The present invention provides means for treatment diseases associated with the presence of amyloid deposits, which include Alzheimer's disease.

Certain facts are known about the biochemical and metabolic phenomena associated with the presence of Alzheimer's Disease (AD). Two structural and histopathological changes observed in the brains of those with AD are neurofibrillar tangles (NFT) and amyloid deposits. Intraneuronal neurofibrillar tangles are also present in other neurodegenerative diseases, but the presence of amyloid deposits both in the intraneuronal spaces (neuritic plaques) and close to the microvasculature (vascular plaques) seems to be characteristic of AD. Of these, neuritic plaques seem to be the most common (Price, D.L., and co-workers, Drug Development Research (1985) 5:59-68).

The main component of these amyloid plaques is a peptide of 40-42 amino acids denominated amyloid peptide Aβ4.

The amyloid peptide Aβ4 is a polypeptide that originates from proteolysis from membrane glycoproteins denominated amyloid peptide Aβ4 precursor proteins (βAPP). These proteins, precursors of amyloid peptide, consist of 695 to 770 amino acids, all of them being coded by the same gene.

Two main variants of amyloid peptide Aβ4 have been identified, peptide Aβ40 and Aβ42, containing 40 and 42 amino acids, respectively, which present different tissue distributions in both physiological and pathological conditions. The variant of 42 amino acids is the predominant form in the amyloid plaques located in the brains of patients with AD.

Until present, different possible solutions have been proposed to provide a possible vaccine against AD.

In EP526511 , the administration of homeopathic doses of Aβ to patients with pre-established AD is proposed. However, due to the doses used, the levels of circulating endogenous Aβ in plasma hardly vary, and so no therapeutic benefit is expected.

Schenk et al., (Nature, 1999; 400: 173-177) described immunization of transgenic mice PDAPP with Aβ42, which over expressed human mutant APP, thus preventing the formation of amyloid plaques, neuritic dystrophy and astrogliosis.

In WO9927944 (Schenk D.), a treatment for AD is described by administration to a patient of Aβ42.

A phase III clinical trial in 360 patients diagnosed with medium to moderate AD in 4 European countries and the United States, in which amyloid peptide Aβ42 was used as an antigen, was discontinued after encephalitis was reported in some of the patients (Scrip Daily Online, 25 Feb 2002, S007455320, The Scientist 16 [7]: 22, April 1, 2002).

The problem of using an endogenous protein as a vaccine (or a protein present naturally in the animal that is being vaccinated), as is the case of peptide Aβ42, the organism responds by making antibodies against Aβ42 and against smaller fractions that may also have as yet unknown physiological functions, among some of the possible problems we could mention is the possible development of autoimmune diseases due to the generation of antibodies against the endogenous protein, difficulty in the generation of a immune response due to failure of the immune system for recognizing endogenous antigens, and possible development of an acute inflammatory response.

The present invention is aimed at treatment of Alzheimer's disease and other amyloid diseases by administration of a peptide, of the C-terminus part of Aβ, conjugated with a protein, which in a preferred embodiment of the present invention said protein is the keyhole limpet hemocyanin.

The present invention relates to a vaccine for the treatment of Alzheimer's disease and other related amyloid diseases.

In particular, the present invention is directed to a pharmaceutical composition comprising a peptide with an amino acid sequence comprising SEQ ID No 2, that acts as immunogen to induce antibodies that react with Aβ for use in the treatment of an amyloid disease. The present invention is also directed to a pharmaceutical composition comprising an antibody able to specifically recognize any of the predominant variants of the peptide beta amyloid Aβ40 and Aβ42, for use in the treatment of an amyloid disease, wherein the antibody is obtainable by immunization of mammals or birds, with an amino acid sequence comprising SEQ ID No 2, wherein the amino acid sequence is conjugated to a protein.

According to a preferred embodiment, a vaccine is provided for the treatment of Alzheimer's disease and other related diseases, which overcomes the disadvantages associated with using peptides, proteins or endogenous immunogens.

Examples of other diseases characterized by amyloid deposits are Islandic hereditary syndrome, multiple myeloma, and spongiform encephalitis, including Creutzfeldt-Jakob disease.

The introduction of an immune response can be active such as when an immunogen is administered to induce antibodies that react with Aβ in a patient, or passive, such as when an antibody is administered that reacts by itself with Aβ in a patient.

For the aims of the present invention, the following terms are defined as follows:

The term "related amyloid diseases" includes diseases associated with the accumulation of amyloid which can be restricted to one organ, localized amyloidosis, or spread throughout several organs, systemic amyloidosis. Secondary amyloidosis can be associated with chronic infections (such as, for example, tuberculosis) or chronic inflammation (for example, rheumatoid arthritis), familial Mediterranean fever (FMF) and other types of systemic amyloidosis found in patients in the long-term treatment of hemodialysis. Localized forms of amyloidosis include, but are not limited to, type II diabetes and any other disease related thereto, neurodegenerative diseases with scrapie, bovine spongiform encephalitis, Creutzfeldt-Jakob disease, Alzheimer's disease, cerebral amyloid angiopathy.

The term "passive immunization" is used to relate to the administration of antibodies or fragments thereof to an individual with the intention of conferring immunity on that individual.

In the first aspect, there is described the use of either a peptide that acts as an immunogen or as an antibody, in the preparation of a medication for the treatment of a disease characterized by the accumulation of amyloid deposits. Said methods consist of the induction of an immune response against a peptide component of the amyloid deposits in the patient. Said induction could be active through administration of an immunogen or passive through administration of an antibody or an active fragment or derivative of an antibody.

In a preferred embodiment of the present invention, the disease is Alzheimer's disease.

The medication obtained can be used both in asymptomatic patients such as those who show symptoms of the disease.

The compositions able to provoke an immune response directed against certain components of the amyloid plaques are effective for treatment of diseases related to amyloid deposits. In particular, it is possible to prevent the progress of, reduce the symptoms of and/or reduce the deposition process of amyloid in an individual, when an immunostimulatory dose of a peptide or of an antibody obtained therefrom, is administered to the patient.

In accordance with the present invention, the antibodies are obtained by immunization of mammals or birds by use of a peptide conjugated to a protein as an immunogen.

According to a preferred embodiment of the present invention, the mammals used for immunization can be ruminants, equines, lagomorphs, carnivores, primates, or any other animal that allows adequate quantities of serum to be extracted therefore for antibody. Among the birds used for immunization, we can mention, but in no way limit to, Galliformes, Anseriformes and Columbiformes, among others.

The present invention, provides the use of a peptide with an aminoacid sequence comprising SEQ ID No 2 that acts as an immunogen to induce antibodies that react with Aβ for use in the treatment of an amyloid disease.

The present invention also provides the use of an antibody able to specifically recognize any of the predominant variants of the beta amyloid peptide, Aβ40 and Aβ42 in the preparation of a medicament for the treatment of an amyloid disease, wherein the antibody is obtainable by immunization of mammals or birds, with an amino acid sequence comprising SEQ ID No 2, wherein the amino acid sequence is conjugated to a protein.

According to the most preferred embodiment of the present invention, the protein used for conjugation with the peptide is keyhole limpet protein.

In this application, the amino acids are abbreviated using the single-letter codes accepted in the field, as indicated below: A = Ala = alanine C = Cys = cysteine D = Asp = aspartic acid E = Glu = glutamic acid F = Phe = phenylalanine G = Gly = glycine H = His = histidine I = Ile = isoleucine K = Lys = lysine L = Leu = leucine M = Met = methionine N = Asn = asparagine P = Pro = proline Q = GIn = glutamine R = Arg = arginine S = Ser = serine T = Thr = threonine V = Val = valine W = Trp = tryptophane Y = Tyr = tyrosine

The sequences described in the present invention, and identified as SEQ ID No 2, correspond to the following amino acid sequence:

• SEQ ID NO 2 GLMVGGVV

The antibody obtained from the previous peptide is given the code SAR-3 corresponding to the one shown below:

• SEQ ID NO 2 SAR-3

The information relating to identification of the peptide sequence, described in the present invention, that accompanies the present document in a computer-readable format, is indicated in the list of sequences that is presented along with this document.

The present invention is illustrated by means of the following examples.

The polyclonal antibody against the peptide conjugated with KLH that was used as immunogen was generated by immunization in New Zealand white rabbits.

The immunogen was injected into two rabbits, with five injections in each rabbit: the first intradermal injection of the peptide-KLH conjugate in PBS and emulsified in complete Freud adjuvant and four more intramuscular injections, as a booster dose on days 14, 28, 49 and 80, of the same peptide-KLH conjugate in PBS but this time emulsified in incomplete Freud adjuvant, with the blood letting done at 90 days to detect the presence of antibodies.

After collecting blood, the serum was separated and pre-purified by desalination and then the antibodies were purified by affinity in a matrix comprising 1.5 ml of EMD-Epoxy activated material (Merck) to which 5 mg of the corresponding peptide was added. The purified fractions were packed in 0.1 % BSA (Sigma) and stored at 4°C, and glycerol 20-50% could be added as a cryoprotector.

The Laemmli method was used, described in Current Protocols in Molecular Biology, John Wiley and Sons, New York, 1998, modified by improve the separation of small peptides.

The apparatus used was a Miniprotean 3 from Bio-Rad.

A 15% acrylamide gel was used, mixed with the following components: STOCK SOLUTIONS SEPARATING GEL (15%) STACKING GEL 40% acrylamide 3.75 ml 500 µl Tris 3 M, pH = 8.45 3.3 ml 250 µl Glycerol 1.05 ml - Water 1.9 ml 4.2 µl SDS 20% 50 µl 18.6 µl APS 10% 50 µl 25 µl TEMED 10 µl 5 µl

Initial stock solutions of peptide Aβ40 and 42 of 1 mg/ml were used (dissolved in PBS). The volume necessary was taken of these solutions for each one of the samples and made up to 20 µl with SBLT (SBL + Tris base 2 M). The samples were then boiled for 5 minutes to denature the peptides and eliminate possible proteases.

The center of the cuvette was filled with cathode buffer and the outside with anode buffer, the composition of these buffers being as follows:

• Anode buffer
  24.2 g Tris base (0.2 M final concentration)
  Dilute to 1 litre with H₂O
  Adjust of pH 8.9 with concentrated HCl
  Store at 4°C for up to 1 month

• Cathode buffer
  12.11 g Tris base (0.1 M final concentration)
  17.92 g tricine (0.1 M final concentration)
  1 g SDS (0.1 % final concentration)
  Dilute to 1 litre with H₂O
  Do not adjust Ph
  Store at 4°C for up to 1 month

Finally, the samples were loaded into the wells: 20 µl/well. Using the Polypeptide Standard Kaleidoscope from Bio-Rad as a marker, migration started at low voltage (30 V), and then the voltage was raised to 100 V, after approximately 1 hour of electrophoresis.

The proteins separated in the gel were transferred to the PVDF membrane by electroblotting. In the transfer booklets the following were placed

Black side -sponge- 3 Whatmann papers (or filter papers) -gel- - membrane- -3 Whatmann papers- -sponge- -transparent side.

The cuvette was then filled with electroblotting buffer:

• Glycine 38 nM
• Tris base 50 mM
• Methanol 40%

The transfer was done for 2 hours at 200 mA. During the transfer, the buffer was kept stirring with the magnetic stirrer.

The antibody and the powder milk were dissolved in PBS-t (PBS + 0.5% Tween 20), carrying out the washing with PBS-T also.

After the transfer, the surface of the membrane was blocked with 5% solution of powder milk for 1 hour with stirring and at room temperature (RT)

After this, the membrane was washed for 2 x 5 minutes at RT.

Then, it was incubated with primary antibody ( SAR-3 ) for 1 hour at RT at least diluted 1:500 in PBS-T.

The membrane was washed: 3 x 10 minutes at RT. Then, it was incubated with secondary antibody: goat anti-rabbit-HRP for 1 hour at RT (1:10,000 in all cases).

The washing of the membrane was repeated once again: 3 x 10 minutes at RT.

After the last washing, the membrane was incubated with the solution of the chemoluminescence kit, using the ECL kit+Plus from Pharmacia.

The membrane was wrapped in cellophane and exposed to double-emulsion film (Hyperfilm MP from Amersham), for different times of between 30 seconds and 2 minutes.

The sections of tissue were fixed in paraffin following the following steps:

1. a) fixation in neutral formol at 10%
2. b) dehydration by successive steps in increasing concentrations of alcohol
3. c) passes through xylol and paraffin, this latter step in an oven at 60-62°C.
4. d) carrying out of paraffin blocks, which were cut to 4 microns and mounted in slides.

The sections were then deparaffinized by passing through the following solutions: Xylol 100% 10 minutes Xylol 100% 10 minutes Ethanol 100% 5 minutes Ethanol 100% 5 minutes Ethanol 96% 5 minutes Ethanol 90% 5 minutes Ethanol 70% 5 minutes PBS 5 minutes x 3 times

Afterwards, they were treated in the following way:

1. a) 96% formic acid for 3 minutes in a fume cupboard and with stirring
2. b) Rapid washing with water
3. c) Washing in PBS 2 x 5 minutes
4. d) Block of the endogenous peroxidases for 15 minutes in a solution made up of 70 ml of PBS, 30 ml of methanol and 1 ml of H₂O₂
5. e) Washing in PBS 3 x 5 minutes
6. f) Washing in PBS/T (Triton or Tween-20 at 0.5% in PBS) 3 x 5 minutes
7. g) Block of the non-specific binding with goat serum (Normal Goat Serum) diluted 10:100 in PBS/T for two hours
8. h) Incubation of the primary antibody all night at 4°C in a moisture chamber:
   • Sar-3... Dilution 1:1500 in PBS
9. i) Washing in PBS/T 3 x 5 minutes
10. j) Incubation in secondary antibody (anti-rabbit goat) diluted 1:200 in PBS during 45 minutes
11. k) Washing in PBS 4 x 5 minutes
12. l) Incubation of ABC (avidin-biotin complex) of Vector Labs at a dilution of 1:100 in PBS/T for 45 minutes in darkness, keeping these conditions until development was complete
13. m) Washing in PBS 3 x 5 minutes
14. n) Development in diaminobenzidine (DAB)

The time was controlled empirically under a stereoscopic microscope. For this, first, a washing was done in a solution of Tris-HCl 0.5 M for 10 minutes with shaking, to then continue with incubation with a diaminobenzidine substrate (DAB) diluted in Tris-HCl 0.05M and to which is added 0.5 µ/ml of H₂O₂ at 4°C. Once the reaction was finished, three washes were done in PBS at 4°C for 5 minutes each time and then dehydration in ethanol was done at 70%, 90 % and 100% for 2 minutes each time, passing through xylol for 4 minutes and a further pass through xylol for 2 minutes, until they were mounted with Eukitt for observation under the microscope.

• NUMBER OF SEQUENCES: 1
• INFORMATION ON SEQUENCE 1:
  CHARACTERISTICS OF THE SEQUENCE:
  LENGTH: 8
  TYPE: amino acid
  TYPE OF MOLECULE: peptide
  SOURCE: Chemical synthesis
• SEQUENCE DESCRIPTION:

• <110> ARACLON BIOTECH, S.L.
• <120> Alzheimer's disease treatment method
• <130> EP1513.A.2.div1.div2
• <140> EP10012342.1
  <141> 2010-09-30
• <160> 4
• <170> PatentIn version 3.5
• <210> 1
  <211> 8
  <212> PRT
  <213> Artificial Sequence
• <220>
  <223> A beta fragment of beta amyloid peptide
• <400> 1
• <210> 2
  <211> 8
  <212> PRT
  <213> Artificial Sequence
• <220>
  <223> A beta fragment of beta amyloid peptide
• <400> 2
• <210> 3
  <211> 10
  <212> PRT
  <213> Artificial Sequence
• <220>
  <223> A beta fragment of beta amyloid peptide
• <400> 3
• <210> 4
  <211> 12
  <212> PRT
  <213> Artificial Sequence
• <220>
  <223> A beta fragment of beta amyloid peptide
• <400> 4