TREATMENT OF ALZHEIMER'S DISEASE (AD) WITH AN ALUMINUM SALT

Abstract

Disclosed is a method for the treatment of AD, wherein an immune stimulating pharmaceutical composition comprising an aluminium salt is administered to a patient having AD or having a risk to develop AD in an effective amount.

Claims

1-21. (canceled)

22. A method for treating Alzheimer's Disease (AD) comprising administering to a patient having AD a pharmaceutical formulation comprising from about 1.2 mg to about 5.0 mg of an aluminum salt.

23. The method according to claim 22, wherein the method comprises the step of administering to the patient a diagnostic test that is capable of detecting early stage AD.

24. The method according to claim 23, wherein the diagnostic test comprises a mini-mental state examination (MMSE).

25. The method according to claim 22, wherein the diagnostic test comprises PET imaging, MRI imaging or measuring a cerebrospinal fluid marker.

26. The method of claim 25, wherein the cerebrospinal fluid marker is selected from tau, amyloidβ42 or phospho-tau.

27. The method according to claim 22, wherein the aluminum salt has the general formula Me.sub.a.sup.+Al.sub.b.sup.3+An.sub.c.sup.−.nH.sub.2O, wherein Me.sup.+ is Na.sup.+, K.sup.+, Li.sup.+, Rb.sup.+, Cs.sup.+ or NH.sub.4.sup.+; An is PO.sub.4.sup.3−, SO.sub.4.sup.2−O(OH).sup.3−, O.sup.2− or OH.sup.−; 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.

28. The method according to claim 22, wherein the aluminum salt comprises aluminum hydroxide, aluminum oxyhydroxide, aluminum phosphate, or aluminum sulfate.

29. The method according to claim 22, wherein the pharmaceutical formulation is administered to the patient at least once monthly in a single administration dose.

30. The method according to claim 29, wherein the pharmaceutical formulation comprises from about 1.5 mg to about 3.0 mg of the aluminum salt.

31. The method according to claim 30, wherein the pharmaceutical formulation comprises about 2.0 mg of the aluminum salt.

32. The method according to claim 22, wherein the pharmaceutical formulation comprises a pharmaceutically acceptable carrier, diluent or excipient.

33. The method according to claim 32, wherein the pharmaceutical formulation comprises an aluminum oxyhydroxide suspension.

34. The method according to claim 33, wherein the aluminum oxyhydroxide suspension has a particle size distribution between about 2 μm and about 10 μm.

35. The method according to claim 34, wherein the pharmaceutical formulation is substantially devoid of sulfate, nitrate, or chloride anions

36. The method according to claim 35, wherein the pharmaceutical formulation has a heavy metal content of less than about 20 ppm.

37. The method according to claim 36, wherein the suspension is isotonic.

38. The method according to claim 22, wherein the aluminum salt is aluminum oxyhydroxide and is administered to the patient subcutaneously, intranodally, intradermally, or intramuscularly.

39. The method according to claim 38, comprising administering the aluminum salt to the patient subcutaneously in the upper arm.

40. The method according to claim 22, wherein the aluminum salt is aluminum oxyhydroxide and is administered to the patient at least once monthly for at least two years.

41. The method according to claim 22, wherein the aluminum salt is aluminum oxyhydroxide and is administered to the patient by an injection device.

42. The method according to claim 22, wherein the aluminum salt is aluminum oxyhydroxide and is administered to the patient in liquid form in a volume of from about 0.1 to about 10 ml.

Description

[0060] The invention is further explained by way of the following examples and the figures, yet without being limited thereto.

[0061] 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.

[0062] 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 pronounced in the cohort of patients in earlier disease stages.

[0063] 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,NS,HC: <0.0001; 2 mg vs. HCADNI,NS,HC: <0.0001).

[0064] FIG. 4 shows development of volume (in mm.sup.3) of right hippocampus for 2 mg and 1 mg aluminium oxyhydroxide treatment group of the mild population of patients (the mild population is defined by a baseline MMSE score of 24 and higher), showing that this effect is most pronounced in the cohort of patients in earlier disease stages.

[0065] FIG. 5 shows the Quality of Life-Alzheimer's disease (QOLAD) for caregivers. Caregivers completed the measure as a questionnaire 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.

[0066] FIGS. 6A and 6B show immune response of the mice tested in the Tg2576 animal model: Tg2576-mice were injected 6×, s.c., at 4-week intervals with either conjugate-vaccine containing 30 μg net peptide, KLH formulated with Alum or Alum only. Alum doses used were equivalent to 2 mg/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 analyzed for their concentration of IgG Abs against specific peptides. Values depicted are the titer calculated as OD max/2 (at 405 nm) plus SEM. IgG response towards 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 Aβ1-40/42 after immunization. SeqID 1 (n=10) and SeqID 2 (n=8), treated animals show anti A1340/42 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.

[0067] 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). Naïve wt animals (n=20) were used as positive controls for Contextual fear conditioning (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.

[0068] FIG. 8 shows amyloid load in the animals tested: Groups of Tg2576 mice (n≤10/group) received 6 monthly injections of KLH/ALUM (n=9) or SeqID 1 (n=10)-, SeqID 2 (n=7)-conjugate vaccines or ALUM only (n=8). Alum dose in all formulations equivalent to 2 mg/ml. Brains were isolated, 8 weeks after the 6th immunization. Quantification of the relative total brain area covered by amyloid deposits (in % of total tissue analyzed) is based on immuno-fluorescence staining using the Aβ 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 Aβ deposition is detectable in Alum only treated vs. KLH-Alum treated animals. (ns) Arrowhead in C indicates unspecific fluorescence from a cerebral vessel. Scale bar: 200 μM; pictures taken at 10× magnification.

EXAMPLES

[0069] 1. Excerpt of an AD clinical trial (AFF006; Eudract: 2009-016504-22)

Materials and Methods:

[0070] 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 into the 2 study arms. Participation of a given patient lasted 18 months.

Study Design:

[0071] 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 defined in the protocol. It was performed in a total of 6 countries: Austria, France, Germany, Slovakia, Czech Republic and Croatia.

[0072] 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 establish the patients' baseline characteristics. Following screening, eligible patients were randomly allocated to the treatment groups. After randomization at week 0, patients received 6 injections 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).

[0073] 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 after 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

[0074] The study was done in patients with early AD. Diagnosis was defined by the following criteria: [0075] probable Alzheimer's disease as defined by NINCDS/ADRDA criteria (1) [0076] MMSE score ≥20 (2) [0077] result of Free and Cued Selective Reminding Test (FCSRT) result of total recall ≤40 or free recall ≤17, indicating hippocampal damage impairing the patient's episodic memory (3) [0078] the result of a centrally read MRI of a patient's brain must be compatible with the diagnosis AD, in particular, presence of a medial temporal lobe atrophy (Scheltens Score ≥2) (4)

[0079] Other in-/exclusion criteria applied (e.g., written informed consent; age between 50 and 80 years, treatment with immunosuppressive drugs (exclusion)).

Administration of Study Drug

[0080] During the study Visits 2, 3, 4, 5, 7 and 9 the patient received 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 presence of an intact regional lymph node station. If the draining lymph node stations of both upper arms were not intact, injection was placed into the thigh close to the inguinal lymph nodes. Two alternating injection sites (e.g. left and right upper arm, left upper arm and left thigh) were used throughout the 6 injections.

[0081] Injections were applied to the subcutaneous tissue (s.c.). Special care was taken to avoid intravasal application by careful aspiration before each injection. All administrations were performed at the trial site.

Volume-Based Morphometry

[0082] 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 determined using a fully-automated method which combines transformations derived from the nonlinear registration of the atlas labels to individual subject scans and subject-specific image information (Collins et al., J. Comput. Assist. Tomogr., 18: 192-205, 1994). Lateral ventricle and hippocampal segmentations that failed post-processing QC review were manually corrected. The total intracranial volume (TIV) was estimated from the brain mask generated during pre-processing and the average TIV (TIV.sub.avg) for each subject was determined by averaging the estimated TIV across visits. The normalization factor (TIV.sub.template/TIV.sub.avg_subject) was used to normalize the hippocampal and ventricular volumes for each subject in order to account for differences in head size.

Safety Assessments:

[0083] Safety evaluations included the following: [0084] adverse events (AEs) and serious adverse events (SAEs) (number of patients who withdrew due to AEs; reason for withdrawal) [0085] Laboratory assessments: hematology, biochemistry, coagulation, serology, urinalysis, APP crossreactivity [0086] vital signs (blood pressure, heart rate, respiratory rate and body temperature) [0087] physical and neurological examination

Efficacy Assessments:

[0088] 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 composite:

[0089] 1. Co-Primary: Adapted ADAS-cog;

[0090] 2. Co-Primary: Adapted ADCS-ADL;

[0091] 3. Combined Primary Outcome: Composite score.

[0092] 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.

[0093] 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 opposite 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 performance. The following items included in the adapted ADAS-cog and combined composite require reverse scoring: Verbal PAL, NTB Category Fluency and CogState ONB.

Secondary Efficacy Outcomes:

Quality of Life (QOL) Caregiver

[0094] 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 excellent. 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

[0095] Subjects were described using demographic information and baseline characteristics recorded during the screening phase (Visit 1).

[0096] 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

[0097] 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 endpoint. This analysis assessed whether or not there is a difference in estimated CFB values between treatment groups.

[0098] 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 following covariates and fixed effects: [0099] Age (covariate); [0100] Level of Education (fixed effect split into categories of 7.2 years, >12 years); [0101] Gender (fixed effect); [0102] Baseline Test Score of Efficacy Parameter (covariate); [0103] Center (fixed effect); [0104] Treatment (fixed effect); [0105] APOEe4 status (fixed effect, positive or negative); [0106] Use of AChE Inhibitors (fixed effect, determined from medications); [0107] Time (covariate, time will be defined in terms of visits); [0108] Time by Treatment Interaction (Time*Treatment);

[0109] The covariance structure for the model was first-order heterogeneous autoregressive (ARH[1]). Least-squares means were estimated at each visit in the study. The LS mean at a particular visit was interpreted as the expected CFB in the efficacy outcome at that time point (Visit) when the specified treatment was administered. Least squares means and standard errors were estimated from the mixed model at each visit and are shown for the various groups.

[0110] 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 cognitive and basic functions. The following equation is used to derive the combined primary outcome, i.e. combined Composite score:

[0111] Combined Composite Score:

[0112] =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 Appointments+0.37*Travel+0.33*Alone+0.35*Appliance+0.49*Clothes+0.36*Read+0.62*Telephone+0.33*Writing

[0113] The percent contribution of each item to the combined Composite score can be found in Table 1 below:

TABLE-US-00001 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

[0114] 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).

[0115] 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 innate immune response.

[0116] 2 mg aluminium group compares favourably even to the 1 mg aluminium group (other groups) with regard to parameters informing 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, exemplified with Adapted ADAS-cog (FIG. 3).

[0117] Results obtained were compared to public datasets. Historical datasets identified were the ADNI 1 mild AD cohort (observational study), the mild placebo patients from the ADCS Homocysteine 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 (HCADNI,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.

[0118] Although the adapted ADAS-cog used some items from the ADAS-cog supplemented with items from the NTB and the CogState Battery, 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 Instructions+0.81*Spoken Language+1.01*Word Finding).

[0119] The adapted ADAS-cog2 shows substantially more decline in the historical control group than the 1 and 2 mg aluminium oxyhydroxide treated groups from the AFF006 study (FIG. 3). The p-values were: 1 mg vs. HC-ADNI, NS, HC: <0.0001; 2 mg vs. HC-ADNI, NS, HC: <0.0001.

[0120] Also the MRI data show a statistically significant disease modifying effect for the 2 mg group of patients and a correlation of the hippocampus volume with clinical endpoints, e.g. right hippocampus with adapADAS: p=0.0006 or Composite score: p=0.0095) (FIG. 4). It has to be specifically mentioned that the present investigation has provided for the first time a parallel development of clinical data with a radiologic biomarker (MRI in the present case)).

[0121] FIG. 4 shows that the patients treated according to the present invention showed almost no AD related reduction 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.).

[0122] FIG. 5 shows that caregivers of patients treated according to the present invention rated the QOL of the patient as significantly improved over a period of 18 months following 2 mg compared to 1 mg Alum and other groups (not shown).

TABLE-US-00002 TABLE 2 Patient Population and Disposition 1 mg 2 mg Patient Disposition (N = 48) (N = 51) Number of Subjects n (%) Completed 41 (85.4%) 45 (88.2%) Discontinued 7 (14.6%) 6 (11.8%) P-value.sup.1 Reason for Discontinuation from the Study: Death 2 (4.2%) 0 (0.0%) Adverse Event 0 (0.0%) 0 (0.0%) Withdrawal by Subject 4 (8.3%) 5 (9.8%) Lost to Follow-up 0 (0.0%) 0 (0.0%) Other 1 (2.1%) 1 (2.0%)

TABLE-US-00003 TABLE 3 Demographics - Race, Gender, Education, Age 1 mg 2 mg Demographics (N = 48) (N = 51) Race Asian/Pacific 0 (0.0%) 1 (2.0%) Islander Caucasian 48 (100.0%) 50 (98.0%) Gender Male 28 (58.3%) 19 (37.3%) Female 20 (41.7%) 32 (62.7%) P-value.sup.1 Education Years Mean (SD) 12.3 (4.03) 11.8 (3.18) Median 12 11 (Q1, Q3)  (9.0, 15.0) (10.0, 13.0) Min, Max  8, 26  6, 22 P-value.sup.1 Age (yrs) n 48 51 Mean (SD) 70.3 (6.56) 68.9 (8.36) Median 71 69 (Q1, Q3) (65.0, 75.5  (64.0, 77.0) Min, Max 57, 80 50, 80 P-value.sup.1 Weight (kg) n 48 51 Mean (SD) 70.45 (10.375) 67.62 (13.700) Median   70.5 65 (Q1, Q3) (64.00, 77.70) (57.00, 78.00) Min, Max  47.5, 101.0  45.0, 100.0 P-value.sup.1 BMI (kg/m.sup.2) n 48 51 Mean (SD) 24.66 (2.903) 24.81 (3.627) Median   24.8   24.2 (Q1, Q3) (22.95, 26.15) (22.30, 27.30) Min, Max 17.8, 31.2 18.2, 35.4 P-value.sup.1

TABLE-US-00004 TABLE 4 Adverse Event Summary of Local Reactions MedDRA System Organ Class 1 mg 2 mg Preferred Term (N = 48) (N = 51) Number of subjects with 31 (64.6%) 42 (82.4%) reported adverse event Number of unique events 96 162 General Disorders and 31 (64.6%), 209 42 (82.4%), 487 Administration 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 2 (4.2%), 2 4 (7.8%), 9 Hypersensitivity 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 0 (0.0%), 0 0 (0.0%), 0 Temperature 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: 0 (0.0%), 0 0 (0.0%), 0 Paraesthesia Nervous System Disorders: 0 (0.0%), 0 0 (0.0%), 0 Dizziness 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 0 (0.0%), 0 1 (2.0%), 1 Tissue Disorders: Pain in Extremity 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 Aβ targeting vaccines SeqID 1-KLH-Alum and SeqID 2-KLH Alum in comparison to KLH-Alum and Alum only

SeqIDs:

SeqID 1: SWEFRTC

SegID 2: SEFKHGC

Animal Experiments:

[0123] All animal experiments were performed in accordance with the Austrian Animal Experiments Act (TVG2012) using Tg2576-mice (Taconic Farms, USA; 12956/SvEvTac). General health was checked by modified Smith Kline Beecham, Harwell, Imperial College, Royal London Hospital, phenotype assessment (SHIRPA) primary observational screen (Rogers D C 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% Paraformaldehyde (PFA, Sigma Aldrich, USA), dehydrated and paraffin-embedded. Brain tissue was sectioned at 7 μM using a sliding microtome (Leitz, Germany) and sections were mounted on Superfrost Plus Slides (Menzel, Germany).

Titer Determination by ELISA:

[0124] Standard enzyme-linked immunosorbent assay (ELISA) technology 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) Aβ1-40/42 (at 5 μg/ml), KLH (1 μg/ml) and peptide-Bovine serum albumin (BSA) conjugates (SegID 1 and SegID 2, 1 μM). Optical density (OD) was measured at 405 nm using a micro-well reader (Tecan, CH). ODmax/2 was calculated.

Behavioral Tests:

[0125] To analyse cognitive dysfunction, immunised Tg2576 animals were subjected to contextual fear conditioning (CFC, Comery T A 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 0.8 mA 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 s120-240 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 Aβ:

[0126] Immunofluorescence (IF) analysis was done as described previously (Mandler M et al. (2012) J Alzheimers Dis 28: 783-794). For Aβ-specific IF-staining, brain sections of immunized Tg2576 were processed for analysis of amyloid load using mAb 3A5 (AFFiRiS AG, Austria). All secondary reagents used were obtained from Vector Labs (USA). For IF, sections were mounted and counterstained 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 determining the relative cerebral area occupied by amyloid deposits using a semi-automated area recognition program (eDefiniens Architect 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 tissue artifacts or aberrant staining were excluded. To assess the number of Aβ-positive vessels, 3A5 stained sections (3 slides/animal covering cortex and hippocampus and up to five individual sections per slide) have been analysed. Aβ-positive vessels were manually counted in sub-regions of the cortex as well as in the hippocampus. Number of positive vessels per mm.sup.2 was determined.

REFERENCES

[0127] Rogers et al., Behav Brain Res 105 (1999): 207-217. [0128] Mandler et al., PLoS ONE 10(1) (2015): e0115237. doi:10.1371/journal.pone.0115237. [0129] Mandler et al., J Alzheimers Dis 28: 783-794. [0130] Comery et al., J Neurosci 25 (2005): 8898-8902.

Results:

[0131] To test the immunogenicity of two Aβ targeting vaccines SeqID 1-KLH-Alum and SeqID 2-KLH Alum in comparison to KLH-Alum and Alum (Aluminium-oxyhydroxide) only, Tg2576-mice were injected 6×, s.c., at 4-week intervals with either conjugate-vaccine containing 30 μg net peptide, equivalent doses of KLH formulated with Alum or Alum only. Alum doses used were equivalent to 2 mg/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 Aβ targeting vaccines, SeqID 1-KLH-Alum and SeqID 2-KLH-Alum, elicited Abs to human Aβ whereas KLH-Alum vaccine and Alum only did not elicit signals above background in treated animals (FIG. 6B).

[0132] To evaluate the effect of Aluminum-oxyhydroxide only (Alum) in comparison to Aβ targeting vaccines (SeqID 1-+SeqID 2-KLH-Alum) and non Aβ specific vaccines (KLH-Alum) on cognitive functions, 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 superior to control animals receiving KLH-Alum (thus not eliciting an Aβ specific immune response) in this AD model of Aβ deposition (FIG. 7). Interestingly, animals receiving Alum only, (without a conjugate eliciting an active immune response against KLH or Aβ, respectively), showed similar effects as detectable with Aβ targeting vaccines in this AD model in the absence of Aβ-specific antibodies.

[0133] To test whether Alum would also significantly influence cerebral 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-injected 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 (FIGS. 8B+D and E, p<0.05 and data not shown). Importantly, 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.

[0134] Thus, FIGS. 7 and 8 also 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 cerebral Aβ levels. This is implying an APP/Aβ independent mechanism underlying beneficial functional effects exerted by aluminium-oxyhydroxide in this AD model and further evidences the lack of scientific plausibility of the “amyloid channel hypothesis”.