( 0.0%), 0 0 ( 0.0%), 0 Nausea

Gastrointestinal Disorders:

0 ( 0.0%), 0 0 ( 0.0%), 0 Vomiting

Nervous System Disorders: Pa-

0 ( 0.0%), 0 0 ( 0.0%), 0 raesthesia

Nervous System Disorders: Diz^¬

0 ( 0.0%), 0 0 ( 0.0%), 0 ziness

Cardiac Disorders: Cyanosis 0 ( 0.0%), 0 0 ( 0.0%), 0

Infections and Infestations:

0 ( 0.0%), 0 0 ( 0.0%), 0 Rash Pustular

Musculoskeletal and Connective

Tissue Disorders: Pain in Ex^¬ 0 ( 0.0%), 0 1 ( 2.0%), 1 tremity

Psychiatric Disorders: Tension 0 ( 0.0%), 0 0 ( 0.0%), 0

Vascular Disorders: Haematoma 0 ( 0.0%), 0 0 ( 0.0%), 0

2. Immunogenicity of two Αβ targeting vaccines SeqID 1-KLH-Alum and SeqID 2-KLH Alum in comparison to KLH-Alum and Alum only

SeqIDs :

SeqID 1: SWEFRTC

SeqID 2: SEFKHGC

Animal experiments :

All animal experiments were performed in accordance with the Austrian Animal Experiments Act (TVG2012) using Tg2576-mice (Ta- conic Farms, USA; 129S 6/SvEvTac) . General health was checked by modified Smith Kline Beecham, Harwell, Imperial College, Royal London Hospital, phenotype assessment (SHIRPA) primary observa^¬ tional screen (Rogers DC et al . (1999) Behav Brain Res 105: 207- 217.) . Mice were injected s.c. 6 times in monthly intervals. Blood was taken in regular intervals, plasma prepared and stored until further use. At study end mice were sacrificed, brains were collected and hemispheres separated. One hemisphere was fixed in 4 "6 Paraformaldehyde (PFA, Sigma Aldrich, USA), dehydrat^¬ ed and paraffin-embedded. Brain tissue was sectioned at 7μΜ us^¬ ing a sliding microtome (Leitz, Germany) and sections were mounted on Superfrost Plus Slides (Menzel, Germany) .

Titer determination by ELISA:

Standard enzyme-linked immunosorbent assay (ELISA) technolo^¬ gy was used to measure levels of vaccine-induced antibodies in plasma and CSF (Mandler M et al . (2012) J Alzheimers Dis 28: 783-794.). Substrates used include human (BACHEM, CH) Αβ1-40/42 (at 5μg/ml) , KLH (^g/ml) and peptide-Bovine serum albumin (BSA) conjugates (SeqID 1 and SeqID 2, ΙμΜ) . Optical density (OD) was measured at 405nm using a micro-well reader (Tecan, CH) . ODmax/2 was calculated.

Behavioral tests:

To analyse cognitive dysfunction, immunised Tg2576 animals were subjected to contextual fear conditioning (CFC, Comery TA et al. (2005) J Neurosci 25: 8898-8902.), analyzed using AnyMaze software (Stoelting Co, USA) . For CFC, on day 1 mice were placed in the conditioning chamber (AFFiRiS AG, Austria) , allowed to habituate for 2 min. and received three 0.8mA foot-shocks in 2 min intervals plus 30s rest. To assess contextual learning on day 2, animals were readmitted to the chamber and monitored for 5 min. with sl20-240 chosen as time frame for analysis (time freezing = lack of movement except for respiration) . The first two minutes of day 1 were considered as baseline-freezing which was subtracted from day 2 values.

Analysis of cerebral Αβ :

Immunofluorescence (IF) analysis was done as described pre^¬ viously (Mandler M et al . (2012) J Alzheimers Dis 28: 783-794). For Αβ-specific IF-staining, brain sections of immunized Tg2576 were processed for analysis of amyloid load using mAb 3A5 (AF- FiRiS AG, Austria) . All secondary reagents used were obtained from Vector Labs (USA) . For IF, sections were mounted and coun- terstained using DAPI-containing VECTASHIELD-HardSet Mounting Medium. Sections were examined using MIRAX-SCAN (Carl Zeiss AG, Germany) . AD-like pathology in animals was assessed by determin^¬ ing the relative cerebral area occupied by amyloid deposits us^¬ ing a semi-automated area recognition program (eDefiniens Archi^¬ tect XD; www . definiens . com, Mandler M. et al (2015) PLoS ONE 10(1): e0115237.). For analysis three slides/animal and ≤ five individual sections/slide were assessed. Sections carrying tis^¬ sue artifacts or aberrant staining were excluded. To assess the number of Αβ-positive vessels, 3A5 stained sections (3 slides/animal covering cortex and hippocampus and up to five in^¬ dividual sections per slide) have been analysed. Αβ-positive vessels were manually counted in sub-regions of the cortex as well as in the hippocampus. Number of positive vessels per mm² was determined.

References :

Rogers et al . , Behav Brain Res 105 (1999): 207-217.

Mandler et al . , PLoS ONE 10(1) (2015): e0115237. doi:10.1371/journal. pone.0115237.

Mandler et al . , J Alzheimers Dis 28: 783-794.

Comery et al . , J Neurosci 25 (2005): 8898-8902.

Results :

To test the immunogenicity of two Αβ targeting vaccines Se- qlD 1-KLH-Alum and SeqID 2-KLH Alum in comparison to KLH-Alum and Alum (Aluminium-oxyhydroxide) only, Tg2576-mice were inject^¬ ed 6x, s.c, at 4-week intervals with either conjugate-vaccine containing 30yg net peptide, equivalent doses of KLH formulated with Alum or Alum only. Alum doses used were equivalent to 2mg/ml. Vaccination induced Abs were measured in plasma samples taken at sacrification (SeqID 1 (n=10), SeqID 2 (n=8), KLH-Alum (n=10) and Alum only (n=8)) . All 3 vaccines elicited strong and comparable IgG titers towards the peptide used for immunization (Fig 6A) . Alum only did not elicit signals above background (Fig 6A) . Both Αβ targeting vaccines, SeqID 1-KLH-Alum and SeqID 2- KLH-Alum, elicited Abs to human Αβ whereas KLH-Alum vaccine and Alum only did not elicit signals above background in treated an^¬ imals (Fig 6B) .

To evaluate the effect of Aluminum-oxyhydroxide only (Alum) in comparison to Αβ targeting vaccines (SeqID 1- + SeqID 2-KLH- Alum) and non Αβ specific vaccines (KLH-Alum) on cognitive func^¬ tions, we applied Contextual Fear Conditioning (CFC) analyzing contextual memory and learning in Tg2576-mice. As expected, CFC demonstrated that SeqID 1- and SeqID 2-treated mice were superi^¬ or to control animals receiving KLH-Alum (thus not eliciting an Αβ specific immune response) in this AD model of Αβ deposition (Fig. 7) . Interestingly, animals receiving Alum only, (without a conjugate eliciting an active immune response against KLH or Αβ, respectively) , showed similar effects as detectable with Αβ tar^¬ geting vaccines in this AD model in the absence of Αβ-specific antibodies .

To test whether Alum would also significantly influence cer^¬ ebral amyloid load, animals undergoing CFC were subsequently sacrificed at 14 months of age. Their brains were assessed for diffuse and dense-cored plaques by IF-staining using monoclonal antibody 3A5. Cortical as well as hippocampal sections of KLH/ALUM-inj ected controls were covered by numerous amyloid plaques (Fig.8A+C) . By contrast, respective brain areas of SeqID 1- and SeqID 2-immunized Tg2576-mice contained significantly less deposits (Fig. 8B+D and E, p<0.05 and data not shown) . Im^¬ portantly, treatment of Tg2576 animals with Alum only did not significantly alter amyloid deposition as compared to KLH-Alum treated animals (Fig.8 E) in this AD model.

Thus, Figs. 7 and 8 also disclose that topically applied al- uminium-oxyhydroxide is able to lower cognitive decline signifi^¬ cantly in an APP-transgenic model for Alzheimer's disease (Tg2576) without significantly changing cerebral Αβ levels. This is implying an ΑΡΡ/Αβ independent mechanism underlying beneficial functional effects exerted by aluminium-oxyhydroxide in this AD model and further evidences the lack of scientific plau^¬ sibility of the "amyloid channel hypothesis". 3. Preclinical analysis

3.1. Effect of aluminium oxyhydroxide containing preparations on bone marrow derived macrophages/monocytes :

To investigate whether aluminium oxyhydroxide containing preparations are contributing to alterations of bone marrow de^¬ rived macrophages/monocytes in vitro studies are performed using these preparations as stimulants for primary murine bone marrow derived macrophages in vitro and subsequently measure the re^¬ spective phagocytic activity (e.g. uptake of fluorescent beads coupled with recombinant human Αβ; uptake of fluorescently la^¬ belled Αβ-aggregates) , and changes in chemokine/cytokine expres^¬ sion of monocytes/macrophages following aluminium oxyhydroxide stimulation by suitable methods (e.g. a flow cytometry based de^¬ tection method or a bead-based multiplexing technology or elec- trochemiluminescence based multi-array technology and Western Blot analyses) .

Material and methods:

Isolation and in vitro differentiation of macrophages is es^¬ sentially performed as described e.g. in Zhang et al . 2008

[PMID: 19016445] . In brief, tibia- and femur-derived bone marrow cells from 8 to 12 weeks old BALB/c mice or 12 months old APP transgenic mice are divided onto 2-3 10cm-cell culture dishes at a density of 20-30xl0⁶ cells per dish, differentiated 4 days in RPMI/10%FCS+Penicilin/Streptamycin (P/S) in the presence of 20ng/ml M-CSF (RD-Systems) , redistributed at day 5 into 24-well tissue culture plates at a density of 150.000 cells/well until day 9. 24hrs before phagocytosis, cells are starved in 500μ1 RPMI-media +10%FCS +P/S in absence of M-CSF but presence of LPS

(l g/ml) . For phagocytosis cells are incubated in the presence or absence of ascending doses of aluminium oxyhydroxide (Alhydro- gel/Brenntag) containing buffer ranging from 0.1 g/ml to 2 mg/ml for 2h.

Phagocytosis is performed using fluorescently labelled Αβ aggregates: For aggregate formation, fluorescent N-terminally- labelled Αβ1-42 (Hilyte488, Anaspec) is used. As described by An^¬ derson and Webb (BMC Biotechnology 2011, 11:125) labelling of Αβ peptides does not prevent amyloid aggregate formation. There^¬ fore, A i-42-Hilyte-488 (Anaspec, detectable in the FITC-channel ; lmg/ml) is used for aggregate-production. Aggregates are assembled by constant rotation (600 rpm) for 24-48h at room tempera^¬ ture on shaker in PBS .

Subsequently these Αβ-aggregates are added to differentiated and primed macrophages (2h Alum) for lhr at 37°C (0.5 g - 5 g Αβ- aggregates/well) allowing for in vitro uptake by phagocytosis. After washing and scraping the cells in ice cold PBS, cells are washed in FACS-Buffer ( lxPBS+l%BSA) and analysed for fluorescence signal by standard FACS procedures. Cell differentiation efficacy was monitored in parallel using anti F4/80 (Biolegend, Cat-No: B123109) and anti CDllb (Biolegend, Cat-No: B101219) marker antibodies based on the protocol suggested by the manu^¬ facturer .

Zhang et al . [PMID: 19016445]; Curr Protoc Immunol. 2008; CHAPTER: Unit-14.1. "The isolation and characterization of murine macrophages . "

Western blot for detection of changes in the M1/M2 specific chemokine profile is done according to Mandler et al . 2014.

Result :

With these experiments it is shown that exposure to differ^¬ ent increasing doses of a monocyte inducing agent or monocyte activating agent or monocyte recruiting agent, e.g. aluminium salts, such as aluminium oxyhydroxide, leads to a significant change in phagocytic activity of Αβ.

3.2. Effect of aluminium oxyhydroxide containing preparations on microglia/ macrophage cell lines :

To investigate whether aluminium oxyhydroxide containing preparations are contributing to alterations of macrophage- and microglial cell lines we perform in vitro studies using these preparations as stimulants for mouse macrophage- and microglial cell lines in vitro and subsequently measure the respective phagocytic activity (e.g. uptake of fluorescently labelled Αβ- aggregates) , and changes in chemokine/cytokine expression of mi^¬ croglia following aluminium oxyhydroxide stimulation by suitable methods (e.g. a flow cytometry based detection method or a bead- based multiplexing technology or electrochemiluminescence based multi-array technology and Western Blot analyses) .

Material and Methods:

For this purpose following primary microglia cell lines are used :

EOC13.31 (ATCC® No. CRL-2468TM) mouse microglia cell lines defective in TLR4 signaling

Macrophage cell line: J774A.1 (mouse, ATCC® No. TIB-67TM)

Microglia and macrophage cell lines are cultured under standard conditions. Cells are seeded at 150.000 cells/well onto a 24-well tissue culture plate and are incubated 24hrs before phagocytosis with LPS.

For phagocytosis cells are incubated in the presence or ab^¬ sence of ascending doses of aluminium oxyhydroxide (Brenntag) containing buffer ranging from 0.1 g/ml to 2 mg/ml for 2h. Subsequently, Αβ-aggregates are added to macrophages/microglia for lhr at 37°C (0.5μg - 5μg Αβ-aggregates/well) allowing for in vitro uptake by phagocytosis.

FACS Analysis:

Cells are harvested after incubation with different increas^¬ ing concentrations of aluminium oxyhydroxide and Αβ1-42 HiLyte Fluor™488 and analysed for phagocytic activity (uptake of fluo- rescently labelled Αβ1-42). Samples are analysed for fluorescence signal by standard FACS procedures.

Multiplex chemokine/cytokine assay:

Cell extracts and supernatant are prepared from cultivated and stimulated microglia/ macrophage cells and analysed for changes in chemokine/cytokine expression with bead-based multi^¬ plexing technology (Luminex, according to manufacturer's proto^¬ col) or electrochemiluminescence based multi-array technology (MSD, according to manufacturer's protocol) .

Results : With the present experiment, it can be shown that exposure to different increasing doses of a monocyte inducing agent or monocyte activating agent or monocyte recruiting agent, such as aluminium oxyhydroxide, leads to a significant change in phago^¬ cytic activity of Αβ in both cell lines in vitro.

3.3. Effect of aluminium oxyhydroxide containing preparations on peripheral monocytes in wt mice (i.e. representative tests for monocyte inducing/activating properties) :

To investigate whether aluminium oxyhydroxide containing preparations are contributing to alterations of overall periph^¬ eral blood mononuclear cells (PBMCs) and peripheral blood mono^¬ cytes, single and repeated local injections of these prepara^¬ tions (sub-cutaneous) into na^'l^'ve, wt mice are performed and the respective changes in the number of PBMC cell types and monocyte number, induction of monocyte sub-populations and monocyte phag^¬ ocytic activity (e.g. uptake of fluorescently labelled Αβ- aggregates) are measured by a flow cytometry based detection method .

Methods :

For this purpose, C57BL/6 and BALB/c female mice (Janvier) are injected once to three times (at Day 0, 14 and 21) with alu^¬ minium oxyhydroxide in ascending doses (0,1 - 10 mg/ml; s.c. or i .p . ) .

For detection of alterations in PBMCs, peripheral blood is withdrawn from injected mice via cardiac puncture with EDTA as anticoagulant. Aliquots of 50 μΐ whole blood are treated with FC blocking cocktail (CD16/CD32; BD Fc Block™ by BD Biosciences) for 10 min on ice. FC-blocked cells are suspended in cell stain^¬ ing buffer (eBioscience) with a combination of the following directly conjugated antibodies at their pre-determined optimal concentration (as described by Brockman et al . , 2009) for 30 min .

CD3-PE, CD4-PerCp-Cy5.5, CD8-FITC, CD19-APC-Cy7 , CD3_PE- Cy7/CD49b-FITC and CD115-APC/Ly6C-FITC (BD Biosciences) . After the 30 min incubation time, red blood cells were lysed with a commercial RBC lysis solution (Red Blood Cell Lysis Solution, Miltenyi. Cells are finally washed three times and re-suspended in cell staining buffer (eBioscience) . Fluorescence minus one (FMO) controls are always included in the assays for fluorescent compensation setting. Samples are acquired on a flow cytometer (BD FACSCanto II) and data analyzed with the FACSDiva software (BD Biosciences) . Monocytes are identified by their Side/Forward scatter properties and gated as CD115+cells.

In the live cell gate, doublet exclusion is sequentially performed for FSC (FSC-W vs FSC-H plot) and SSC (SSC-W vs SSC-H plot) . CD4 T cells are gated as (CD3+CD4+) , CD8 T cells are gat^¬ ed as (CD3+CD8+ ),B cells are gated as (CD3- CD19+ ) and NK cells are gated as (CD3- CD49b+) .

For detection of alteration in monocytes and induction of monocyte sub-populations, inflammatory monocytes

( (CD115+/Ly6Chigh) and patrolling monocytes (CD115+/Ly6Clow) are distinguished by the expression of Ly6C.

For alterations in monocyte phagocytic activity, 24-48 hours after the last injection, 25 g per mouse of fluorescent HiLyte Fluor™ 488-labeled Amyloid β 1-42 (Anaspec, Fremont, CA) is in^¬ jected in the tail vein. Peripheral blood is drawn from treated mice via cardiac puncture with EDTA as anticoagulant, 30 min af^¬ ter i.v. injection of the Αβ1-42 HiLyte Fluor™ 488. Aliquots of 50 μΐ whole blood are treated with FC blocking cocktail (CD16/CD32; BD Fc Block™ by BD Biosciences) for 10 min on ice. FC-blocked cells are suspended in cell staining buffer (eBiosci^¬ ence) and incubated for 30 min with CD115-APC. After the 30 min incubation time, red blood cells were lysed with a commercial RBC lysis solution (Red Blood Cell Lysis Solution, Miltenyi . Cells are finally washed three times and re-suspended in cell staining buffer (eBioscience) .

Αβ uptake is assessed by reporting the percentage and Mean Fluo^¬ rescence Intensity (GeoMean) of positive HiLyte fluor™ 488 Αβΐ- 42 cells among CD115+ cells.

Results :

With the present experiment, an increase in peripheral mono^¬ cytes following injection of aluminium oxyhydroxide (or other agents according to the present invention) application s.c can be demonstrated. As shown in Figure 9, C57B1/6 and Balb/c mice were injected once (s.c.) and blood was withdrawn for PBMC anal^¬ ysis. Analysis of CD115+ Monocytes showed an increase in the number of CD115+ cells 24h after injection (p<0.05 compared to na^'l^'ve, untreated animals) which was still detectable at 48h after the aluminium oxyhydroxide application (similar results in both mouse strains used, (Fig.9 A-C) . No change in the number of T- cells (CD4+ and CD8+ ) , B-cells or NK cells was detectable fol^¬ lowing injection (not shown) .

In addition it can be shown that exposure to different in^¬ creasing doses of aluminium oxyhydroxide leads to a change in phagocytic activity of Αβ, 48h after injection (Fig.9 D) support^¬ ing the role of Aluminium salts as monocyte inducing/monocyte activating agents in vivo.

3.4. Effect of aluminium oxyhydroxide containing preparations on peripheral monocytes in an animal model for AD (i.e. representative tests for monocyte inducing/activating properties) :

To investigate whether aluminium oxyhydroxide containing preparations are contributing to alterations of PMBCs and pe^¬ ripheral blood monocytes in an AD animal model, repeated local injections of these preparations (sub-cutaneous) into na^'l^'ve mice overexpressing human APP were performed and the respective changes in the number of PBMC cell types and monocyte number, induction of monocyte sub-populations and monocyte phagocytic activity (e.g. uptake of fluorescently labelled Αβ-aggregates ) were measured by a flow cytometry based detection method.

Methods :

For this purpose, APP transgenic mice (Tg2576-mice (Taconic Farms, USA; C57BL/6, 6 months at start of the experiment) or C57BL/6-wt mice ( littermates ) are injected six times (monthly intervals) with PBS only, aluminium oxyhydroxide (0,2 - 2 mg/ml; s.c), aluminium phosphate (2 mg/ml; s.c.) or MPLA (5 g/ml, as positive control) .

For detection of alterations in PBMCs, peripheral blood is withdrawn from injected mice via cardiac puncture with EDTA as anticoagulant. Aliquots of 50 μΐ whole blood are treated with FC blocking cocktail (CD16/CD32; BD Fc Block™ by BD Biosciences) for 10 min on ice. FC-blocked cells are suspended in cell stain- ing buffer (eBioscience) with a combination of the following directly conjugated antibodies at their pre-determined optimal concentration (as described by Brockman et al . , 2009) for 30 min .

CD3-PE, CD4-PerCp-Cy5.5, CD8-FITC, CD19-APC-Cy7 , CD3_PE- Cy7/CD49b-FITC and CD115-APC/Ly6C-FITC (BD Biosciences) . After the 30 min incubation time, red blood cells were lysed with a commercial RBC lysis solution (Red Blood Cell Lysis Solution, Miltenyi. Cells are finally washed three times and re-suspended in cell staining buffer (eBioscience) . Fluorescence minus one

(FMO) controls are always included in the assays for fluorescent compensation setting. Samples are acquired on a flow cytometer

(BD FACSCanto II) and data analyzed with the FACSDiva software

(BD Biosciences) . Monocytes are identified by their Side/Forward scatter properties and gated as CD115+cells.

Results :

The results of the present example can demonstrate an in^¬ crease in peripheral monocytes following injection after aluminium oxyhydroxide and aluminium phosphate (or another agent ac^¬ cording to the present invention) application s.c (Fig.lOA) supporting the role of Aluminium salts as monocyte induc- ing/monocyte activating agents in a model of AD. No change in the number of T-cells (CD4+ and CD8+) , B-cells or NK cells was detectable following injection (not shown).

3.5. Effect of aluminium oxyhydroxide containing preparations on CNS macrophages (i.e. brain derived microglia and bone marrow derived monocytes) in an animal model for AD (i.e. representative tests for monocyte inducing/activating/recruiting proper- ties ) :

To investigate whether aluminium oxyhydroxide containing preparations are contributing to alterations of CNS macrophages, especially also on recruitment of peripheral monocytes to the brain, single and repeated local injections of these prepara^¬ tions (e.g. sub-cutaneous) into na^'l^'ve, APP transgenic mice are performed and subsequently brains of these animals are isolated. The brains are then subjected to an analysis of changes in mac^¬ rophage number (i.e. total number of brain derived microglia and bone marrow derived monocytes) , differential changes in number of microglia, bone marrow derived macrophages/monocytes, and in sub-populations of microglia/monocytes (e.g. M1/M2 status) by suitable methods (e.g. a flow cytometry based detection method or a bead-based multiplexing technology or electrochemilumines- cence based multi-array technology and Western Blot analyses) .

Method:

At study end (4 weeks after the last injection) mice were sacrificed, and perfused. For flow cytometry analysis perfused brain is dissected, and mononuclear cells are separated over discontinuous 70%/30% percoll gradients as previously described in Pino PA and Cardona AE 2011. Cellular pellets are resuspended in cell staining buffer (eBioscience) . Isolated cells are incu^¬ bated on ice for 5 min with anti-mouse CD16/CD32 (BD Fc Block™ by BD Biosciences) to block Fc receptors, and then incubated on ice for 30 min with the following mix of fluorochrome-conj ugated anti-mouse antibodies: for microglia identification: CD45-PeCy7 (eBioscience), CX3CR1-Alexa Flour 488 (R&D Systems) and CCR2-APC (R&D Systems; for MHCII activation and neutrophil detection: CD45-PeCy7 (eBioscience, CDllb-APC (BD Pharmingen) , Ly6G-PE (BD Pharmingen) and MHCII-FITC (eBioscience); for M1/M2 activation: CD45-PeCy7 (eBioscience, CDllb-PE (BD Pharmingen), CD86-APC (BD Pharmingen) and CD206-Alexa Flour 488 (R&D Systems) . Cells are finally washed three times and re-suspended in cell staining buffer (eBioscience) . Fluorescence minus one (FMO) controls are always included in the assays for fluorescent compensation set^¬ ting. Samples are acquired on a flow cytometer (BD FACSCanto II) and data analyzed with the FACSDiva software (BD Biosciences) .

Brain microglia are analyzed for CD45 expression and periph^¬ erally derived myeloid cells (CX3CR1-/CCR2+) were distinguished from resident CD45+ cells (CX3CR1+/CCR2-) via expression of their respective markers. Enhanced mononuclear cell activation is determined by increased expression of MHCII on CD45+ brain mononuclear cells.

For evaluation of the expression of M1/M2 markers, the expression level of CD86 (Ml) or CD206(M2) on CD45+CDllb+ micro^¬ glia has been analyzed.

Result :

Administration of a monocyte inducing agent or monocyte ac^¬ tivating agent or monocyte recruiting agent, such as aluminium oxyhydroxide and aluminium phosphate, leads to a slight change in the number of infiltrating CCR2+ monocytes in the brain. In addition, specific changes in the activation- as well as M1/M2 status are detectable in cerebral macrophages upon aluminium ox^¬ yhydroxide and aluminium phosphate injection:

As shown in Fig.lOB, Tg2576 mice (background: C57BL/6) do not show differences in the number of CX3CR1+ microglia as com^¬ pared to wt littermates at 12 months of age. In addition, Tg2576 mice (background: C57BL/6) undergoing 6 monthly injections of aluminium oxyhydroxide and aluminium phosphate (using doses of 2mg/ml or 0.2mg/ml) or MPLA, a TLR4 agonist (5 g/ml) , do not develop changes in CX3CR1+ cells as compared to wt or PBS-treated Tg2576 mice, respectively.

Analysis of CCR2+, infiltrating monocytic cells (Fig. IOC) revealed that 12 months old Tg2576 mice only develop a slight reduction in the number of CCR2+ cells in the brain as compared to wt littermates in this experiment. As expected, treatment with the well-known monocyte activating/recruiting agent MPLA leads to a reversion to wt levels in transgenic animals. Inter^¬ estingly, aluminium oxyhydroxide and aluminium phosphate treat^¬ ment also changes CCR2+ numbers and treated animals show a slightly increased level, similar to wt littermates in this ex^¬ periment supporting a role of Aluminium salts as monocyte re^¬ cruiting agents in vivo.

Analysis of activation- and M1/M2 status of brain-derived macrophages revealed that aluminium salt treatment using doses suitable for human use, leads to a mild increase in activation of cerebral mononuclear cells (Fig. 10D) in Tg2576 mice, and a mild change in Ml-positive cells (Fig. 10E) . M2-status is not changed in these animals irrespective of genotype or treatment (Fig. 10F) supporting the role of Aluminium salts as monocyte activating agents in vivo.

It follows that the present invention discloses the follow^¬ ing individually preferred embodiments:

1. Monocyte inducing agent and/or a monocyte activating agent and/or a monocyte recruiting agent for use in the treatment and prevention of dementias associated with β-amyloid deposition, preferably Alzheimer's Disease (AD).

2. Monocyte inducing agent and/or a monocyte activating agent and/or a monocyte recruiting agent for use according to embodiment 1, wherein the monocytes are Grl ⁽⁺⁾ /CD1 lb ⁽⁺⁾ cells, CDllb⁺CX₃CRl^lowCCR2⁺CXCR4^high cells or CD14⁺⁺CD16^" cells.

3. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to embodiment 1, se^¬ lected from the group consisting of M-CSF, GM-CSF, IFN-gamma, TGF-beta, TNF-alpha, IL-l,IL-2, IL-4, IL-5, IL-6, 11-10, IL-13, Fractalkine/CX3CL1 , members of the Chemokine (CC-motif) ligand family (CCL) , especially CCL2, CC13, CCL4, CCL12, members of the IL-8 family, especially CXCl-8 (11-8), Toll-like receptor ago^¬ nists, especially TLR4 agonists, preferably LPS or MPLA and an aluminium salt.

4. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of embodi^¬ ments 1 to 3, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is administered subcutaneously, intra-cranially or into the bone marrow. 5. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to any one of embodi^¬ ments 1 to 3, wherein the monocytes are human phagocytes, espe^¬ cially autologous phagocytes of the AD patient to whom the mono^¬ cytes are administered for treating AD.

6. Monocyte inducing agent and/or a monocyte activating agent and/or a monocyte recruiting agent for use according to embodiment 5, wherein said phagocytes have been obtained from an AD patient, treated ex vivo with a monocyte inducing agent or a monocyte activating agent or a monocyte recruiting agent and then administered to the AD patient from whom said phagocytes have been obtained.

7. Monocytes for use according to embodiment 6, wherein said monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is an aluminium salt, especially aluminium oxy- hydroxide .

8. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to any one of embodi^¬ ments 1 to 7, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is an aluminium salt with the general formula Me_a ⁺Al_b ³⁺An^c~ ·η¾0, wherein

Me⁺ is Na⁺, K⁺, Li⁺, Rb⁺, Cs⁺ or NH₄ ⁺;

An is P0₄ ^3", SO₄ ^2", O(OH)^3", 0₂ ^~ or OH^";

a is 0, 1, 2, or 3;

b is 1 or 2 ;

c is 1, 2, 3, 4, 5, or 6; and

n is 0 to 48.

9. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to embodiment 8, where^¬ in the aluminium salt is selected from aluminium oxyhydroxide, aluminium phosphate, or aluminium sulphate.

10. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to any one of embodi^¬ ments 1 to 9, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is a cytokine or a mixture of cytokines, preferably a mixture of at least three, especially at least five, cytokines.

11. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of embod^¬ iments 1 to 10, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is administered in an amount of at least 1.2 mg per dose, preferably at least 1.5 mg per dose, especially at least 1.8 mg per dose.

12. Monocyte inducing agent or monocyte activating agent or a monocyte recruiting agent for use according to any one of embod^¬ iments 1 to 11, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is an aluminium salt in an amount of 1.2 to 10.0 mg per dose, preferably 1.5 to 5 mg per dose, especially 1.8 to 2.5 mg, (given as A1₂0₃ equiva^¬ lent) per dose.

13. Monocyte inducing agent or monocyte activating agent or a monocyte recruiting agent for use according to any one of embod^¬ iments 1 to 12, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is administered intracranially or into the cerebrospinal fluid.

14. Monocyte inducing agent or monocyte activating agent or a monocyte recruiting agent for use according to any one of embod^¬ iments 1 to 13, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is free of pro- teasome based adjuvants.

15. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to any one of embodi^¬ ments 1 to 14, wherein the monocyte inducing agent or a monocyte activating agent or a monocyte recruiting agent are administered in an amount effective for obtaining an astrocytosis reduction and/or a blood brain barrier breakdown and/or nitric oxide oxidative stress and/or neuronal death in the AD patient.

16. Monocyte inducing agent or monocyte activating agent or mon- ocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 15, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is an alu^¬ minium salt, preferably aluminium oxyhydroxide, and is present in a ready-to-use form to be directly applied to a patient, es^¬ pecially in a prefilled syringe.

17. Monocyte inducing agent or a monocyte activating agent or a monocyte recruiting agent for use according to any one of embod^¬ iments 1 and 6 to 16, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is an al^¬ uminium salt, preferably aluminium oxyhydroxide, and is con^¬ tained in a pharmaceutical preparation.

18. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 17, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is an alu^¬ minium salt, preferably aluminium oxyhydroxide, and is contained in a pharmaceutical preparation wherein said preparation contains the aluminium salt as the single effective ingredient.

19. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 18, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is contained in a pharmaceutical preparation, wherein said preparation comprises auxiliary substances, especially stabilisators , deter^¬ gents, antioxidants, complexing agents for mono- or divalent metal ions, carbohydrates and/or buffer substances.

20. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 19, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is contained in a pharmaceutical preparation wherein said preparation is sterilised and, optionally, liquid, frozen or lyophilised, pref^¬ erably liquid.

21. Monocyte inducing agent or monocyte activating agent or mon- ocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 20, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is contained in a pharmaceutical preparation wherein said preparation is liquid and has a pH of 5 to 9, preferably of 5.5 to 8.0, especially from 6 to 7.5.

22. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 21, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is an alu^¬ minium salt and is present in a medicament as single effective ingredient (active substance) .

23. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 22, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is an alu^¬ minium oxyhydroxide suspension, preferably European Pharmacopoe^¬ ia grade aluminium-oxyhydroxide (monograph 1664), especially Alhydrogel .

24. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 23, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered in an amount of at least 1.2 mg (given as A1₂0₃ equivalent) to an AD patient.

25. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 24, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered in an amount of 1.2 mg to 5.0 mg (given as A1₂0₃ equivalent) to an AD patient.

26. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 25, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered in an amount of at least 1.5 mg (given as A1₂0₃ equivalent) to an AD patient.

27. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 26, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered in an amount of 1.5 mg to 5.0 mg, preferably 1.5 to 3.0 mg, especially 1.5 to 2.5 mg, (given as A1₂0₃ equivalent) to an AD patient.

28. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 27, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered in an amount of 1.6 mg to 2.5 mg, preferably 1.8 to 2.2 mg, especially 1.9 to 2.0 mg, (given as A1₂0₃ equivalent) to an AD patient.

29. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 28, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and additionally contains one or more stabilisa- tors, especially thiomersal, detergents, antioxidants, complex- ing agents for mono- or divalent metal ions, especially eth- ylenediaminetetraacetic acid (EDTA) , sugars, sugar alcohols, glycerol, and/or buffer substances, especially TRIS or phosphate buffer substances.

30. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 29, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered to a patient in a suspension with a pH of 4 to 10, preferably of 5 to 9, more preferred of 6 to 8, especially from 7.0 to 7.5.

31. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to any one of embodi- ments 1 and 6 to 30, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered to a patient in an isotonic suspension .

32. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 31, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered subcutaneously, intranodally, intradermally, or intramuscularly, especially subcutaneously, to an AD patient.

33. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 32, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered at least once monthly for at least two months to an AD patient.

34. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 33, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered at least once monthly for at least six months to an AD patient.

35. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 34, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered at least twice a month for at least six months, preferably for at least twelve months, espe^¬ cially at least 24 months, to an AD patient.

36. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 37, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered to an AD patient subcutaneously in the upper arm, preferably alternating in the left and in the right upper arm.

37. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 36, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered in split doses to an AD pa^¬ tient, especially at the same site of administration.

38. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 37, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered in split doses of 0.8 to 5.0 mg, preferably of 1.0 to 3.0, especially from 1.0 to 1.5 mg, (given as A1₂0₃ equivalent) to an AD patient.

39. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 38, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered at least monthly for at least two years, preferably at least four years, especially at least 8 years, to an AD patient.

40. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 39, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered by an injection device, espe^¬ cially a syringe, to an AD patient.

41. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 40, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered in an amount of at least 1.8 mg (given as A1₂0₃ equivalent) to an AD patient. 42. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 41, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered to the AD patient in liquid form in an application volume of 0.1 to 10 ml, preferably of 0.2 to 5 ml, especially of 0.4 to 3 ml.

43. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 42, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is contained in a pharmaceutical preparation, wherein said preparation is devoid of sulphate, nitrate, or chloride anions.

44. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 43, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is contained in a pharmaceutical preparation, wherein said preparation has a heavy metal content of less than 20 ppm.

45. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to any one of embodi^¬ ments 1 and 6 to 44, wherein the monocyte inducing agent or mon^¬ ocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is contained in a pharmaceutical preparation, wherein said preparation is a suspension of aluminium oxyhydroxide and has a particle size distribution between 2 ym and ap^¬ proximately 10 ym, said particles being aggregates, composed of smaller fibers of preferably about 2 nm x 4.5 nm x lOnm.

Claims

Claims :

2. Monocyte inducing agent and/or a monocyte activating agent and/or a monocyte recruiting agent for use according to claim 1, wherein the monocytes are Grl ⁽⁺⁾ /CD1 lb ⁽⁺⁾ cells, CDllb⁺CX₃CRl^lowCCR2⁺CXCR4^high cells or CD14⁺⁺CD16^" cells.

3. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to claim 1, selected from the group consisting of M-CSF, GM-CSF, IFN-gamma, TGF-beta, TNF-alpha, IL-l,IL-2, IL-4, IL-5, IL-6, 11-10, IL-13, Frac- talkine/CX3CLl , members of the Chemokine (CC-motif) ligand fami^¬ ly (CCL) , especially CCL2, CC13, CCL4, CCL12, members of the IL- 8 family, especially CXCl-8 (11-8), Toll-like receptor agonists, especially TLR4 agonists, preferably LPS or MPLA and an alumini^¬ um salt.

4. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of claims 1 to 3, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is administered subcutaneous- ly, intra-cranially or into the bone marrow.

5. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to any one of claims 1 to 3, wherein the monocytes are human phagocytes, especially au^¬ tologous phagocytes of the AD patient to whom the monocytes are administered for treating AD.

6. Monocyte inducing agent and/or a monocyte activating agent and/or a monocyte recruiting agent for use according to claim 5, wherein said phagocytes have been obtained from an AD patient, treated ex vivo with a monocyte inducing agent or a monocyte ac^¬ tivating agent or a monocyte recruiting agent and then adminis- tered to the AD patient from whom said phagocytes have been ob^¬ tained .

7. Monocyte inducing agent and/or a monocyte activating agent and/or a monocyte recruiting agent for use according to claim 6, wherein said monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is an aluminium salt, espe^¬ cially aluminium oxyhydroxide .

8. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to any one of claims 1 to 7, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is an aluminium salt with the general formula Me_a ⁺Al_b ³⁺An^c~ ·η¾0, wherein

Me⁺ is Na⁺, K⁺, Li⁺, Rb⁺, Cs⁺ or NH₄ ⁺;

An is P0₄ ^3~, SO₄ ^2", O(OH)^3-, 0₂ ^~ or OH^";

a is 0, 1, 2, or 3;

b is 1 or 2 ;

c is 1, 2, 3, 4, 5, or 6; and

n is 0 to 48.

9. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to claim 8, wherein the aluminium salt is selected from aluminium oxyhydroxide, aluminium phosphate, or aluminium sulphate.

10. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to any one of claims 1 to 9, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is a cytokine or a mixture of cytokines, preferably a mixture of at least three, especially at least five, cytokines.

11. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of claims 1 to 10, wherein the monocyte inducing agent or monocyte acti^¬ vating agent or monocyte recruiting agent is administered in an amount of at least 1.2 mg per dose, preferably at least 1.5 mg per dose, especially at least 1.8 mg per dose.

12. Monocyte inducing agent or monocyte activating agent or a monocyte recruiting agent for use according to any one of claims 1 to 11, wherein the monocyte inducing agent or monocyte acti^¬ vating agent or monocyte recruiting agent is an aluminium salt in an amount of 1.2 to 10.0 mg per dose, preferably 1.5 to 5 mg per dose, especially 1.8 to 2.5 mg, (given as A1₂0₃ equivalent) per dose.

13. Monocyte inducing agent or monocyte activating agent or a monocyte recruiting agent for use according to any one of claims 1 to 12, wherein the monocyte inducing agent or monocyte acti^¬ vating agent or monocyte recruiting agent is administered intra- cranially or into the cerebrospinal fluid.

14. Monocyte inducing agent or monocyte activating agent or a monocyte recruiting agent for use according to any one of claims 1 to 13, wherein the monocyte inducing agent or monocyte acti^¬ vating agent or monocyte recruiting agent is free of proteasome based adjuvants.

15. Monocyte inducing agent or monocyte activating agent or mon^¬ ocyte recruiting agent for use according to any one of claims 1 to 14, wherein the monocytes or a monocyte inducing agent or a monocyte activating agent or a monocyte recruiting agent are ad^¬ ministered in an amount effective for obtaining an astrocytosis reduction and/or a blood brain barrier breakdown and/or nitric oxide oxidative stress and/or neuronal death in the AD patient.