WO2015165971A1 - Treatment and prevention of alzheimer's disease (ad) - Google Patents

Treatment and prevention of alzheimer's disease (ad) Download PDF

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Publication number
WO2015165971A1
WO2015165971A1 PCT/EP2015/059351 EP2015059351W WO2015165971A1 WO 2015165971 A1 WO2015165971 A1 WO 2015165971A1 EP 2015059351 W EP2015059351 W EP 2015059351W WO 2015165971 A1 WO2015165971 A1 WO 2015165971A1
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Prior art keywords
vaccine
per dose
especially
αβ
mg per
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PCT/EP2015/059351
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French (fr)
Inventor
Markus Mandler
Achim Schneeberger
Wolfgang Zauner
Vera BÜRGER
Frank Mattner
Walter Schmidt
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Affiris Ag
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1712Not used, see subgroup
    • A61K38/1716Amyloid plaque core protein
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K33/00Medicinal preparations containing inorganic active ingredients
    • A61K33/06Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/0005Vertebrate antigens
    • A61K39/0007Nervous system antigens; Prions
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/39Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/505Medicinal preparations containing antigens or antibodies comprising antibodies
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505Inorganic adjuvants

Abstract

The invention discloses a vaccine for use in the treatment and prevention of dementias associated with β-amyloid deposition, preferably Alzheimer's Disease (AD), wherein the vaccine contains an aluminium salt 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 (given as Al2O3 equivalent) per dose.

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 vaccine for use in the treatment and prevention of dementias associated with β- amyloid deposition ( β-amyloidoses ) , preferably AD, wherein the vaccine contains an aluminium salt 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.

In the course of the present invention it has surprisingly turned out that aluminium salts, especially aluminium oxyhydrox- ide, have proven in clinical trials to be effective in real dis¬ ease modifying effects in AD patients leading to clinical effi¬ cacy hitherto not seen in any of the clinical trials for AD med¬ ication so far. The present invention therefore provides a breakthrough technology for this disease. For the first time, a significant disease modifying effect could be detected in AD pa¬ tients. 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.

This improvement according to the present invention is achieved by combining the disease-modifying effect of aluminium salts with the immunotherapy approach for dementias associated with β-amyloid deposition. Active or passive amyloid-targeted immunotherapy, especially amyloid-targeted active vaccination, is a strategy that has been pursued with considerable effort, yet without significant breakthrough, however, with numerous drawbacks, such as the failure of the first active vaccine (AN1792, consisting of preaggregate Αβ and an immune adjuvant, QS-21) that was abandoned because it caused meningoencephalitis in approximately 6% of treated patients. Further studies have identified significant risks in the immunotherapy based on na¬ tive antigens, such as Αβ42. Also the passive immunotherapy ap¬ proach has not yet resulted in positive results from clinical trials. As also noted above, reports from clinical trials of bapineuzumab and solanezumab were disappointing as well.

As already noted above, current preclinical and clinical re¬ search in active and passive immunisation is focused on two pos¬ sible targets in this regard. One is the inhibition of accumula¬ tion and deposition of amyloid beta 1-42 (Αβ42), which is one of the major peptides found in senile plaques, and the second tar¬ get is hyperphosphorylated tau, which forms neurofibrillary tan¬ gles inside the nerve cell and shows association with the pro¬ gression of dementia. Mouse models have shown that immunotherapy targeting Αβ42 as well as tau with the respective anti-Αβ or an- ti-tau antibodies can provide significant improvements in these mice. While anti-Αβ immunotherapy (active and passive immuniza¬ tions) is already in several stages of clinical trials, tau- based immunizations have been analyzed only in mouse models. Re¬ cently, as a significant correlation of progression of dementia and levels of phosphorylated tau have been found, high interest has again focused on further development of tau-based therapies. While Αβ immunotherapy might delay the onset of AD, immunothera¬ py targeting tau might provide benefits in later stages of this disease. Last but not least, targeting Αβ and tau simultaneously with immunotherapy might provide additional therapeutic effects, as these two pathologies are likely synergistic.

With the present invention, however, the active and passive vaccination approach for diseases like AD is combined with the clinical breakthrough of administration of an immune stimulating pharmaceutical composition comprising aluminium salts, especially aluminium oxyhydroxide (Alhydrogel) .

Therefore, a preferred embodiment of the present invention is the combination of an active or passive vaccine against Αβ, especially Αβ1-42, and/or against tau, especially hyperphosphor- ylated tau with an effective amount of an aluminium salt.

The positive clinical outcome for aluminium salts according to the present invention is specifically highlighted when an ad¬ ministration dose of at least 1.2 mg aluminium salt, especially alhydrogel/dose is applied. If the aluminium salt component of the present invention is combined with the active or passive vaccine, it is, however, preferred to use higher doses, because of the protein binding property of aluminium salts (also depend¬ ing on the p i of the protein and the pH of the pharmaceutical preparation) . Accordingly, the aluminum salt concentrations are preferably increased when proteins or polypeptides are present in the pharmaceutical preparation to be administered to a pa¬ tient .

For example, aluminium phosphate (Adju-Phos) has a maximal binding capacity (in mg protein/mg aluminum at pH 7.4) to Lyso- zyme (p i 11.0) of 1.4 ± 0.1; aluminium oxyhydroxide (Alhydrogel) to Ovalbumin (p i 4.6) of 1.6 ± 0.1 and to BSA (p i 4.9) of 2.2 ± 0.1 (Jones et al . , JBC 280, (2005), 13406-13414). In order to account for the aluminium that is bound to such proteins, more aluminium salt has to be provided in such admixed pharmaceutical preparations than in preparations that contain the aluminium salt as the single effective ingredient or - at least in the ab¬ sence of proteins or polypeptides in the pharmaceutical prepara¬ tion. For example, if a pharmaceutical composition according to the present invention should provide the effectiveness of a dose of 2 mg aluminium salt, especially aluminium oxyhydroxide, and contains a specific amount of proteins or polypeptides (antigens or antibodies) which binds to the aluminium salt, an amount equal to the aluminium salt portion binding to such proteins has to be additionally included in the pharmaceutical preparation to provide 2 mg "free" aluminium salt. It may therefore be neces¬ sary to provide considerably more than 2 mg Alhydrogel into such a protein (or polypeptide) containing vaccine, e.g. at least 2.5 mg, preferably at least 3 mg, especially at least 4 mg, of alu¬ minium salt, especially Alhydrogel (as further explained below, the mg amounts of aluminium salts, especially aluminium oxyhy¬ droxide are always given as AI2O3 equivalent) .

Preferably, the vaccine according to the present invention is an active or passive vaccine against Αβ, especially Αβ1-42, and/or against tau, especially hyperphosphorylated tau.

The active vaccine is preferably containing an antigen that elicits an immune response against Αβ, Αβ aggregates, especially oligomeric Αβ aggregates, tau protein, phospho-tau protein, ag¬ gregated tau protein, hyperphosphorylated tau protein or a natu¬ rally occurring fragment thereof, preferably Αβ1-40/42, Αβ 2- 40/42, Αβ 3-40/42, Αβ 4-40/42, Αβ 1-38, or Αβ 1-39. The antigen may also be an Αβ or tau antigen containing modifications, pref¬ erably racemisation of aspartate and serine residues, isomerisa- tion of aspartate residues, and pyroglutamate formation at the glutamate residues. Such modifications are e.g. disclosed by Shapira et al . , 1988; Roher et al . , 1993a; or Mori et al . , 1992) . More recent work has added to the list of Αβ variants, with phosphorylated and nitrated Αβ being described (Kumar et a1. , 2011; Kummer et al., 2011).

The "aggregate" contained in the active vaccine according to the present invention refers to the aggregate as such, but also to preparations containing the aggregate, especially prepara¬ tions suitable for diagnostic purposes in human medicine, i.e. preparations made according to GMP and standardised procedures. Preferably, the aggregate-forming Tau protein variant and the aggregate-forming Αβ1-42 variant are, independently of each other, selected from the following groups:

Hyperphosphorylated Tau, abnormally phosphorylated Tau (as referred to and defined in Shahani et al . J. Neurosci. 26 (2006), 6103-6114), Tau protein variants that have marker func¬ tion for a disease (either isoform of all 6 naturally occurring isoforms (Tau441, Tau412, Tau411, Tau383, Tau381, Tau352) or mutant forms thereof (e.g. P301L, P301S, V337M, etc.), e.g.: forms preferably simultaneously phosphorylated at amino acids 181, 202, 205, 212, 214, 231, 396 and, optionally at additional resi¬ dues present in Tau like 18, 153, 175, 189, 235, 262, 394, 404 and 422, of Tau441, Tau412, Tau410, Tau383, Tau381, Tau352 (a more detailed analysis of Tau phosphorylation is disclosed e.g. in Hanger et al . , Trends in Mol. Med. 15 (2009), 112-119); and Αβ1-40, Αβ1-42, Αβ1-43, Αβ1-37, Αβ1-38, Αβ1-39, Αβ3-40/42/43, Αβ11-40/42/43, Αβρ (Ε) 3-40/42 and Αβρ (Ε) 11-40/42, especially Αβΐ- 40, Αβ1-42, Αβ3-42 and Αβρ (Ε) 3-40/42 ; an Αβ variant selected from the group consisting of

a mutant of the Αβ-1-40- or the Αβ-Ι-42-peptide containing at least one mutation at an amino acid position other than amino acids 16 to 20, 25 to 28 and 31 to 35,

a truncated form of the Αβ-1-40- or the Αβ-Ι-42-peptide, wherein the truncation is at amino acid positions other than amino acids 16 to 35 and wherein the truncation is at least one amino acid and at most 20 amino acids; or

combinations thereof, especially combinations of at least one mutation and at least one truncation of the Αβ-1-40- or the Αβ-Ι-42-peptide .

Preferably, the aggregate has a size of 50 nm to 15 ym, preferably from 100 nm to 10 ym, especially from 200 nm to 5 ym.

Preferably, the aggregate is obtainable by incubating the protein species at a pH of 2 to 9 for at least 20 min, prefera¬ bly at least 1 h, especially at least 4 h (see also: e.g. WO 2013/050249 Al) .

Preferred passive vaccines to be improved according to the present invention are vaccines containing monoclonal antibodies directed against the Αβ and tau proteins and variants listed above. Preferred monoclonal antibodies are selected from the group consisting of solanezumab, bapineuzumab, gantenerumab, crenezumab, ponezumab, and aducanumab. Although these antibodies have not been successful in clinical trials yet, their potein- tial is, at least partially, still believed to be present. The present invention is a strategy to provide to such antibodies clinical efficiency in humans, if combined with an effective amount of the aluminium salt, especially aluminium oxyhxdroxide (Alhydrogel) .

The combination of the active or passive vaccine with the aluminium salt according to the present invention can be provided in a single vaccine preparation. It can also be applied by a combination of separated agents (as a "kit") so that both compo¬ nents can be administered separately to a patient.

Accordingly, a preferred embodiment of the present invention is a vaccine comprising two components,

- an active or passive vaccine against Αβ, especially Αβ1-42, and/or against tau, especially hyperphosphorylated tau; and

- an immune stimulating pharmaceutical composition comprising an aluminium salt, especially aluminium oxyhydroxide, (given as AI2O3 equivalent) .

The two components may be provided together (as "kit of parts", optionally together with other components, such as ad¬ ministration devices, especially syringes, instructions for use, diluents, mixing devices (for mixing dried components with dilu¬ ents) , etc.) or separately. They may be administered in paral¬ lel, subsequently or alternating.

Suitable active and passive vaccines are well available in the art and have also been subject to intensive clinical testing (such as ACC-001 and AN-1792 with a vaccine against Αβ) . Pre¬ ferred vaccines to be improved according to the present inven¬ tion for the treatment and prevention of β-amyloidoses , espe¬ cially AD, are disclosed in many patent documents, such as WO 1993/011231 A, WO 1995/017429 A, US 2009/0238831, WO 99/27944 A, WO 00/72880 A, WO 01/62801 A, WO 02/46237 A, WO 02/088306 A, WO 02/088307 A, WO 00/77178 A, WO 03/070760 A, WO 03/016466 Al , WO 2007/068429 A, EP 2 009 104 A, US 2012/0244146 Al, WO

2014/008404 A, EP 0 772 634 A, (monoclonal antibodies, especial¬ ly humanised and human antibodies binding to epitopes on the Αβ peptide and on tau/tau variants), WO 99/27944 A, WO 00/72880 A, WO 2012/149365 A, WO 2004/007547 A, (active immunisation with Αβ and tau protein epitopes, conjugates and constructs) , WO 2006/005707 A, WO 2004/062556 A, WO 2009/149485 A, WO 2009/149486 A, and WO 2009/149487 A (relating to mimotope vac¬ cines of Αβ which are specifically preferred to be further im¬ proved according to the present invention.

In the case of a combined vaccine, the aluminium salt, espe¬ cially aluminium oxyhydroxide is preferably provided in a higher amount as 1.2 mg/dose (mainly because of the protein binding ca¬ pacity of the aluminium salts) . Accordingly such a preferred em¬ bodiment is characterised by the vaccine containing the alumini¬ um salt in an amount of 1.2 to 10.0 mg per dose, preferably 1.5 to 8 mg per dose, especially 1.8 to 5 mg per dose (given as AI2O3 equivalent) . It is also preferred to provide such an improved vaccine having the aluminium salt in an amount of 1.9 to 9.0 mg per dose, preferably 2.5 to 8.0 mg per dose especially 3.0 to 7.0 mg per dose (given as AI2O3 equivalent) . Another preferred embodiment is characterised by the vaccine containing the alu¬ minium salt in an amount of 1.2 to 4.0 mg per dose, preferably 1.5 to 3 mg per dose, especially 1.8 to 2.5 mg per dose (given as AI2O3 equivalent) . Preferably, the vaccine contains the alu¬ minium salt in an amount of 2.5 to 10.0 mg per dose, preferably 3.5 to 8 mg per dose, especially 4.5 to 7.5 mg per dose (given as AI2O3 equivalent) . Since passive vaccines usually have a high¬ er protein content, passive vaccines should contain higher amounts of the aluminium salts according to the present inven¬ tion, compared to active vaccine preparations.

If provided in a separate form and intended to be adminis¬ tered separately from the active or passive vaccine, the alumin¬ ium salt is provided in an amount of 1.5 to 2.5 mg per dose, preferably of 1.9 to 2.1 mg per dose, especially 2.0 mg per dose (given as AI2O3 equivalent) .

The vaccine may further contain adjuvants, preferably con¬ tains a proteasome adjuvant.

Preferably, the vaccine is provided in lyophilised, dried or frozen form, preferably in a prefilled syringe (or kit compris¬ ing a pair of prefilled syringes) .

According to another aspect, the present invention also re¬ lates to a kit for use in the treatment and prevention of demen¬ tias associated with β-amyloid deposition, preferably AD, com¬ prising

- an active or passive vaccine against Αβ, especially Αβ1-42, and/or against tau, especially hyperphosphorylated tau; and

- an immune stimulating pharmaceutical composition comprising an aluminium salt, especially aluminium oxyhydroxide, wherein the aluminium salt is present 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 (given as AI2O3 equivalent) per dose (given as AI2O3 equiv¬ alent) .

The active or passive vaccine is then provided and adminis¬ tered separately from the immune stimulating pharmaceutical com¬ position containing the aluminium salt, especially aluminium oxyhydroxide .

As already mentioned, the most preferred embodiment of the present invention comprises the effective administration of alu¬ minium oxyhydroxide (particularly as Alhydrogel) to AD patients

(separately or combined with an active or passive AD (or β- amyloidase) vaccine) .

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 (SO4) 2 · 12¾0, was often used) to alkaline conditions, upon which a suspension is formed. Characterisation with X-ray crys- tallography 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.

Preferred aluminium salts according to the present invention have the general formula Mea +Alb 3+Anc~-nH20, 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.

Preferred examples of such aluminium salts are those that have been studied, examined and verified in and for human use, such as aluminium hydroxide, aluminium oxyhydroxide, aluminium phosphate, aluminium sulphate, or any kind of "alum" (wherein "al¬ um" is usually referred to a class of chemical compounds, includ¬ ing the "classical alum", a hydrated potassium aluminium sulfate (potassium alum) with the formula KA1 (SO4) 2'12¾0, and - more gen¬ erally double sulfate salts, with the formula AA1 (SO4) 2'12¾0, where A is a monovalent cation such as potassium or ammonium) .

The most preferred embodiments of the aluminium salts ac¬ cording to the present invention are selected from aluminium hydroxide, aluminium oxyhydroxide, aluminium phosphate, or alumin¬ ium sulphate, especially aluminium oxyhydroxide, which has been intensively investigated in the course of the present invention, since it is the preferred adjuvant in human use (as Alhydrogel adjuvant in various vaccines) .

Aluminium oxyhydroxide preparations have a point of zero charge at a pH of ~ pH 11, while aluminium hydroxyphosphate 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 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 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 AI2O3 equivalent; this metric (Al as "AI2O3 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 AI2O3 equivalents (of aluminium oxyhydroxide (Alhydrogel) ) ) , even more preferably at a concentration of ≥ 1.5 mg/ml Alhydro¬ gel (given as AI2O3 equivalent) , most preferable at a concentra¬ tion of ≥ 2.0 mg/ml Alhydrogel (given as AI2O3 equivalent) . The amount of aluminium salt for Alhydrogel is given as AI2O3 equiva¬ lent in line with the strength as stated by the manufacturer (i.e. 2% Alhydrogel equates to 2% AI2O3, 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 AI2O3 (Mw 101 , 96) _corresponds to 10.6 mg/mL aluminium (molecular mass 26, 98)) . Depending on the salt used this value can 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 patient (separately or combined with an active or passive AD (or β-amyloidosis ) vac¬ cine) is an aluminium oxyhydroxide suspension, preferably Euro¬ pean Pharmacopoeia grade aluminium-oxyhydroxide (monograph 1664), especially Alhydrogel. The aluminium oxyhydroxide is ad¬ ministered together with the active or passive vaccine 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 procedure 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, specifically if adminis¬ tered separately. A preferred range of amount to be administered to a patient (if administered separately) is an amount of alu¬ minium oxyhydroxide of 1.2 mg to 5.0 mg. The AD ameliorating effect of aluminium oxyhydroxide administration is even more pro¬ nounced 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, espe¬ cially 1.5 to 2.5 mg, to an AD patient. Another preferred embod¬ iment 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 an AD patient.

According to another preferred embodiment, the aluminium ox¬ yhydroxide is administered in amount of 2.2 mg or higher, spe¬ cifically, if administered in combination (i.e. admixture) with the active or passive vaccine. 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 .

The aluminium salt preparation according to the present in- vention may, 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 purpos¬ es, 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 stabili- sators, especially thiomersal, detergents, antioxidants, com- plexing 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 (optionally together with the active or passive vaccine) 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 sus¬ pension .

Preferably, the aluminium salt (optionally together with the active or passive vaccine) 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 effective treatment routes of aluminium oxyhydroxide (optionally together with the active or passive vaccine) according to the present invention are subcutaneous, intranodal, intradermal, or intramuscular ad¬ ministration, especially subcutaneous administration. Subcutane¬ ous administration is performed as a bolus into the subcutis, the layer of skin directly below the dermis and epidermis, espe¬ cially in the fatty tissue in the subcutis. Administration regimes can be optimised individually for each patient, depending on the treatment success, as measured by various parameters, especially by cognitive and functional per¬ formances and by biomarkers, especially MRI 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 (optionally together with the active or passive vaccine) according to the present in¬ vention may also be performed at least twice a month (for exam¬ ple 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. If the administration of the aluminium salt is performed separately from the active or passive vaccine, such separate administration may be performed at the same session as the administration of the active or passive vaccination; however, it may be preferred to alternate the administrations (week¬ ly, bi-weekly or monthly) or at least have at least one day, preferably two days, especially at least one week between the administration of the active or passive vaccine and the alumini¬ um salt. For example, the aluminium salt is administered first, then the active or passive vaccine follows in a week and then the next aluminium administration follows in another week or a month later (and so on), or vice versa.

According to a preferred embodiment aluminium oxyhydroxide is administered to an AD patient (optionally together with the active or passive vaccine) 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 (or the ac¬ tive/passive vaccine) into the right (or left) upper arm and the second dose (the aluminium salt) into the left (right arm) , and so on), or vice versa. 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(s) to be administered can also be split into two (or more) split doses that are administered sim¬ ultaneously (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, pref¬ erably, 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 oxyhydrox¬ ide in an amount of 0.8 to 5.0 mg, preferably of 1.0 to 3.0, es¬ pecially 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 active or passive vaccine and the alu¬ minium salt, especially the aluminium oxyhydroxide, according to the present invention may be performed by any suitable admin¬ istration device. For convenience reasons, the vaccines accord¬ ing to the present invention are administered by an injection device, especially a syringe, to an AD patient. However, passive immunisation is also often performed by way of infusion (which makes the use of separate administration of passive vaccine and aluminium salt often a preferred embodiment) . 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, lyoph- ilisation, etc. Preferably, combinations of such means are used to enhance storage stabilities of such preparations. When alumi¬ num-salt containing agents, such as aluminium oxyhydroxide con¬ taining pharmaceutical formulations, are frozen or lyophilized, an aggregation of adjuvant particles during processing may be observed. By cooling such formulations, especially aluminium ox¬ yhydroxide (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 buff¬ er salts induces modifications in surface chemistry and crystal- Unity 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 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 (optionally to¬ gether with the active or passive vaccine) in an amount of at least 1.8 mg to an AD patient.

Preferred patients to whom the vaccine preparations or kit according to the present invention is administered are β- amyloidose patients, especially AD patients, that are early stage patients, including those patients that are often also re¬ ferred to as "patients with mild cognitive impairment" (MCI) . The concept of MCI was developed in the 1990s to capture pa¬ tients with early clinical signs of Alzheimer disease (AD) who did not yet fulfil the criteria for dementia. The amnestic vari¬ ant of MCI features the following: memory complaints, 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 re¬ call, word list) ; preserved general cognitive function (for ex- ample, 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 remaining one- third have non-progressive or very slowly progressive causes of cognitive impairment (for example, depression or age-related cognitive impairment) . Proposed new diagnostic criteria 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 follow¬ ing four markers: medial temporal atrophy on MRI; temporoparie¬ tal cortical hypometabolism on 18F-fluorodeoxyglucose PET; ab¬ normality 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≤l 0 /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 6th 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.

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 (TIVavg) for each subject was determined by averaging the estimated TIV across visits. The normalization factor (TIVtemPiate/TIVavg_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:

= 1.67*Word recall + 1.35*Orientation + 1.42*Word Recogni¬ tion + 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

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 oxo- hydroxide 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 patients and a correla¬ tion 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 radiologic biomarker (MRI in the present case)) . Fig. 4 shows that the patients treated ac¬ cording to the present invention showed almost no AD related re¬ duction in hippocampus 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 . ) . These results show that the effect of the immune stimulating pharmaceutical formulation comprising an aluminum salt, especially aluminium oxyhydroxide, is also plausible in combination administration with active or passive immunisation, if the dose of the aluminium salt is sufficiently high.

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

Figure imgf000033_0001

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 SmithKline 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".

Claims

Claims :
1. Vaccine for use in the treatment and prevention of dementias associated with β-amyloid deposition, preferably Alzheimer's Disease (AD) , wherein the vaccine contains an aluminium salt 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 (given as AI2O3 equiva¬ lent) per dose.
2. Vaccine for use according to claim 1, wherein the vaccine is an active or passive vaccine against Αβ, especially Αβ1-42, and/or against tau, especially hyperphosphorylated tau.
3. Vaccine for use according to claim 1 or 2, wherein the vaccine contains an antigen that elicits an immune response against Αβ, Αβ aggregates, especially oligomeric Αβ aggregates, tau pro¬ tein, phospho-tau protein, aggregated tau protein, hyperphos¬ phorylated tau protein or a naturally occurring fragment there¬ of, preferably Αβ1-40/42, Αβ 2-40/42, Αβ 3-40/42, Αβ 4-40/42, Αβ 1-38, or Αβ 1-39.
4. Vaccine for use according to any one of claims 1 to 3, wherein the Αβ or tau antigen contains modifications, preferably racemisation of aspartate and serine residues, isomerisation of aspartate residues, and pyroglutamate formation at the glutamate residues .
5. Vaccine for use according to claim 1 or 2, wherein the vaccine contains an antibody against Αβ or tau protein or a natu¬ rally occurring fragment thereof, especially wherein the anti¬ body is selected from the group consisting of solanezumab, bapi- neuzumab, gantenerumab, crenezumab, ponezumab, and aducanumab.
6. Vaccine for use according to any one of claims 1 to 5, wherein the vaccine comprises two components,
- an active or passive vaccine against Αβ, especially Αβ1-42, and/or against tau, especially hyperphosphorylated tau; and
- an immune stimulating pharmaceutical composition comprising an aluminium salt, especially aluminium oxyhydroxide .
7. Vaccine for use according to any one of claims 1 to 6, wherein the vaccine contains the aluminium salt in an amount of 1.2 to 10.0 mg per dose, preferably 1.5 to 8 mg per dose, espe¬ cially 1.8 to 5 mg per dose (given as AI2O3 equivalent) .
8. Vaccine for use according to any one of claims 1 to 7, wherein the vaccine contains the aluminium salt in an amount of 1.9 to 9.0 mg per dose, preferably 2.5 to 8.0 mg per dose espe¬ cially 3.0 to 7.0 mg per dose (given as AI2O3 equivalent) .
9. Vaccine for use according to any one of claims 1 to 8, wherein the vaccine contains the aluminium salt in an amount of 1.2 to 4.0 mg per dose, preferably 1.5 to 3 mg per dose, espe¬ cially 1.8 to 2.5 mg per dose (given as AI2O3 equivalent) .
10. Vaccine for use according to any one of claims 1 to 9, wherein the vaccine contains the aluminium salt in an amount of 2.5 to 10.0 mg per dose, preferably 3.5 to 8 mg per dose, espe¬ cially 4.5 to 7.5 mg per dose (given as AI2O3 equivalent) .
11. Vaccine for use according to any one of claims 1 to 10, wherein the vaccine contains the aluminium salt in an amount of 1.9 to 2.1 mg per dose, especially 2.0 mg per dose (given as AI2O3 equivalent) .
12. Vaccine for use according to any one of claims 1 to 11, wherein the vaccine contains a proteasome adjuvant.
13. Vaccine according to any one of claims 1 to 12, provided in lyophilised, dried or frozen form, preferably in a prefilled sy¬ ringe .
14. Kit for use in the treatment and prevention of dementias as¬ sociated with β-amyloid deposition, preferably AD, comprising
- an active or passive vaccine against Αβ, especially Αβ1-42, and/or against tau, especially hyperphosphorylated tau; and
- an immune stimulating pharmaceutical composition comprising an aluminium salt, especially aluminium oxyhydroxide, wherein the aluminium salt is present 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 (given as AI2O3 equivalent) per dose (given as AI2O3 equiv¬ alent) .
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Citations (3)

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Publication number Priority date Publication date Assignee Title
WO1994016327A1 (en) * 1993-01-14 1994-07-21 Pollard Harvey B Methods and compositions for blocking amyloid protein ion channels
WO1999027944A1 (en) * 1997-12-02 1999-06-10 Neuralab Limited Prevention and treatment of amyloidogenic disease
WO2011120924A1 (en) * 2010-03-29 2011-10-06 Novartis Ag Composition comprising the amyloid beta 1-6 peptide coupled to a virus-like particle and an adjuvant

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Title
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MICHAEL T. HENEKA ET AL: "NLRP3 is activated in Alzheimer's disease and contributes to pathology in APP/PS1 mice", NATURE, vol. 493, no. 7434, 19 December 2012 (2012-12-19), pages 674 - 678, XP055125448, ISSN: 0028-0836, DOI: 10.1038/nature11729 *

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