WO2015185602A1

WO2015185602A1 - Treatment and prevention of parkinson's disease (pd)

Info

Publication number: WO2015185602A1
Application number: PCT/EP2015/062340
Authority: WO
Inventors: Markus Mandler; Achim Schneeberger; Arne Von Bonin; Frank Mattner; Walter Schmidt
Original assignee: Affiris Ag
Priority date: 2014-06-04
Filing date: 2015-06-03
Publication date: 2015-12-10

Abstract

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

Description

Treatment and prevention of Parkinson's Disease (PD)

The present invention relates to means and methods for the treatment and the prevention of neurodegenerative diseases associated with alpha-Synuclein (aSyn) deposition, preferably Parkinson's Disease (PD) .

Various neurodegenerative diseases are characterized by the aberrant accumulation of aSyn resulting in aSyn deposition. Such diseases are also summarised under the term synucleopathies . Synucleinopathies are a diverse group of neurodegenerative disorders that share a common pathologic characteristic: in neuropathologic examinations characteristic lesions can be detected containing abnormal aggregates of alpha-synuclein (alpha-syn, aSyn) protein in selected populations of neurons and glia cells. aSyn (initially identified as PARK1 and PARK4) is widely expressed in the central nervous system but can be detected in hematopoietic cells including B-, T-, and NK cells as well as monocytes and platelets. The exact role in these cells is not known but it has been implicated in the differentiation of megakaryocytes (platelet precursors) . The most common synucleinopathies include but are not limited to Lewy body disorders (LBDs) like Parkinson's disease (PD) , Parkinson's disease with dementia (PDD) and dementia with Lewy bodies (DLB) , as well as Multiple System Atrophy (MSA) or Neurodegeneration with Brain Iron Accumulation type I (NBIA Type I) .

Parkinson's disease (PD) is the second most common neurodegenerative disorder of the elderly (behind Alzheimer's disease (AD) (1); in people aged >65, its prevalence ranges between 100 and 250 cases per 100,000 in Western countries and 1, 700 per 100, 000 in China. It was long considered a motor disease characterized by akinesia/bradykinesia, rigidity and rest tremor. Its non-motor symptoms (neuropsychiatric- , gastrointestinal-, autonomous symptoms) are now well established. PD treatments available to date primarily address the disease's motor component. As they deliver symptomatic benefit only, they ultimately lose their clinical activity and fail. Moreover, long-term use is associated with complications such as treatment induced dyskinesia. There are several areas of medical need. First, we lack a disease-modifying agent. Second, measures to combat treatment-induced dyskinesia are limited. Third, there are only a few therapeutic options for non-motor symptoms .

Obviously, rational development of a disease-modifying therapeutic approach primarily requires understanding the underlying pathology. In case of PD, there is mounting evidence for a causal and essential pathophysiological role of alpha- synuclein (aSyn) . This field was pioneered by Spillantini and colleagues when they identified aSyn, by means of immunohistochemistry, to be a component of Lewy bodies [Spillantini MG; Schmidt ML; Lee VM et.al. Alpha-synuclein in Lewy bodies. Nature 1997;388:839-840]. Further arguments supporting a key role of aSyn in the pathogenesis of PD include the demonstration that certain dominantly inherited PD forms are caused by mutations in/duplications of the aSyn gene [Lansbury PT Jr; Brice A. Genetics of Parkinson's disease and biochemical studies of implicated gene products. Curr. Opin. Genet. Dev. 2002; 12 (3) :299-6. Singleton AB; Farrer M; Johnson J et.al. alpha-Synuclein locus triplication causes Parkinson's disease. Science 2003; 302 : 841. Polymeropoulos MH; Lavedan C; Leroy E et.al. Mutation in the alpha-synuclein gene identified in families with Parkinson's disease. Science 1997;276:2045-2047. Ross OA; Braithwaite AT; Skipper LM et.al. Genomic investigation of alpha-synuclein multiplication and parkinsonism. Ann. eurol. 2008; 63 (6) : 743-50. Eriksen JL; Dawson TM; Dickson DW et.al. Caught in the act: alpha-synuclein is the culprit in Parkinson's disease. Neuron 2003;40:453-56.], the observation that aSyn represents the major component of the neuropathological signature lesions in patients (Lewy bodies and Lewy neurites) [Braak H; Del Tredici K; Rub U et.al. Staging of brain pathology related to sporadic Parkinson's disease. Neurobiol Aging, 2003; 24 : 197-11. Jellinger KA. Synuclein deposition and non-motor symptoms in Parkinson disease. J. Neurol. Sci. 2011; 15; 310 (1-2) : 107-11. Dickson DW; Fujishiro H; Orr C et.al. Neuropathology of non-motor features of Parkinsons disease. Parkinsonism Rel Disord, 2009; 15 (S3) : S1-S5.] , the GWAS finding that certain aSyn gene variants contain the highest risk for sporadic PD [Gandhi S; Wood NW. Genome-wide association studies: the key to unlocking neurodegeneration? Nat.

Neurosci.2010;13 (7) :789-94. Simon-Sanchez, J; Schulte C; Bras JM et.al. Genome-wide association study reveals genetic risk underlying Parkinson's disease. Nat. Genet. 2009; 41 (12) : 1308- 1312. Satake, W; Nakabayashi Y; Mizuta I et.al. Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease. Nat. Genet. 2009; 41 (12) : 1303-1307. Edwards TL; Scott WK, Almonte C et.al. Genome-wide association study confirms SNPs in SNCA and the MAPT region as common risk factors for Parkinson disease. Ann Hum Genet 2010 ; 74 (2 ) : 97-109. ] , the demonstration that overexpression of human aSyn recapitulates certain features of the disease in experimental animals [Masliah E; Rockenstein E; Veinbergs I et al . Dopaminergic loss and inclusion body formation in alpha- synuclein mice : implications for neurodegenerative disorders. Science 2000;287:1265-1269. Rockenstein E; Crews L; Masliah E. Transgenic animal models of neurodegenerative diseases and their application to treatment development. Adv Drug Deliv Rev 2007; 59 (11) : 1093-102.] , and, finally, the fact that clinical symptoms and their progression correlate with the localization and development of aSyn pathology [Jellinger KA. Synuclein deposition and non-motor symptoms in Parkinson disease. J. Neurol. Sci. 2011 ; 15 ; 310 ( 1-2 ): 107-11. Lim S-Y; Fox S.H.; Lang A.E. Overview of the extranigral aspects of Parkinson Disease. Arch Neurol. 2009 ; 66 (2 ) : 167-172 ] . The latter is further supported by studies demonstrating pathological aSyn to be transmitted from cell to cell in a prion-like propagation pathway [Luk KC et al . Pathological alpha-synclein transmission initiates Parkinson-like neurodegeneration in nontransgenic mice. Science 2012; 338:949-53.]. While all these studies suggest aSyn to be the toxic culprit, downstream events such as mitochondrial dysfunction and oxidative stress are likely to mediate and even modulate its toxicity.

Beyond PD, aSyn was identified in hallmark lesions of other entities including multiple system atrophy (MSA) and dementia with Lewy bodies defining a group of neurodegenerative disorders designated as synucleopathies [Spillantini MG, Goedert M. The alpha-synucleopathies: Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. Ann NY Acad Sci 2000; 920: 16-27. ] .

It is currently believed that one potential treatment strategy to exhibit a disease-modifying effect for PD and related disorders could be based on immunotherapy to prevent or reduce the accumulation of neurotoxic agents like aSyn. Indeed, Masliah and colleagues were the first to test this hypothesis [Masliah E, Rockenstein E, Adame A, Alford M, Crews L, Hashimoto M, Seubert P, Lee M, Goldstein J, Chilcote T, Games D, Schenk D (2005) Effects of alpha-synuclein immunization in a mouse model of Parkinson's disease. Neuron 46 ( 6) : 857-868. ] . They treated mice overexpressing the human aSyn gene with an aSyn-based vaccine. Mice developing high affinity Abs were found to have reduced amounts of aggregated aSyn both within their cell bodies and at the synapses. Experimental evidence provided suggests degradation of aSyn to occur via the lysosomal pathway. Importantly, the reduction of aSyn oligomers was found to correlate inversely with the extent of neurodegeneration.

The above investigators obtained similar effects when using an aSyn-specific Ab in the sense of passive immunotherapy. In addition, we also recently applied immunotherapy using AFFITOPE^® vaccines directed against aSyn in vivo [Mandler et al . 2014] . AFFITOPE^® vaccination resulted in high target-specific (aSyn) antibody titers in plasma and antibodies were able to cross the blood brain barrier as evidenced by different techniques (in situ staining and CSF analysis) . Antibodies elicited were shown to primarily recognize aSyn aggregates and induction of these antibodies was associated with a lowering of cerebral aSyn burden, especially of its oligomeric form. This was accompanied by a reduction of the degeneration of tyrosine hydroxylase expressing nerve cells. Most importantly, successfully immunized mice were also shown to exhibit improvements in motor as well as memory deficits. Studies to the mode of action indicate that clearance of aSyn involves the activation of microglia in the context of a milieu of anti-inflammatory cytokines. Despite these promising approaches, so far no effective, disease modifying treatment is available to stop the progressive neurodegeneration and associated functional decline in human patients. 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 PD enabling a cure to PD in the meaning that the status of the diseased patient is not further developing or even ameliorated. Another object is to provide means and methods for preventing the development of PD in persons having or being at risk of developing PD. More specifically, it is an object of the present invention to provide efficient PD treatment, as proven with respect to at least one significant biomarker.

Therefore, the present invention provides monocytes and/or a monocyte inducing agent and/or a monocyte activating agent and/or a monocyte recruiting agent for use in the treatment and prevention of neurodegenerative diseases associated with aSyn deposition, preferably PD.

The present invention provides monocytes and/or a monocyte inducing agent and/or a monocyte activating agent and/or a monocyte recruiting agent for use in the treatment and prevention of neurodegenerative diseases associated with aSyn deposition, preferably Parkinson's Disease (PD) , but also Dementia with Lewy bodies, Multiple System Atrophy or other synucleopathies .

According to the present invention, either the crucial cells itself that are needed for the treatment or inducing/recruiting/activating agent (s) for these cells 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 peripheral monocytic cells entering the CSF/brain. In the mouse, at the site of action (in the brain) they are CDllb⁺CX₃CRl^lowCCR2⁺CXCR4^high cells, as distinct from

CDllb+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 understood. Human monocytes that are CD14^highCDl 6^~ are considered classical monocytes. Together with the CD14^highCDl 6⁺ (i.e. intermediate monocytes) these are considered inflammatory monocytes. In contrast, CD14⁺CD16⁺ cells are non-classical monocytes (also considered patrolling monocytes) . According to the present invention, 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 invention 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,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 agonists, especially TLR4 agonists, preferably LPS or MPLA and an aluminium salt.

The monocytes or monocyte inducing agent or monocyte activating 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 PD patient to whom the monocytes are administered for treating PD. These phagocytes can be obtained from a PD patient, treated ex vivo with a monocyte inducing agent or a monocyte activating agent or a monocyte recruiting agent and then administered to the PD patient from whom said phagocytes have been obtained (as autologous cells) .

According to another preferred embodiment, the monocyte inducing 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", S0₄ ^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 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.

Preferably, 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.

Preferably, the monocytes or 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 AI₂O₃ equivalent) per dose.

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

Preferably, the monocytes or monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is free of proteasome based adjuvants.

It is also preferred to administer the monocytes or 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 reduction of a blood brain barrier breakdown and/or nitric oxide oxidative stress and/or neuronal death in the PD patient.

So far all active immunotherapeutic approaches tested in humans have used stimulation of the adaptive immune system to exert its therapeutic effect mainly mediated by humoral immune responses specific for the target in patients.

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 immune mediators that are expressed by a variety of immune and non-immune cells [Crack PJ et al . , Immunol Cell Biol 2007; 85:476-480] .

In PD, no approaches using TLR-agonists as potential treatment modality have been disclosed so far. This is in contrast to AD, where several TLR-agonists (functioning as PAMPs) have been used as novel drug candidates for prevention and therapy. However, the importance of the TLR system for PD has been supported by findings by Kim et al, who could show that oligomeric a-synuclein can act as an endogenous agonist of TLR2 leading to paracrine activation of microglia in the brain associated with the activation of inflammatory responses [Kim, C. et al . Neuron-released oligomeric a-synuclein is an endogenous agonist of TLR2 for paracrine activation of microglia. Nat. Commun. 2013 4:1562.] . A recent study by Stefanova et al . [Stefanova, N. et al . Toll-like receptor 4 promotes alpha-synuclein clearance and survival of nigral dopaminergic neurons. Am. J. Pathol. 179, 954-963 (2011)] also suggested that TLR4 is involved in aSyn clearance in aSyn transgenic mice, however, these authors also suggested in another report that microglial activation mediates neurodegeneration in the same mouse model [Stefanova, N. et al . Microglial activation mediates neurodegeneration related to oligodendroglial alpha-synucleinopathy : implications for multiple system atrophy. Mov. Disord. 22, 2196-2203 (2007).].

Along these lines WO 2009/105641 A2 is directed to a method of preventing or reducing 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 stimulation using a detoxified ligand (TLR4 agonist: monophosphoryl lipid A) improves Alzheimer's disease-related pathology in transgenic mice. Accordingly, WO 2012/055981 Al is directed to compositions and methods for preventing and/or reducing amyloid deposition 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 aminoalkyl 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 similar TLR4 agonists can exert differential effects in vivo as LPS has been demonstrated to exacerbate whereas MPL ameliorated amyloid 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 exposure of TLR4 agonists (i.e. LPS) exacerbated the Αβ plaque load in an APP_swe/PSl transgenic AD model. It is believed that this exacerbation is mainly caused by excessive and sustained inflammation 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 proteasome derived Protollin™ was tested for prevention and therapy in animal models. Protollin™ is an adjuvant comprising Proteosomes™ non-covalently complexed with the TLR4 agonist LPS for intranasal application. Protollin™ application in APP Tg mice significantly improved cognitive function and stimulated the microglia 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 signal through different pathways and induce the production of distinct cytokines and chemokines that play a key role in the priming, expansion and polarization of the immune responses. Activation of the NLR family members NLRP3 and NLRC4 triggers the formation 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 induction 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 underlying mechanism of action for treating PD 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. circulating monocytes, to the brain could help to ameliorate defects in immune cell function detectable in the degenerating nervous system in PD 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 mesodermal/ mesenchymal progenitors and have migrated into the brain during development. After invading the CNS, microglial precursors disseminate homogeneously throughout the neural tissue. Microglia 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). Davalos, 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 PD. Usually such changes lead to the induction of gliosis exerting an important role in the pathogenesis of various CNS diseases including PD [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, develop 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 precursors for such macrophages that continuously develop in the bone marrow, circulate in the blood and migrate into tissues

(e.g. the PD brain), where they become local macrophages or dendritic 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. exerting neuroprotection and restoration) for PD disease progression. Interestingly, recent data from AD research implies that such resident and bone marrow-derived cells (i.e. CCR2⁺ positive monocytes) have distinct neuroprotective function in neurodegenerative diseases [Naert et al . , J Mol Cell Biol (2013) 5 (5) : 284-293] . AD patients show CCR2⁺ monocytopenia 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 resulting 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]. Recent work by Gardai and colleagues [Gardai SJ, Mao W, Schu^" le B, Babcock M, Schoebel S, et al . (2013) Elevated Alpha-Synuclein Impairs Innate Immune Cell Function and Provides a Potential Peripheral Biomarker for Parkinson's Disease. PLoS ONE 8(8): e71634.] demonstrates that increased aSyn protein impairs cytokine release and phagocytosis in a transgenic PD model and in idiopathic PD cases. These processes were affected in vivo in peritoneal macrophages and microglia in the CNS . In humans similar results were obtained with monocytes and fibroblasts isolated from idiopathic or familial Parkinson' s disease patients compared to age-matched controls. Along these lines, Hasegawa et al showed that PD patients show impaired cytokine production by peripheral blood mononuclear cells and monocytes/macrophages [Hasegawa, Y., Inagaki, T., Sawada, M. and Suzumura, A. (2000), Impaired cytokine production by peripheral blood mononuclear cells and monocytes/macrophages in Parkinson's disease. Acta Neurologica Scandinavica, 101: 159-164]. In addition, the central role of CCR2+ mononuclear cells for PD is also supported by the finding that PD patients and asymptomatic high-risk individuals show a strong upregulation in the percentage of monocyte precursors in the peripheral blood as well as a strong CCR2 upregulation on classical monocytes in Parkinson's patients [Funk N et al . Characterization of peripheral hematopoietic stem cells and monocytes in Parkinson's disease. Mov Disord. 2013 Mar; 28 (3) : 392-5] .

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 ] . This could also be corroborated for PD by the recent finding that bone marrow-derived macrophages transporting the antioxidant enzyme catalase to the brain were eliciting a substantial decrease in nigrostrital degeneration as well as neuroinflammation in murine PD models [Batrakova EV, Li S, Reynolds AD, Mosley RL, Bronich TK, et al . A macrophage-nanozyme delivery system for Parkinson's disease. Bioconjug Chem. 2007; 18:1498-1506. Brynskikh AM, Zhao Y, Mosley RL, Li S, Boska MD, et al . Macrophage delivery of therapeutic nanozymes in a murine model of Parkinson's disease. Nanomedicine (Lond) . 2010; 5:379- 396. ] .

Thus CCR2⁺ monocyte recruitment and increase of phagocytic activity may constitute an important line of defense against PD associated degeneration including aSyn deposition, synaptic dysfunction, and functional decline. Thus, one potential treatment paradigm for PD could be based on the specific induction and recruitment of peripheral monocytes to the PD brain in order to enhance microglial activity in the brain and thereby to alleviate aSyn deposition, synaptic and neuronal dysfunction and functional decline in PD 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 compounds (e.g. aluminium oxyhydroxide, the hydrated form of aluminium oxide (AI₂O3) . These particulate compounds exert TLR independent activity and play an important role as activators of innate 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 discussed controversially whether particulate aluminium compounds activates innate immune cells via the cytoplasmic NLRP3, which associates with ASC and caspase 1 to form the inf1ammasome .

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 uptake 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 alternative 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 response 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 antigen 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 aluminium compounds (e.g. Alhydrogel, Alhydrogel adsorbed with biological macromolecules ) and their local (i.e. at the site of injection) as well as systemic effects (i.e. activation and recruitment of peripheral immune cells to the target organ, e.g. the PD brain) can be harnessed to treat or prevent pathophysiologic alterations in PD patients leading to an amelioration or prevention of clinical disease manifestation.

The most preferred embodiment of the present invention comprises the effective administration of aluminium oxyhydroxide (particularly as Alhydrogel) to PD 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 preparations, namely Alhydrogel (aluminium-oxyhydroxide) and AdjuPhos (aluminiumhydroxyphosphate) , onto which antigens are adsorbed for vaccine preparations (reviewed in E. B. Lindblad (2004) Vaccine 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 their 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 exposing a soluble aluminium salt (historically potassium alum, i.e. KA1 (SO₄) 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 structure (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 aluminium hydroxyphosphate have an opposite surface charge at neutral pH, with the latter being negatively charged. It has to be 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 oxyhydroxide .

For aluminium oxyhydroxide, the preparation is devoid of an^¬ ions such as sulphates, nitrates, or chlorides and has a specified 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 invention relates to the use of European Pharmacopoeial grade (Aluminium-oxyhydroxide, monograph 1664), more specifically to the product manufactured by Brenntag Biosector (2% Alhydrogel) tested towards EP compliance. Alhydrogel is available in three varieties: Alhydrogel 1.3%; Alhydrogel 2% and Alhydrogel "85". Alhydrogel 2% was elected as the International Standard Preparation for aluminium hydroxide gels. The pharmaceutical preparation according to the present invention is aseptically formulated into a suitable buffer, preferably a isotonic phosphate buffer (ImM to 100 mM) , preferably at a concentration of ≥ 1.0 mg/ml Alhydrogel (given as AI₂O₃ equivalent; this metric (Al as "AI₂O₃ equivalent") is used generally for the present invention; accordingly, all doses and amounts referred to in the present application, as far they are relating to aluminum salts (especially as far as they are relating to aluminium oxyhydroxide) refer to AI₂O₃ equivalents (of aluminium oxyhydroxide (Alhydrogel) ) ) , even more preferably at a concentration of ≥ 1.5 mg/ml Alhydrogel (given as AI₂O₃ equivalent), most preferable at a concentration of ≥ 2.0 mg/ml Alhydrogel (given as AI₂O₃ equivalent) .

Alhydrogel 2%, often also referred to as alum, is an aluminium oxyhydroxide wet gel suspension.

In the most preferred embodiment of the present invention, the aluminium salt to be administered to the PD patient is an aluminium oxyhydroxide suspension, preferably European Pharmacopoeia grade aluminium-oxyhydroxide (monograph 1664), especially Alhydrogel. The aluminium oxyhydroxide is administered in an amount effective to achieve a PD ameliorating effect . Accordingly, any administration procedure or dosage regimen for the aluminium salt formulation, especially aluminium- oxyhydroxide formulation, according to the present invention that is suitable to achieve the PD modifying effect as provided by the present invention is subject to the present invention. Although it is possible to deliver the preparation 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 a PD patient. A preferred range of amount to be administered to a patient is an amount of aluminium oxyhydroxide of 1.2 mg to 5.0 mg. The PD ameliorating effect of aluminium oxyhydroxide administration is even more pronounced at an amount of at least 1.5 mg. According to another preferred embodiment 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 a PD patient. Another preferred embodiment comprises administration 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 a PD patient.

According to another preferred embodiment, the aluminium oxyhydroxide 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 substance 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 administered, be it for administration purposes, storage purposes, or other purposes. According to a preferred embodiment, the aluminium oxyhydroxide preparation to be applied according to the present invention contains a pharmaceutically acceptable carrier, diluent or excipient, for example water for injection. Preferably, the aluminium oxyhydroxide preparation according to the present invention additionally contains one or more stabilisators , especially thiomersal, detergents, antioxidants, complexing agents for mono- or divalent metal ions, especially ethylenediaminetetraacetic 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 suspension, e.g. with a volume of between 0.1 and 10 ml, more preferred 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 according to pharmaceutical Good Manufacturing Practice (GMP) , as required and defined by the European and/or US Pharmacopeia.

According to a preferred embodiment, the aluminium oxyhydroxide 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. Preferably, the suspension is an isotonic suspension.

Preferably, the aluminium salt is administered by a route that is as convenient as possible for the PD patient but is still effective to achieve a PD modifying effect. Most effective 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 PD patient, depending on the treatment success, as measured by various parameters, especially by motoric, cognitive and functional performances and by biomarkers. According to the course of the present invention, at least monthly administrations of aluminium oxyhydroxide to a PD patient are successful in ameliorating PD. In order to achieve a long lasting therapeutical effect, such at least monthly administrations should be continued for at least three months, especially 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 a PD 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 a PD 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 circle) ) , 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 simultaneously (at the same physician date; at least at the same day) to the PD 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, preferably, at the same site of administration. The "same site of administration" 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, especially from 1.0 to 1.5 mg.

In order to achieve a very long lasting effect of the PD 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 administered at least monthly for at least two years, preferably at least four years, especially at least 8 years, to a PD patient .

Administration of the aluminium oxyhydroxide according to the present invention may be performed by any suitable administration device. For convenience reasons, the aluminium oxyhydroxide dose is administered by an injection device, especially a syringe, to a PD 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 sterilisation, 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 lyophilized, an aggregation of adjuvant particles during processing may be observed. By cooling such formulations, especially aluminium oxyhydroxide (Alhydrogel) formulations, at faster rates or by the addition of sufficient amounts of a glass forming excipient, 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 minimized 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 PD patients according to the present invention are manufactured (and finished) 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 invention, the aluminium oxyhydroxide is administered in an amount of at least 1.8 mg to a PD patient. Preferred patients to which aluminium oxyhydroxide preparations according to the present invention is administered are PD patients that are early stage patients, selected by a suitable score, e.g. staging according to MDS-UPDRS. The Unified Parkinson's Disease Rating Scale (UPDRS) , a rating scale used to follow the longitudinal course of Parkinson's disease. The UPDRS is the most commonly used scale in the clinical study of Parkinson's Disease . [Goetz, Christopher G.; Fahn, Stanley, Martinez-Martin, Pablo, Poewe, Werner, Sampaio, Cristina, Stebbins, Glenn T., Stern, Matthew B., Tilley, Barbara C., Dodel, Richard, Dubois, Bruno, Holloway, Robert, Jankovic, Joseph, Kulisevsky, Jaime, Lang, Anthony E., Lees, Andrew, Leurgans, Sue, LeWitt, Peter A., Nyenhuis, David, Olanow, C. Warren, Rascol, Olivier, Schrag, Anette, Teresi, Jeanne A., Van Hilten, Jacobus J., LaPelle, Nancy (1 January 2007) . "Movement Disorder Society-sponsored revision of the Unified Parkinson's Disease Rating Scale (MDS-UPDRS) : Process, format, and clinimetric testing plan". Movement Disorders 22 (1): 41-47. doi:10.1002/mds.21198. ] .

Preferred patients to which the compositions according to the present invention (especially aluminium oxyhydroxide preparations according to the present invention) is administered are PD patients that are early stage patients, selected by a suitable score, preferably according to the staging according to the Hoehn and Yahr scale. The Hoehn and Yahr scale is a commonly used system to describe disease progression including stages 1-5 [Hoehn M, Yahr M (1967) . "Parkinsonism: onset, progression and mortality.". Neurology 17 (5): 427-42.]. Early stage patients can be defined as Hoehn and Yahr stages 1-2. Accordingly, a preferred group of patients to be treated according to the present invention are patients with Hoehn and Yahr stages 1-2, more preferred stage 1-1.5, especially stage 1.

Early stage PD patients can also be selected by other scores, preferably scores that combine motoric, cognitive and functional parameters (and numerical limits) for limiting PD population to be (effectively treated) .

The present invention is further described by the following examples and the figures, yet without being restricted thereto. Figures :

Fig. 1 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 number of peripheral monocytes was assessed by FACS analysis 24h (A.. C57B1/6; B.. Balb/c) and 48h after injection (C. Balb/c) . *..p<0.05 compared to na^'l^'ve animals)

Fig. 2 shows neuronal function of the mice tested: Groups of TASD61 mice (n≤15/group) received 7 monthly injections of PBS (n=8) or ALUM only (n=14) . ALUM only treated non-transgenic littermates (WT, n=15) were used as positive controls for assessing neuronal function. Odor choice testing using the latency to first stimulus contact (i.e. attractive (urine) or neutral (water) ) as a measure for olfactory function was performed. Parameter depicted is the latency to start sniffing. Values depicted represent means +/- standard error

Examples :

Preclinical analysis

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 derived 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 respective phagocytic activity (e.g. uptake of fluorescent beads/microspheres coupled with recombinant human alpha Synuclein (aSyn) or uncoupled; uptake of fluorescently labelled aSyn-aggregates) , and changes in chemokine/cytokine expression of monocytes/macrophages 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:

Isolation and in vitro differentiation of macrophages is essentially 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

(Alhydrogel/Brenntag) containing buffer ranging from 0.1 g/ml to 2 mg/ml for 2h.

Phagocytosis is performed using fluorescently labelled aSyn aggregates or fluorescent microspheres: For aggregate formation, fluorescent N-terminally-labelled aSyn (Hilyte488, Anaspec) is used. aSyn aSyn-Hilyte-488 (Anaspec, detectable in the FITC- channel; 1mg/ml) is used for aggregate-production. Aggregates are assembled by constant rotation (600 rpm) for 24-48h at room temperature on shaker in PBS .

Subsequently these aSyn-aggregates or fluorescent microspheres are added to differentiated and primed macrophages (2h Alum) for lhr at 37°C (0.5 g - 5 g aSyn-aggregates/well or fluorescent microspheres (Bang Laboratories) /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 manufacturer.

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 different 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 bone marrow derived macrophages/monocytes in vitro.

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 fluorescent beads/microspheres coupled with recombinant human alpha Synuclein (aSyn) or uncoupled; uptake of fluorescently labelled aSyn-aggregates) , and changes in chemokine/cytokine expression of microglia 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 (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 absence of ascending doses of aluminium oxyhydroxide (Brenntag) containing buffer ranging from 0. lmg/ml to 2 mg/ml for 2h.

Subsequently, aSyn-aggregates or fluorescent microspheres are added to differentiated and primed macrophages (2h Alum) for lhr at 37°C (0.5μg - 5μg aSyn-aggregates/well or fluorescent microspheres (Bang Laboratories) /well)

FACS Analysis:

Cells are harvested after incubation with different increasing concentrations of aluminium oxyhydroxide and aSyn HiLyte Fluor™488 or fluorescent microspheres and analysed for phagocytic activity (uptake of fluorescently labelled aSyn or fluorescent microspheres) . 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 multiplexing technology (Luminex, according to manufacturer's protocol) 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 phagocytic activity of both cell lines in vitro.

3. Effect of aluminium oxyhydroxide containing preparations on peripheral monocytes in mice :

To investigate whether aluminium oxyhydroxide containing preparations are contributing to alterations of overall peripheral blood mononuclear cells (PBMCs) and peripheral blood monocytes, single and repeated local injections of these preparations (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 phagocytic activity (e.g. uptake of fluorescently labelled aSyn-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 aluminium 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 staining 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 gated 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, 50 g per mouse of fluorescent HiLyte Fluor™ 488-labeled aSyn (Anaspec, Fremont, CA) is injected in the tail vein. Peripheral blood is drawn from treated mice via cardiac puncture with EDTA as anticoagulant, 120 min after i.v. injection of the aSyn 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 (eBioscience) 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) .

aSyn uptake is assessed by reporting the percentage and Mean Fluorescence Intensity (GeoMean) of positive HiLyte fluor™ 488 aSyn cells among CD115+ cells.

Results :

With the present experiment, an increase in peripheral monocytes following injection of aluminium oxyhydroxide (or other agents according to the present invention) application s.c can be demonstrated. As shown in Figure 1, C57B1/6 and Balb/c mice were injected once (s.c.) and blood was withdrawn for PBMC analysis. 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.l A-C) supporting the role of Aluminium salts as monocyte inducing/monocyte activating agents in vivo. No change in the number of T-cells (CD4+ and CD8+) , B-cells or NK cells was detectable following injection (not shown) .

4. Effect of aluminium oxyhydroxide containing preparations on neuronal function in an animal model for PD : Animal experiment :

The experiment was performed in accordance with the Austrian Animal Experiments Act (TVG2012) using mThyl ha-syn Tg mice (TASD61) (Background: C57BL/6 x DBA/2) (Rockenstein et al . 2002) starting with 2 months of age. General health was checked by modified SmithKline Beecham, Harwell, Imperial College, Royal London Hospital, phenotype assessment (SHIRPA) primary observational screen (Rogers DC et al . (1999) Behav Brain Res 105: 207-217.) . Mice were injected s.c. 7 times in monthly intervals and subjected to behavioral analysis at ca. 9 months.

Behavioral tests:

To analyse neuronal function, immunised TASD61 animals were subjected to a test assessing olfactory function (odor choice test) , analyzed using AnyMaze software (Stoelting Co, USA) .

For the odor choice test single mice were put to a clean cage without a lid (Type II: 36x21x13) provided with fresh bedding (~3cm) . Fresh pooled urine from untreated, unfamiliar wildtype females (age: 2-8 months) was used as odor stimulus. Either 30μ1 of urine pool (stimulus) or water (control) was applied to a filter paper. Each filter paper was placed in the corner of one of two opposite zones in the experimental cage and covered with bedding.

Each mouse was placed individually in the middle ( ^xNeutral Zone' ) of the fresh cage and recording started immediately thereafter. The latency to first stimulus contact (i.e. attractive (urine) or neutral (water)) was recorded (max. 180sec) (ANYmaze ©, Stoelting) as a measure for olfactory function .

Results :

To evaluate the effect of Aluminum-oxyhydroxide (Alum) on PD associated neuronal dysfunction function, i.e. olfactory dysfunction, in a PD mouse model, we applied odor choice testing (OCT) in TASD61 mice. Olfactory dysfunction is among the earliest non-motor features of PD with approximately 90% of early-stage PD patients developing signs of this dysfunction. Olfactory dysfunction can precede the onset of the classical motor symptoms of PD by years (Doty RL . Nat Rev Neurol. 2012 May 15 ; 8 ( 6) : 329-39. Olfactory dysfunction in Parkinson disease.). As reported by Fleming et al . (2008), TASD61 animals also show olfactory dysfunction already at 3 months of age, 1 month after initiation of Alum treatment in this experiment.

Analysis of OCT (Figure 2) demonstrated that male, Alum treated TASD61 mice (n=14) were superior to control animals receiving PBS only (n=8) in this testing paradigm. Control TASD61 animals showed severely impaired olfactory function at 9 months of age as n=5/8 animals failed to react to the attractive stimulus. In Alum treated TASD61 mice n=13/14 mice were still able to react to the attractive stimulus at 9 months and the overall latency to react was similar to wt littermate controls (Figure 2, WT) treated with Alum (n=15) as well, indicating that the cohort of animals tested were largely retaining olfactory function. All three groups were only limitedly exploring the neutral stimulus.

Claims

Claims :

1. Monocytes and/or 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 associated with alpha- Synuclein (aSyn) deposition, preferably Parkinson's Disease (PD) .

2. Monocytes for use according to claim 1, wherein the monocytes are Grl ⁽⁺⁾ /CD1 lb ⁽⁺⁾ cells, CD1 lb⁺CX₃CRl^lowCCR2⁺CXCR4^high cells or CD14⁺⁺CD16^" cells.

3. Monocytes or monocyte inducing agent or monocyte activating agent or monocyte 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, 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 agonists, especially TLR4 agonists, preferably LPS or MPLA and an aluminium salt.

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

5. Monocytes or monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of claims 1 to 3, wherein the monocytes are human phagocytes, especially autologous phagocytes of the PD patient to whom the monocytes are administered for treating PD.

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

7. Monocytes for use according to claim 6, wherein said monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is an aluminium salt, especially aluminium oxyhydroxide .

8. Monocyte inducing agent or monocyte activating agent or monocyte 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°^~ ·ηΗ₂0, wherein

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

An is P0₄ ^3", S0₄ ^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 monocyte recruiting agent for use according to claim 8, wherein the aluminium salt is selected from aluminium oxyhydroxide, aluminium phosphate, or aluminium sulphate.

10. Monocytes or monocyte inducing agent or monocyte activating agent or monocyte 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. Monocytes or monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of claims 1 to 10, wherein the monocytes or 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. Monocytes or 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 monocytes or 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 AI₂O₃ equivalent) per dose.

13. Monocytes or 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 monocytes or monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is administered intracranially or into the cerebrospinal fluid.

14. Monocytes or 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 monocytes or monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is free of proteasome based adjuvants.

15. Monocytes or monocyte inducing agent or monocyte activating agent or monocyte 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 administered in an amount effective for obtaining an astrocytosis reduction and/or a reduction of blood brain barrier breakdown and/or nitric oxide oxidative stress and/or neuronal death in the PD patient.

16. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of claims 1 and 6 to 15, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is an aluminium salt, preferably aluminium oxyhydroxide, and is present in a ready-to-use form to be directly applied to a patient, especially 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 claims 1 and 6 to 16, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is an aluminium salt, preferably aluminium oxyhydroxide, and is contained in a pharmaceutical preparation.

18. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of claims 1 and 6 to 17, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is an aluminium 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 monocyte recruiting agent for use according to any one of claims 1 and 6 to 18, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is contained in a pharmaceutical preparation, wherein said preparation comprises auxiliary substances, especially stabilisators , detergents, antioxidants, complexing agents for mono- or divalent metal ions, carbohydrates and/or buffer substances.

20. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of claims 1 and 6 to 19, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is contained in a pharmaceutical preparation wherein said preparation is sterilised and, optionally, liquid, frozen or lyophilised, preferably liquid.

21. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of claims 1 and 6 to 20, wherein the monocyte inducing agent or monocyte 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 claims 1 and 6 to 21, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is an aluminium salt and is present in a medicament as single effective ingredient (active substance) .

23. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of claims 1 and 6 to 22, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is an aluminium oxyhydroxide suspension, preferably European Pharmacopoeia 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 claims 1 and 6 to 23, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered in an amount of at least 1.2 mg (given as AI₂O₃ equivalent) to a PD patient.

25. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of claims 1 and 6 to 24, wherein the monocyte inducing agent or monocyte 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 AI₂O₃ equivalent) to a PD patient.

26. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of claims 1 and 6 to 25, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered in an amount of at least 1.5 mg (given as AI₂O₃ equivalent) to a PD patient.

27. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of claims 1 and 6 to 26, wherein the monocyte inducing agent or monocyte 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 AI₂O₃ equivalent) to a PD patient.

28. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of claims 1 and 6 to 27, wherein the monocyte inducing agent or monocyte 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 AI₂O₃ equivalent) to a PD patient.

29. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of claims 1 and 6 to 28, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and additionally contains one or more stabilisators , especially thiomersal, detergents, antioxidants, complexing agents for mono- or divalent metal ions, especially ethylenediaminetetraacetic 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 monocyte recruiting agent for use according to any one of claims 1 and 6 to 29, wherein the monocyte inducing agent or monocyte 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 monocyte recruiting agent for use according to any one of claims 1 and 6 to 30, wherein the monocyte inducing agent or monocyte 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 claims 1 and 6 to 31, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered subcutaneously, intranodally, intradermally, or intramuscularly, especially subcutaneously, to a PD patient.

33. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of claims 1 and 6 to 32, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered at least once monthly for at least two months to a PD patient.

34. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of claims 1 and 6 to 33, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered at least once monthly for at least six months to an PD patient.

35. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of claims 1 and 6 to 34, wherein the monocyte inducing agent or monocyte 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, especially at least 24 months, to a PD patient.

36. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of claims 1 and 6 to 35, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered to a PD 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 claims 1 and 6 to 36, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered in split doses to a PD patient, especially at the same site of administration.

38. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of claims 1 and 6 to 37, wherein the monocyte inducing agent or monocyte 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 AI₂O₃ equivalent) to a PD patient.

39. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of claims 1 and 6 to 38, wherein the monocyte inducing agent or monocyte 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 a PD patient.

40. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of claims 1 and 6 to 39, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered by an injection device, especially a syringe, to a PD patient.

41. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of claims 1 and 6 to 40, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered in an amount of at least 1.8 mg (given as AI₂O₃ equivalent) to a PD patient.

42. Monocyte inducing agent or monocyte activating agent or monocyte recruiting agent for use according to any one of claims 1 and 6 to 41, wherein the monocyte inducing agent or monocyte activating agent or monocyte recruiting agent is aluminium oxyhydroxide and is administered to the PD 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 monocyte recruiting agent for use according to any one of claims 1 and 6 to 42, wherein the monocyte inducing agent or monocyte 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 claims

1 and 6 to 43, wherein the monocyte inducing agent or monocyte 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 monocyte recruiting agent for use according to any one of claims 1 and 6 to 44, wherein the monocyte inducing agent or monocyte 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 approximately 10 ym, said particles being aggregates, composed of smaller fibers of preferably about 2 nm x 4.5 nm x lOnm.

46. Method for the treatment of Parkinson's Disease (PD), wherein an immune stimulating pharmaceutical composition comprising an aluminium salt is administered to a patient having PD or having a risk to develop PD in an effective amount.

47. Method according to claim 46, wherein the aluminium salt has the general formula Me_a ⁺Al_b ³⁺An°^~ ·ηΗ₂0, wherein

Me⁺ is Na⁺, K⁺, Li⁺, Rb⁺, Cs⁺ or NH₄ ⁺; An is P0₄ ^3", S0₄ ^2" O(OH)^3", O₂ ^"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.

48. Method according to claim 46 or 47, wherein the aluminium salt is selected from aluminium hydroxide, aluminium oxyhydroxide, aluminium phosphate, or aluminium sulphate.

49. Method according to any one of claims 46 to 48 wherein the aluminium salt is administered as the single effective ingredient in said immune stimulating pharmaceutical composition .

50. Method according to any one of claims 46 to 49, wherein the immune stimulating pharmaceutical composition comprises auxiliary substances, especially stabilisators , detergents, antioxidants, complexing agents for mono- or divalent metal ions, carbohydrates and/or buffer substances.

51. Method according to any one of claims 46 to 50, wherein the immune stimulating pharmaceutical composition is liquid and has a pH of 5 to 9.

52. Method according to any one of claims 46 to 51, wherein the immune stimulating pharmaceutical composition is liquid and has a pH of 5.5 to 8.0.

53. Method according to any one of claims 46 to 52, wherein the immune stimulating pharmaceutical composition is liquid and has a pH of 6 to 7.5.

54. Method according to any one of claims 46 to 53, wherein the aluminium salt is an aluminium oxyhydroxide suspension.

55. Method according to any one of claims 46 to 54, wherein the aluminium salt is European Pharmacopoeia grade aluminium- oxyhydroxide (monograph 1664).

56. Method according to any one of claims 46 to 55, wherein the aluminium salt is Alhydrogel.

57. Method according to any one of claims 46 to 56, wherein the aluminium salt is aluminium oxyhydroxide and is administered in an amount of at least 1.2 mg (given as AI₂O₃ equivalent) to a PD patient .

58. Method according to any one of claims 46 to 57, wherein the aluminium salt is aluminium oxyhydroxide, and wherein the effective amount is between 1.5 and 4 mg aluminium oxyhydroxide, per application, especially per dose.

59. Method according to any one of claims 46 to 58, wherein the aluminium salt is Alhydrogel, and wherein the effective amount is between 1.5 and 4 mg Alhydrogel, per application, especially per dose.

60. Method according to claim 58 or 59, wherein the application is an injection, especially a double or triple injection at about the same injection site.

61. Method according to claim 60, wherein the same injection site is defined as being within an area of 10 cm² of the skin.

62. Method according to claim 60, wherein the same injection site is defined as being within an area of 5 cm² of the skin, especially within 1 cm² of the skin.

63. Method according to any one of claims 58 to 62, wherein the aluminium salt is administered to a patient in two or three doses within two days and in an amount of at least 1.2 mg of at least one dose.

64. Method according to any one of claims 46 to 63, wherein the aluminium salt is administered to a patient at least once monthly in an amount of at least 1.5 mg.

65. Method according to any one of claims 46 to 64, wherein the aluminium salt is administered to a patient at least once monthly for a duration of at least three months, preferably of at least six months, especially for at least one year, in an amount of at least 1.5 mg.

66. Method according to any one of claims 46 to 65, wherein the aluminium salt is administered to a patient at least once monthly for a duration of at least one year, preferably of at least three years, especially for at least five years, in an amount of at least 1.5 mg.

67. Method according to any one of claims 46 to 66, wherein the aluminium salt is administered to a patient at least once monthly for a duration of at least three months, preferably of at least six months, especially for at least one year, in an amount of at least 1.8 mg, preferably in an amount of 1.8 to 2.5 mg, especially of about 2.0 mg.

68. Method according to any one of claims 46 to 67, wherein the aluminium salt is administered to a patient at least twice monthly for a duration of at least one month, preferably of at least two months, especially for at least one year, in an amount of at least 1.8 mg, preferably in an amount of 1.8 to 2.5 mg, especially of about 2.0 mg.

69. Method according to any one of claims 46 to 67, wherein the aluminium salt is administered to a patient at least four times monthly for a duration of at least one month, preferably of at least two months, especially for at least one year, in an amount of at least 1.0 mg, preferably in an amount of 1.2 to 3 mg, especially of 1.5 to 2.5 mg.

70. Method according to any one of claims 46 to 69, wherein the aluminium salt is administered to a patient at least twice within one week at about the same injection site, in an amount of at least 1.0 mg, preferably in an amount of 1.2 to 3 mg, especially of 1.5 to 2.5 mg.

71. Method according to any one of claims 46 to 70, wherein the aluminium salt is aluminium oxyhydroxide and is administered in an amount of 1.2 mg to 5.0 mg to a PD patient.

72. Method according to any one of claims 46 to 71, wherein the aluminium salt is aluminium oxyhydroxide and is administered in an amount of at least 1.5 mg to a PD patient.

73. Method according to any one of claims 46 to 72, wherein the aluminium salt is aluminium oxyhydroxide and additionally contains one or more stabilisators , especially thiomersal, detergents, antioxidants, complexing agents for mono- or divalent metal ions, especially ethylenediaminetetraacetic acid (EDTA) , sugars, sugar alcohols, glycerol, and/or buffer substances, especially TRIS or phosphate buffer substances.

74. Method according to any one of claims 46 to 73 wherein the aluminium salt 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.

75. Method according to any one of claims 46 to 74 wherein the aluminium salt is aluminium oxyhydroxide and is administered to a patient in an isotonic suspension.

76. Method according to any one of claims 46 to 75, wherein the aluminium salt is aluminium oxyhydroxide and is administered subcutaneously, intranodally, intradermally, or intramuscularly, especially subcutaneously, to a PD patient.

77. Method according to any one of claims 46 to 76, wherein the aluminium salt is aluminium oxyhydroxide and is administered at least once monthly for at least two months to a PD patient.

78. Method according to any one of claims 46 to 77, wherein the aluminium salt is aluminium oxyhydroxide and is administered at least once monthly for at least six months to a PD patient.

79. Method according to any one of claims 46 to 78, wherein the aluminium salt is aluminium oxyhydroxide and is administered at least twice a month for at least six months, preferably for at least twelve months, especially at least 24 months, to a PD patient .

80. Method according to any one of claims 46 to 79, wherein the aluminium salt is aluminium oxyhydroxide and is administered to a PD patient subcutaneously in the upper arm, preferably alternating in the left and in the right upper arm.

81. Method according to any one of claims 46 to 80, wherein the aluminium salt is aluminium oxyhydroxide and is administered in split doses to a PD patient, especially at the same site of administration .

82. Method according to any one of claims 46 to 81, wherein the aluminium salt 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, to a PD patient.

83. Method according to any one of claims 46 to 82, wherein the aluminium salt 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 a PD patient.

84. Method according to any one of claims 46 to 83, wherein the aluminium salt is aluminium oxyhydroxide and is administered by an injection device, especially a syringe, to a PD patient.

85. Method according to any one of claims 46 to 84, wherein the aluminium salt is aluminium oxyhydroxide and is administered and is administered in an amount of at least 1.8 mg to a PD patient.

86. Method according to any one of claims 46 to 85, wherein the aluminium salt is preferably aluminium oxyhydroxide and is administered to the PD 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.

87. Method according to any one of claims 46 to 86 contained in a pharmaceutical preparation, wherein said preparation is devoid of sulphate, nitrate, or chloride anions.

88. Method according to any one of claims 46 to 87 contained in a pharmaceutical preparation, wherein said preparation has a heavy metal content of less than 20 ppm.

89. Method according to any one of claims 46 to 88 contained in a pharmaceutical preparation, wherein said preparation is a suspension of aluminium oxyhydroxide and has a particle size distribution between 2 ym and approximately 10 ym, said particles being aggregates, composed of smaller fibers of preferably about 2 nm x 4.5 nm x lOnm.

90. Method according to any one of claims 46 to 89, wherein the patient is an early stage patient, preferably a patient with Hoehn and Yahr stage 1-2, more preferred a patient with Hoehn and Yahr stage 1-1.5, especially a patient with Hoehn and Yahr stage 1.