Vaccine against intermediate with amyloid coagulation

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biochemistry, in particular to cyclic peptide, capable of inducing antibody response. Peptide consists of formula X2X3VGSNK-Z or X3VGSNKG-Z, where X2 represents E, G, Q or K, X3 represents D or N and Z represents agent, stabilising bent, present inside peptide sequence, with peptide being cyclised by covalent bonding of N-end amino acid with Z, where Z represents peptide fragment YNGK. Conjugate, containing said cyclic peptide, conjugated with immunogenic carrier molecule is also claimed. peptide and conjugate, which contains it, can be applied for production of medication or as vaccine against Alzheimer's disease. Methods of obtaining cyclic peptide and conjugate are also claimed.

EFFECT: invention makes it possible to obtain improved, less toxic vaccine against Alzheimer's disease.

8 cl, 11 dwg, 1 tbl, 2 ex

 

Area of technology

The invention relates to an improved vaccine that can be used for the treatment of Alzheimer's disease.

Art

According to the world Health Organization around the world from Alzheimer's disease suffers 18 million people (Vas et al. 2001). In the Netherlands, the Alzheimer's disease identified in approximately 250,000 people. The problem increases with the average age of the population. Estimated costs for patient care in the medical-rehabilitation center for the aged are 30000-60000 euros per year (McDonnell et al. 2001). Vaccination would be cost-effective.

Alzheimer's disease is a neurodegenerative disorder (Sadowski & Wisniewski 2004, Blennow et al. 2006, Editorials, Nature Med. 2006). Characteristic of the disease is the formation of plaques in the brain or in the blood vessels of the brain. These plaques originate from neural membrane-associated protein, amyloid protein precursor. α-Helical fragment from 38-43 (usually 42) amino acid residues enzymatically cleaved from the protein, thus forming a peptide called "soluble Aβ", which probably initially adopt a stretched conformation and is present in all body fluids. If soluble Aβ reaches a high concentration, it undergoes conformational changes and obraze� units. Many aggregates have been detectedin vitroorin vivoincluding many conformers of the monomer, different types of oligomers resulting from soluble Aβ ligands, protofibrils, fibrils and spheroids (taken from Klein et al. 2004). Fibrillar Aβ has a cross-beta conformation (Sawaya et al. 2007) and eventually is deposited in the brain with the formation of neurodegenerative plaques.

As it turned out, immunization of transgenic mice (Schenk et al. 1999) and humans in phase I clinical trials (Hock et al. 2002) using suspension "pre-aggregated" Aβ1-42 showed a therapeutic effect. Antibodies in human immune serum recognized plaques, Aβ-deposition and β-amyloid structure in the blood vessels of the brain. The antibody does not recognize amyloid protein precursor or soluble Aβ.

The lack of "pre-aggregated" suspension Aβ1-42 is that the physical properties of this material is unclear. But a much more serious problem is the induction of meningoencephalitis in the form associated with the vaccine side effect, manifested in 6% of patients in phase II clinical trials (Check 2002, Gilman et al. 2005). This side effect is caused by a cellular inflammatory reaction, attributed to ThI cell response to epitopes localized in the Central and C-terminal part of Aβ 1-42 (McLaurin et al. 2002, Gelinas et al. 204). It was demonstrated that providing therapeutic effect of the antibodies induced by Aβ1-42, directed against the N-Terminus (McLaurin et al. 2002, (Lee et al. 2005). Thus, it was proposed the use of truncated C-Terminus Aβ-peptides as immunogens (Sigurdsson et al. 2004, Lemere et al. 2006, Gevorkian et al. 2004, Lemere et al. 2007). Such short peptides are slaboimmunogennymi. With the aim of increasing the immunogenicity of multiple copies of the peptide were to be condensed with a non-immunogenic carriers (to induction of IgM) or with carriers that provide heterologous T-cell epitopes (Agadjanyan et al. 2005, Ghochikyan et al. 2006, Maier et al. 2006 (Movsesyan et al. 2008)). It was expected that in none of these conjugates the peptide does not adopt a conformation residues 4-10 when exposed with the help of β-amyloid oligomers or pre-fibrils. Thus, it is expected that the antibodies induced by truncated peptide conjugates are subspecific for oligomers or pre-fibrils.

Thus, there still exists the need for an effective drug, preferably a vaccine against Alzheimer's disease. The present invention provides an improved vaccine that has all of the shortcomings of existing vaccines: is less or no toxic and able to induce an effective antibody response to immune�ation. The vaccine is proposed in the present invention, is a new analogue of β-amyloid peptide.

Description of the invention

The peptides of the invention

In the first aspect of the invention proposes a peptide comprising the following sequence X1X2X3VGSN-Z, X2X3VGSNK-Z or X3VGSNKG-Z, where X1represents A or G, X2is an E, G, Q or K, X3represents D or N, and Z is an agent stabilizing the bend present within the sequence of peptide X1X2X3VGSN, X2X3VGSNK or X3VGSNKG. The peptides of the invention are modified peptides are herein to be understood as peptides that are not natural Aβ 1-42. Z can also be defined as an agent that stabilizes a conformation X1X2X3VGSN (SEQ ID NO:1), X2X3VGSNK (SEQ ID NO:2) or X3VGSNKG (SEQ ID NO:3), as the most acceptable in Aβ 1-42, preferably acceptable in Aβ 1-42. Peptide sequence X1X2X3VGSN, X2X3VGSNK or X3VGSNKG identified above correspond to amino acids 22 to 28 and 23 to 29 respectively of Aβ 1-42. The preferred peptide sequence represents X2X3VGSNK, which corresponds to amino acids 22-28 �W Aβ 1-42. Different possible identities for X1, X2and X3as indicated in this document was the result of the presence of several known mutations within the human population in the sequence of Aβ 1-42: X2is a 22 amino acid and primarily in populations of E. However it is also known mutations Arctic (Arctic) (E22G), Dutch (Dutch) (E22Q) and Italian (E22K). Was recently identified another mutation (E22Δ) (Tomiyama et al. 2008). X3is a 23 amino acid, and advantageously, it is D. However, mutation of Iowa (Iowa) has already been identified (D23N). Thus, the person skilled in the art it is clear that if later will be identified other mutations in a particular part of Aβ 1-42, as identified in this document; i.e., amino acids 21-27, 22-28 23-29 or, the sequence of the peptide according to the invention can be possibly adjusted to account for this later identified mutation.

It was tested multiple overlapping peptide sequences (see example). As far as we know, it was discovered that two of the tested peptide sequences (AK 22-28 23-29 or) able to induce antibody response in mice, and the antibody was able to recognize specific conformational epitope Aβ 1-42, expressio�himself in the form of a monomer, soluble oligomer (Haass and Selkoe 2007, Lambert et al., 2007, and Wash and Selkoe, 2007) fibrils or neurodegenerative plaques. Apparently, recognition of oligomeric Ab is even more critical than the detection of fibrils or plaque, as of oligomeric Ab is more toxic to neurons. The excretion of soluble oligomers rapidly improves the recognition, while the plaque is still present. The functionality of the peptide according to the invention are preferably tested as shown in example 2: ELISA. The use of conjugates peptide-BSA as coating antigens in ELISA allows the determination of anti-peptide titer, whereas oligomeric or fibrillar Aβ 1-42 enables the detection of specific cross-reactivity. The person skilled in the art would understand that any other peptide sequence resulting from Aβ 1-42 and incorporated into the peptide according to the invention and which is able to adopt a conformation X1X2X3VGSN, X2X3VGSNK or X3VGSNKG probably accept Aβ 1-42, also covered by the present invention.

In one embodiment the peptide of the invention contains a formula X1X2X3VGSN-Z, X2X3VGSNK-Z or X3VGSNKG-Z, where X1represents A or G, X2is an E, G, Q or K, X3is the FDS�th D or N, and Z is an agent, stabilizing the bend present within the sequence of peptide X1X2X3VGSN, X2X3VGSNK or X3VGSNKG.

The peptide according to the invention it is critical that the bend present within the X1X2X3VGSN, X2X3VGSNK or X3VGSNKG, stabilizing, since our aim is the design of a peptide that mimics a conformational epitope that is present in collapsed Aβ 1-42, which is expressed in the form of a monomer, soluble oligomer, fibrils or neurodegenerative plaques. Any way to achieve this stabilization covered by the present invention. Specialist after the synthesis of such a peptide of the invention may test his conformation using a method known in the art, for example by NMR, as described in example 1.

In one preferred embodiment of the first way of achieving this stabilization is a cyclization of the peptide according to the invention. Thus, a preferred peptide according to the invention is a cyclic peptide. The specialist knows how to cilitate peptide. Real cyclization reaction can be carried out between any satisfying the provisions in the sequence, including Z. in addition, the actual cyclization reaction can be carried out on a sequence of predecessor�nice, not containing Z, but with getting Z as a result of cyclization. Cyclization can be carried out by linking, preferably by covalent binding of N-terminal amino acid of the peptide sequence, preferably X1, X2or X3respectively in the X1X2X3VGSN-Z, X2X3VGSNK-Z or X3VGSNKG-Z with Z. Thus, the C-terminal amino acid of the peptide sequence X1X2X3VGSN, X2X3VGSNK or X3VGSNKG not employed in the cyclization. Convenient to carry out the cyclization in the solid phase. For example, D23, linked through the side chain with solid phase can cilitates with E22, leading to cyclo-E22-D23 with the linker of the carboxylic acids. In another case, D27, linked through the side chain with solid phase can cilitates with K28, leading to cyclo-D27-K28 with amide linker. Preferably the cyclization is carried out between amino acids in the loop, opposite Z, as, for example, from D to G (N-G) or from G to K*, if Z is a YNGK. It is also possible to cyclization in solution, for example, from G25 to S26 or from G to K*, if Z is a YNGK. It is believed that the cyclization is important for stabilizing the bend present within the X1X2X3VGSN, X2X3VGSNK or X3VGSNKG.

Another preferred�th method of cyclization of the peptide represents the addition of cysteine in the N - and C-terminal region of the peptide sequence or method by adding a cysteine in the N-end peptide sequence and another cysteine in Z. the Presence of two cysteines will give the possibility of disulfide cyclization, a well-known specialist. In another preferred embodiment the second way of achieving this stabilization is the use of z As previously indicated herein, Z is an agent stabilizing the bend present within the X1X2X3VGSN, X2X3VGSNK or X3VGSNKG in the peptide according to the invention. In a preferred embodiment Z stabilizes the bend present within the X1X2X3VGSN, X2X3VGSNK or X3VGSNKG, to ensure that the peptide is likely to adopt a coiled conformation Aβ 1-42. In a more preferred embodiment Z stabilizes the bend present within the X1X2X3VGSN, X2X3VGSNK or X3VGSNKG, to ensure that these peptides are expected to adopt a coiled conformation Aβ 1-42. Based on research it is believed that there is a bend in the coiled conformation Aβ 1-42, and a bend forecast is present in a position corresponding to the position between S and N in the X1X2X3VGSN, X2X3VGSNK or X3VGSNKG.

Z can be any agent known to the specialist, which stabilizes the bend, turn or loop. Z can be defined as "anti-torque" agent with high probability about�of adowania β-folds (Hutchinson et al., 1998; Woolfson et al., 1993). Z can be an amino acid, Oligopeptide, peptide, polypeptide, protein, antigen, mono - or oligosaccharide and/or steroid. In a preferred embodiment Z represents a peptide fragment of 8, 7, 6, 5 or 4 amino acids, with the more preferred less than its length. Preferably the peptide fragment of 4-8 amino acids is a "protivolodochnyi agent with a high predisposition to the conformation of β-folds. Preferred peptide fragment is a tetrapeptide selected from the group consisting of YNGK, TCGV, CGNT, LCGT, LKGT, GAIK, GAIC, AIIK and AIIC. More preferably, the tetrapeptide is selected from the group consisting of YNGK, TCGV, CGNT, LCGT and LKGT. Most preferably the tetrapeptide selected from the group consisting of YNGK, TcGV, CGNT, LcGT and LkGT, where c=D-Cys, and where k=D-Lys (see, Oomen et al. 2003; Oomen et al. 2005). Examples of proteins that can be used for Z, represent HSA, IgG and other serum proteins. Examples of antigens are (bacterial) toxins and virus-like particles. Z may also be a steroid structure, such as described, for example, in Bode et al. (2007, J. Pept.Sci., 13:702-708). Suitable steroid structure for use as Z includes, for example, bile acids and their derivatives, such as, for example, cholic acid, deoxycholic acid and methyl-7-α-acetoxy-3-amine�-12α-amino-5β-cholan-24-oat. Preferably the peptides according to the invention peptide sequence is connected with the provisions of C-3 and C-12 steroid structure, e.g., as described in Bode et al. (2007, above).

Z may be connected to the peptide sequence before cyclization and optional cyclized with the remainder of the peptide sequence. In this embodiment, Z preferably represents a relatively short molecule, such Oligopeptide: amino acid, a dipeptide, a Tripeptide, a tetrapeptide, a Pentapeptide. In this preferred embodiment the total number of amino acids (peptide sequence derived from Aβ 1-42 and Z) is preferably ten or eleven. Even more preferably this amount is eleven. Z may include or consist of amino acids present in the corresponding sequence of Aβ 1-42 to align with the corresponding sequence of X1X2X3VGSN, X2X3VGSNK or X3VGSNKG.

Alternatively, Z may be connected to cyklinowanie peptide sequence. In this embodiment Z may represent a relatively larger molecule than in the previous embodiment: for example, the polypeptide or protein.

The best results were obtained when combining both methods (cyclization and the presence of Z) for stub�ing peptide. Even more preferably, Z binds to the peptide sequence and then cyclized with the remaining peptide sequence. Alternatively, Z is formed in the reaction of cyclization. In this preferred embodiment, the best results were obtained when Z was a tetrapeptide, as defined above, such as, for example, YNGK. More preferably, the tetrapeptide comprises at least one cysteine and lysine, to allow selective conjugation of the peptide with a molecule carrier, as described below. Lysine, preferably is a modified lysine, such as Nε-(S-acetylmercaptosuccinic)lysine (Lys-SAMA). The presence of at least one cysteine and residue Lys-SAMA, the tetrapeptide enables selective conjugation of the peptide according to the invention with sulfhydryl-reactive carrier such as a protein carrier.

In the most preferred embodiment it is proposed a peptide that contains the formula is X2X3VGSNK-Z, where X2is an E, G, Q or K, X3represents D or N, and Z is an agent stabilizing the bend present in the X2X3VGSNK. Preferably Z represents YNGK, where even more preferably K in YNGK is a modified lysine (Lys-SAMA), to allow selective conjugation of pepti�.

In another most preferred embodiment proposes a peptide comprising the following sequence X3VGSNKG-Z, where X3represents D or N, and Z is an agent stabilizing the bend present in the X3VGSNKG. Preferably Z represents YNGK, where even more preferably K in YNGK is a modified lysine (Lys-SAMA), to allow selective conjugation of the peptide.

It was found that both peptide, the peptide comprising a sequence X2X3VGSNKGAI-Z, where X2represents E, X3is a D, and Z is a modified lysine (Lys-SAMA), and the peptide comprising the sequence VGSNKG-Z, where Z is a modified lysine (Lys-SAMA), generate antibody responses to immunization peptides, however, the thus generated antibodies are unable to cross-reaction with oligomeric or fibrillar Aβ 1-42.

The peptide of the invention may be present in the form of an individual peptide, or may be incorporated in the crosslinked molecule, such as crosslinked protein. The peptide may be further modified by deletion or substitution of one or more amino acids, by lengthening the N - and/or C-end with additional amino acids or functional groups that can improve the bioavailability,�elenast on T-cells, or include or release immunomodulating agents, which provide the adjuvant or (co)-stimulatory functions. The impact of these modifications preferably are tested on the conformation of the synthesized peptide. This can be done, for example, using NMR. It is important that the thus obtained peptide conformation X1X2X3VGSN, X2X3VGSNK or X3VGSNKG, such as, probably, was adopted by Aβ 1-42, preferably such as is accepted Aβ 1-42, not modified. Optional additional amino acids at the N - and/or C-end is preferably absent in the corresponding positions in the amino acid sequence of the protein from which the peptide, i.e., in the amino acid sequence of Aβ 1-42. Thus, in an even more preferred embodiment to improve the immunogenicity of the peptide according to the invention, this peptide, preferably a cyclic peptide as described above, conjugated to immunogenic carrier molecule, preferably by selective connection and Z immunogenic carrier molecules. Such a peptide is called a conjugated peptide. Thus, in a preferred embodiment of the peptide according to the invention is a conjugated peptide, more preferably conjugated cyclic peptide. The immunogenic carrier molecule preferred�enforcement is a carrier, which in conjugation with the peptide of the invention induces an immune response to the peptide according to the invention with the introduction of object, such as a mammal. The immunogenic carrier can also possess adjuvant activity as defined in this document later. Specialist know many immunogenic carrier molecules (Hermanson, G. T., 1996, Bioconjugate techniques. Academic Press, San Diego; Drijfhout and Hoogerhout, 2000). Suitable immunogenic carrier molecules include, for example, bacterial toxins or toxoids, such as exotoxins and their variants with reduced toxicity. Preferred immunogenic carrier molecules include diphtheria toxoid CRM197, serum albumin (e.g. human serum albumin) and tetanus toxoid (Beuvery et al., 1986; Claesson et al., 2005).

Composition

The following aspect of the proposed composition comprising a peptide as defined herein. The composition can be a pharmaceutical composition or drug.

In the following a preferred embodiment of the peptide or peptide composition further comprises a pharmaceutical excipient and/or pharmaceutically acceptable carrier, and/or immunomodulator. To the composition can be added any inert pharmaceutically acceptable carrier and/or excipient. SOS�AB medicines and the use of pharmaceutically acceptable auxiliary media are known and are based on the prior art and, for example, described in the publication Remington; The Science and Practice of Pharmacy, 21ndEdition 2005, University of Sciences in Philadelphia.

The pharmaceutical composition may further comprise pharmaceutically acceptable stabilizing agents, osmotic agents, buffering agents, dispersing agents, etc. Preferred form of the pharmaceutical composition depends on the assigned route of administration and therapeutic application. The pharmaceutical carrier can be any compatible, nontoxic substance suitable to deliver the active ingredients, i.e. the peptide, the patient. Pharmaceutically acceptable carriers for intranasal delivery are, as an example, water, tabularinline saline solutions, glycerin, Polysorbate 20, cremophor EL and water mixture Caprylic/capric glycerides and can be sabotinova to provide a neutral pH environment. Pharmaceutically acceptable carriers for parenteral delivery are the example of sterile subverionary 0.9% NaCl or 5% glucose optionally with a 20% albumin. Preparations for parenteral administration must be sterile. Parenteral route of administration of active ingredients is consistent with known methods, e.g. injection or infusion via subcutaneous route, votive�tion, intraperitoneal, intramuscular, intra-arterial or introduction into the affected tissues. The composition according to the invention is preferably administered using a bolus injection. A typical pharmaceutical composition for intramuscular injection should be made so as to contain, for example, 1-10 ml of phosphate-buffered salt solution, and 1-100 μg, preferably 15-45 μg of the modified conjugated peptide. For oral administration the active ingredient may be administered in liquid dosage forms, such as elixirs, syrups and suspensions. Liquid dose forms for oral administration can contain coloring or flavoring to increase the acceptability for the patient.

Methods of obtaining compositions for parenteral, oral or intranasal introduction well known in the art and are described in more detail in various sources, for example in the publication Remington's Pharmaceutical Science (15th ed., Mack Publishing, Easton, PA, 1980) (introduced by reference in full for all purposes).

To the composition can be added to any known immunomodulator, specifically, modulator, resulting in obtaining a balanced Th2/Th1 response, similar to the aluminum phosphate or aluminum hydroxide. Preferably the immunomodulator is an adjuvant. More preferably, the composition includes a peptide defined� earlier in this document and at least one adjuvant. Defined herein adjuvant includes any substance or compound that when used in combination with peptide immunizes the mammal, preferably human, stimulates the immune system, thus inducing, enhancing or facilitating the immune response against the peptide, preferably without generating a specific immune response to the adjuvant itself. Preferred adjuvants enhance the immune response against the antigen in the number of times corresponding to at least a factor of 1.5, 2, 2,5, 5, 10, or 20, compared with the immune response generated against the peptide under the same conditions but in the absence of adjuvant. Tests to determine the statistical average of strengthening the immune response against the peptide produced by using an adjuvant in the group of animals or people, are available in the art. Adjuvant used in the present document, as a rule, is a compound that is alien to the mammal, thereby eliminating immune-boosting compounds that are endogenous to mammals, such as, for example, interleukins, interferons and other hormones.

The composition of the present invention may contain at least one adjuvant. Adjuvant, used�creating in the present invention, be chosen so that ingibirovala the effect of the peptide. Adjuvants used in the present invention are those that are physiologically compatible with humans, they include, in particular, aluminum hydroxide, aluminum phosphate, emulsion adjuvants based on oil/surfactant, such as MontanicTMin which different surfactants (especially mannitol oleate) are combined with mineral oil, squalene-containing emulsions, such as MF59TM, monophosphoryl lipid A, or Neisseriae-mutant liposaccharide (described in PCT/NL98/0063).

The medicine may be administered in a single injection. Alternatively, the introduction of the peptide defined earlier in this document and/or adjuvant may be repeated as needed, and/or various peptides, and/or various adjuvants may be administered sequentially. The peptide composition and a medicament according to the invention preferably comprise a composition suitable for intravenous or subcutaneous, or intramuscular injection, although there may be other routes of administration, such as crosslisted introduction or intradermal and/or intradermal injection, for example, by injection.

According to a preferred embodiment, the peptide described herein is intended for use as a medicament. More preferred�enforcement this medicine is a vaccine against Alzheimer's disease. Even more preferably the drug is intended to prevent, delay the onset and/or treatment of Alzheimer's disease. The vaccine, as defined herein, can be used for preventive protection against Alzheimer's disease or for the treatment of this disease.

In the context of the invention, an organism, or an individual or object can be animal or human. Preferably the organism is a human. Preferably the organism is exposed to treatment predisposed to high risk of developing Alzheimer's disease due to, for example, a potential genetic predisposition, and/or age of the object, and/or lifestyle of the object (for example, eating habits and/or lack of physical activity), and/or due to any other known parameter that indicates that the object has a high risk of developing Alzheimer's disease.

It should be understood that the term "prevention" includes preventing, prevention, and reducing the risk of the individual falling ill specified disease or reducing the risk of a pathological condition and delay the onset of the disease or pathological condition. The term "prevention", thus, also includes treatment facilities, predisposed to the risk of getting sick listed for�yevanim or risk of a pathological condition. It should also be understood that the term includes the relief of already developed symptoms.

The term "delay" as used herein refers to the introduction into the organism of the peptide, i.e., the patient undergoes treatment in the stage preceding pathological condition, the pathological condition is diagnosed in the patient using methods known in the art.

The term "therapy" or "treatment" should be understood as providing treatment and monitoring the patient for the purpose of combating the disease, pathological condition, or disorder. In the context of the invention, the term "treatment of Alzheimer's disease and/or delay its progression" preferably denotes that adds a therapeutically effective amount of the peptide. The number is effective at dosages and for periods of time necessary to achieve the target therapeutic result, i.e. the treatment of Alzheimer's disease and/or slowing its progression.

The amount of the peptide may vary according to factors such as disease stage, age, sex and weight of the individual and the ability of the pharmacological agent to cause the target response from the individual.

Therapeutic effect (resulting in the treatment of Alzheimer's disease and/or slowing its progression) preferably represents an EF�the project which results in, at least, to:

- reduction in the number of beta plaques present in the brain;

- reduce the amount of soluble Aβ-oligomers pre-fibrils present in the brain; and

- reduce the severity of a symptom associated with Alzheimer's disease.

Reducing the number of beta plaques present in the brain of a patient subjected to treatment, preferably means that the amount of added peptide will be able to prevent the de novo formation of a beta plaques, at least to some extent and/or that existing plaques will be, at least to some extent also be inhibited their ability to reproduce. Preferably in this context, the deposition of a beta plaques will not increase in patients subjected to treatment, in terms of the amount of beta-plaques, identified using the method of image reproduction, such as PET-scan (Henriksen, Yousefi et al., 2008) and/or magnetic resonance imaging (MRI) (O'brien, 2007). In PET scanning, the number of beta plaques in proportion to the absorption of the label. Preferably, the amount of beta-plaques will be reduced, at least 2%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. Even more preferably beta plaques will not be identified. Specialist known that V�alizatio beta plaques should be approximately at least one day, at least, through one week, at least one month after vaccination or more. (Meyer-Luehmann, Spires-Jones et al., 2008). If necessary, the specialist may decide to vaccinate several times and regularly observe the amount of beta-plaques.

Reducing the amount of soluble Aβ-oligomers or pre-fibrils present in the patient's brain exposed to treatment indicates that the amount of added peptide will be able to prevent the de novo formation of soluble Aβ-oligomers or pre-fibrils, at least to some extent and/or that existing soluble Aβ-oligomers or pre-fibrils are at least to some extent also be inhibited in their ability to reproduce. Preferably in this context, the amount of soluble Aβ-oligomers or pre-fibrils will not increase the patient subjected to treatment, in terms of the surface identified using the method of image reproduction, as defined above. Preferably, the amount of soluble Aβ-oligomers or pre-fibrils will decrease, at least 2%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. More preferably, the amount of soluble Aβ-oligomers or prefinal will not be detected by using such �Yoda.

In the context of the invention, soluble Aβ-oligomer, pre-fibril or protofibrillar is a symptom of Assembly 2-24 β-monomers (Haas and Selkoe 2007). In the context of the invention, the term "reducing severity of a symptom associated with Alzheimer's disease" preferably refers to the improvement of cognitive ability, measured using a physiological test to evaluate the improvement of cognitive abilities in patients with Alzheimer's disease.

Application

Accordingly, in the following aspect proposed the application of a peptide or composition as defined herein, for the production of drugs against Alzheimer's disease. Preferably the medicament is a vaccine. More preferably, the vaccine is intended to prevent, delay the onset and/or for the treatment of Alzheimer's disease.

Accordingly, in another the next aspect provides a method for the prevention, delay of onset and/or for the treatment of Alzheimer's disease through the introduction of the needy in this patient a peptide or composition as defined herein.

Method for the synthesis of peptide

From the present level of technology there are many ways of generating a peptide according to the invention. The invention is not limited in any way �of everyoane peptide provided what generated the peptide comprises, consists of or overlaps with any of these modified sequences identified herein, and has required conformation, defined earlier in this document.

Accordingly, in the following aspect provides a method for producing a modified cyclic peptide, as defined herein, wherein said method comprises the following steps:

a) synthesis of a cyclic peptide comprising the sequence X1X2X3VGSN-Z, X2X3VGSNK-Z or X3VGSNKG-Z, where X1represents A or G, X2is an E, G, Q or K, X3represents D or N, and Z is an agent stabilizing the bend present within the peptide sequence X1X2X3VGSN, X2X3VGSNK or X3VGSNKG; and

(b) optional conjugation of immunogenic molecule-carrier with cyclic peptide obtained in (a), preferably by connecting Z with the immunogenic carrier molecule.

Each stage of this method known to the skilled worker and described in detail in the example.

Antibody

The following aspect of the proposed antibody directed against a modified (cyclic) peptide according to the invention, defined in the present document.Specialist known how to obtain the antibody from the animal. Methods of generating antibodies or fragments of antibodies that are specific contact with the polypeptide, as described, for example, in the publications by Harlow and Lane (1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and WO 91/19818; WO 91/18989; WO 92/01047; WO 92/06204; WO 92/18619; and US 6420113 and in the references cited herein. The term "specific binding" as defined herein includes specific binding simultaneously with low and high affinity. Specific binding may occur, for example, by using antibodies with low affinity or with a fragment of an antibody having a Kd comprising at least about 10-4M. Specific binding also can occur using antibody or antibody fragment with high affinity, e.g., using antibody or antibody fragment having a Kd equal to at least about 10-7M, at least about 10-8M, at least about 10-9M, at least about 10-10M, or they may have a Kd equal to at least about 10-11M or 10-12M or above.

Diagnostic methods

The next aspect provides a method for diagnosing a neurodegenerative disease or a pathological condition such as disease Alzh�of ymer. The method comprises determining the presence or absence of beta-amyloid plaques (ie. neurodegenerative plaques) in the brain of the patient with the use of antibodies, defined in the present document. Preferably in the method the presence of beta-amyloid plaques in the brain of the object (or sample) is an indication that the object has the risk of developing a neurodegenerative disease or condition, such as Alzheimer's disease. Preferably this method is used to predict or diagnose Alzheimer's disease in a patient. In the context of the invention diagnosis indicates or projected assessment of risk for the later development of the object of Alzheimer's disease, or preferably to the development of Alzheimer's disease in the subject. In the context of the invention, the object can be animal or human. Preferably the object is a person.

According to a preferred embodiment the method is performed in vitro or ex vivo in a sample obtained from the subject. The sample preferably includes brain tissue, taken from the object. More preferably, the tissue is a blood vessel of the brain.

Preferably, the detection of the presence of beta-amyloid plaques are detected by the binding of the antibodies according to the invention with a sample of the brain, which is analyzed using ELISA, �AK explained in the example. The method of diagnosis may be subsequently applied to the object to monitor the development of the disease.

In this document and in its claims, the verb "include" and its forms are used in its non-limiting sense, to denote the inclusion of items following the word, but the items not specifically mentioned are excluded. In addition, the verb "to consist" may be replaced by "to consist essentially of" means that the peptide or composition as defined herein may include additional component(s) other than specifically identified, and the specified component(s) does not alter the unique characteristics of the invention. In addition, a link to the item using an indefinite article "a" or "an" does not exclude the possibility that there is more than one element until such time as the context will follow a clear requirement that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

All links to the patents and literature cited in the present description, are included in full by reference. The following examples are for illustrative purposes only and are not intended to any way limit the scope of the invention.

Description h�of Raja

Figure 1. Model of fibrillar Aβ1-42. A and B represent dimeric cross-β unit and C assembled fibril [Credit: Olofsson et al. 2006 J. Biol. Chem. 281, 477-483].

Figure 2. The sequences of the cyclic peptides of amyloid origin (K* is a modified lysine residue for conjugation purposes).

Figure 3: IgG titers of total antibody of mouse serum against homologous conjugates peptide-BSA. The titer is10log of the corresponding serum dilution at 50% maximum optical density at 450 nm in the ELISA analysis.

Figure 4: OD450 nm total mouse serum as a function from breeding with oligomeric or fibrillar Aβ 1-42 in the form of a coating.

Figure 5: the Titers of IgG antibodies to individual mouse serum groups 8 and 9 against oligomeric or fibrillar Aβ 1-42. The titer represents the corresponding serum dilution at 50% maximum optical density at 450 nm in the ELISA analysis.

Figure 6: Immunohistochemical staining of sections of human brain-donor 99-30 (Braak 6) together with mouse serum (1:300), immunized with conjugate cyclo[Aβ(22-28)-YNGK]/tetanus toxoid (a) and with a control monoclonal antibody 6E10 (1:15000) in (b). Such a profile of the plaques recognize mouse serum as a positive control. (Microscope: Leica DMRE equipped with.�Ohm DC300).

EXAMPLES

EXAMPLE 1: strategy synthesis

We tested truncated Aβ peptides that do not include the immunodominant N-terminal B-cell epitope. Our goal was targeting antibody response in previously incorrectly Packed protein Aβ.

The sequence of Aβ 1-42:

DAEFR5HDSGY10EVHHQ15KLVFF20AEDVG25SNKGA30IIGLM35VGGAAZ40IA

(SEQ ID NO:4).

The structure of Aβ1-42 fibrils was resolved by NMR spectroscopy (Olofsson et al. 2006). Experiments on fibrils with the replacement of the hydrogen/deuterium showed that areas Glu-Gly and Lys-Ala in the sequence Aβ protected from the solvent, whereas the N-end of the Asp1-Tyr10and a fragment of two residues of Ser26-Asn27are accessible to the solvent. Consistent with this NMR data: the Asp1-Tyr10and a fragment of two residues of Ser26-Asn27are accessible to the solvent. NMR data are consistent with the model of the fibrils shown in Figure 1C. The predicted structure is a twisted cross-β conformation. Figure IA and IB shown section, dimeric cross-β unit. Within the dimer, each monomer contains two antiparallel β-sheet that are associated with folds, consisting of Ser26-Asn27.

On the basis of the amyloid fibrillar model (Figure 1), we decided to obtain�boron from YNGK*-stable cyclic 10 - and 11-dimensional amyloid peptides. Figure 2 shows target the amyloid Decapeptide. We hypothesized that could stabilize the conformation of short Aβ-peptides by adding artificial sequence YNGK*, where K* is a modified lysine residue for selective conjugation with a carrier protein, with subsequent amide cyclization of the main chain ("head to tail") (Oomen et al. (2005)). Similarly, we received a small panel cyclic decameric and undeclared peptides spanning six or seven residues from section 21-31 of Aβ and YNGK* (see Table 1).

Table 1
The synthesized peptides
GroupAntigenCode of peptideMN+peptide
Detected/calculated
1Oligomeric Aβ 1-42--
2Fibrillar Aβ 1-42--
3Linear Aβ(22-28)/TTdS070-07
4Cyclo-Aβ(25-30)/TTdS060-08
5Cyclo-Aβ(24-29)/TTdS060-09
6Cyclo-Aβ(23-28)/TTdS060-10
7Cyclo-Aβ(24-30)/TTdS061-56
8Cyclo-Aβ(23-29)/TTdS060-05
9Cyclo-Aβ(22-28)/TTdS060-06
10Cyclo-Aβ(21-27)/TTdS076-08

EXAMPLE 2: specificity of some of the modified cyklinowanie peptides to a conformational epitope of the coiled Aβ 1-42

Conjugate of tetanus toxoid and cyclic peptide [Aβ(22-28)-YNGK*], i.e., cyclo[EDVGSNKYNGK*], or peptide that is defined as a group 9 in Table 1, called antibodies that cross-react in vitro with Aβ (l-42) oligomers (Figure or 5) and Aβ(l-42) fibrils (Figure 4 or 5). These antibodies also recognize Aβ-deposition in an Alzheimer's patient brain tissue (hippocampus), obtained after death, see Figure 6. The corresponding conjugate linear N-acetylated amide peptide Ac-K*EDVGSNKYNG-NH2induced good titles antipeptide antibodies, but antibodies are not generated recognize oligomeric or fibrillar Aβ. Thus, the cyclic peptide mimics a conformational epitope in a collapsed Aβ 1-42, which usually induces antibody response. During system testing antibody response to conjugate the peptide is defined as a group of 10, a cyclic peptide [Aβ(21-27)- YNGK*] was still in operation.

Materials and methods:

Peptide synthesis, purification and conjugation

α-(2,4-dimethoxybenzoyl) ether Nα-fluorenylmethoxycarbonyl-L-aspartic acid (Fmoc-Asp-ODmb) condensible through its side chain to the polymer for the synthesis of peptide amides (for further conversion of the original Asp to Asn). Associated with the resin consistency with protected side chain GK*EDVGSNKYD(resin) in which K* is an Nε-(S-acetylmercaptosuccinic)lisil, then gathered as described previously (Brugghe et al., 1994). Associated with the resin, the linear peptide was converted into cyclo[GK*EDVGSNKYD(resin)]. After removing the protection of the side chain, except for Lys(SAMA), and the split-off�I from resin cyclo[GK*EDVGSNKYN] ≡ cyclo[EDVGSNKYNGK*] received peptide from group 9 (table 1). Peptides from groups 4-8 were obtained similarly. Peptide from group 10, cyclo [AEDVGSNYNGK*], received from associated with the linear resin precursor YNGK*AEDVGSD(resin) with protected side chain. Peptides were purified using reversed-phase chromatography and okharakterizovali using mass spectrometry with ion sputtering (MH+detected/calculated, see Table 1). Purified peptides were condensible or bromotetradecane with tetanus toxoid or with maleimide-modified bovine serum albumin (BSA) (modifying reagent: NHS-PEO2-Maleimide, Pierce) (Drijfhout JW, et al., 2000).

The disaggregation of Aβ(l-42)

Lyophilized Aβ 1-42 (Anaspec) was dissolved in trifluoroacetic acid at a concentration of 1 mm was left at room temperature for 1 h and dried under a stream of nitrogen and then under vacuum (1 mm Hg) for 15 min. Peptide is then re-dissolved in hexafluoroisopropanol at a concentration of 1 mm and after 1 h of incubation at room temperature, dried, as described previously (Zeng et al., 2001). The peptide was stored at -20°C for 18-20 h.

Obtaining oligomeric or fibrillar Aβ(l-42)

Disaggregated Aβ 1-42 was dissolved in dimethylsulfoxide at a concentration of 5 mm, diluted 50 times with or with phosphate-buffered saline (PBS), pH of 7.2, or with 10 mm hydrochloric acid. Solution in PBS were incubated at 4°St for 24 h (to produce oligomers), then as a solution in 10 mM HCl were incubated at 37°C for 24 h (to produce fibrils) (Stine et al.).

Immunization of mice

A group of eight female mice Balb/c aged 6-8 weeks were immunized subcutaneously on days 0 and 28 with either 25 μg of Aβ 1-42 in PBS without adjuvant, or with 50 μg of peptide conjugate-TTd and 75 mcg of AlPO4d PBS. Small serum samples collected on day 0. Mice were bled on day 42.

ELISA

On the microplates (Greiner 655092) was coated in the form of Aβ1-42 or conjugates peptide-BSA. Freshly prepared Aβ 1-42 oligomers or fibrils were diluted to final concentration of 2.5 μm (11,3 mg/ml) 0.04 M buffer carbonate/sodium bicarbonate, pH of 9.7. Conjugates peptide-BSA in phosphate-buffered saline solution, pH of 7.2 (PBS), had a concentration of 0.5 μg total protein/ml. Aliquots (100 μl) of these solutions were transferred into the wells of the plates. The plates were incubated for 90 min at 37°C. the plates then were processed as described previously (Westdijk, Van den Ijssel et al., 1997).

Immunohistochemical staining

Used section of the human brain from the hippocampus of several donors with Alzheimer's disease (Braak 5 or 6) (Brain Bank of the Netherlands (Netherlands Brain Bank)). Cryosection (10 µm) were cut from unfixed frozen tissues directly, defrosted, dried for 1 hour and kept in an airtight box at -20°C. For immunoablative section f�was kirawali in 4% solution of PFA-PBS for 10 min, was washed with 0.05 M phosphate buffer (PB) for 10 min with 2 changes of solution and blocked with normal donkey serum (NDS)+0,4% Triton in 0.05 M PB for 1 hour at CT. The blocking solution was poured and added diluted mouse serum (1:300; primary antibody in 3% NDS+0,4% Triton in 0.05 M PB and incubated O/N at CT in a box with wet tissues. Sections were washed with 0.05 M PB; at least 30 min with one or more changes of solution. Then sections were incubated with Donkey-anti-Mouse antibody ~Cy3 1:1400 in 0.05 M PB for 2 hours. Sections were washed with 0.05 M PB; at least 30 min with one or more changes of solution. Sections were heat-sealed in Vectashield medium with DAPI (Vector). As a positive control was used a mouse monoclonal antibody 6E10 to beta-amyloid peptide 1-17 (Abeam, Cambridge, UK) (1:15000).

The list of references

Agadjanyan MG, Ghochikyan A, Petrushina IVasilevko V, Movsesyan N, Mkrtichyan M, Saing T, and Cribbs DH, 2005. Prototype Alzheimer's disease vaccine using the immunodominant B cell epitope from β-amyloid and promiscuous T cell epitope pan HLA DR-binding peptide. J. Immunol. 174, 1580-1586.

Beuvery, E. C., Roy R., Kanhai V., Jennings H. J., 1986. Characteristics of two types of meningococcal group C polysaccharide conjugates using tetanus toxoid as carrier protein. Dev.Biol. Stand. 65, 197-204.

Blennow K, de Leon MJ, Zetterberg H, 2006. Alzheimer's disease. Lancet 368, 387-403.

Brugghe HF, Timmermans HAM, van Unen LMA, ten Hove GJ, van de Werken G, Poolman JT, and Hoogerhout P, 1994. Simultaneous multiple synthesis and selective conjugation of cyclized peptides, derived from a surface-loop of a meningococcal class 1 outer membrane protein. Int. J. Peptie Protein Res. 43, 166-172.

Check E, 2002. Nerve inflammation halts trial for Alzheimer's drug. Nature 415, 462.

Claesson B. A., Trollfors B., Lagergard T., Knutsson Corporation N., R. Schneerson, J. B. Robbins, 2005. Antibodies against Haemophilus influenzae type b capsular polysaccharide and tetanus toxoid before and after a booster dose of the carrier protein nine years after primary vaccination with a protein conjugate vaccine. Pediatr. Infect. Dis. J. 24, 463-464.

Drijfhout JW and Hoogerhout P, 2000. Methods of preparing peptide-carrier conjugates. In: Fmoc Solid Phase Peptide Synthesis: A Practical Approach (W. C. Chan and P. D. White, eds.). Oxford University Press, p. 229-241.

Editorials Nature Med. 2006, Vol. 12, # 7 July 2006.

Gelinas DS, DaSilva K, Fenili D, St. George-Hyslop P, and McLaurin J, 2004. Immunotherapy for Alzheimer's disease. Proc. Natl. Acad. Sci. USA 101, 14657-14662.

Gevorkian G., Petrushina I, Manoutcharian K, Ghochikyan A, Acero G, Vasilevko V, Cribbs DH, and Agadjanyan MA, 2004. Mimotopes of conformational epitopes in fibrillar β-amyloid. J. Neuroimmunol. 156, 10-20.

Ghochikyan A, Mkrtichyan M, Petrushina I, Movsesyan N, Karapetyan A, Cribbs DH, and Agadjanyan MG, 2006. Prototype Alzheimer's disease epitope vaccine induced strong Th2-type anti-Aβ antibody response with Alum to Quil A adjuvant switch. Vaccine 24, 2275-2282.

Gilman S, Roller M, Black RS, Jenkins L, Griffith SG, Fox NC, Eisner L, Kirby L, Boada Rovira M, Forette F, Orgogozo J-M, for the AN1792(QS-21)-201 study team, 2005. Clinical effects of Aβ immunization (AN 1792) in patients with AD in an interrupted trial. Neurology 64, 1553-1562.

Haass C, Selkoe D. J., 2007. Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide. Nat. Rev. Mol. Cell Biol. 8, 101-112.

Henriksen G., B. H. Yousefi, Drzezga A., Wester H. J., 2008. Development and evaluation of compounds for imaging of beta-amyloid plaque by means of positron emission tomography. Eur. J. Nucl. Med. Mol. Imaging 35 Suppl 1, S75-S81.

Hock C, Konietzko U, Papassotiropoulos A, Wollmer A, Streffer J, von Rotz RC, Davey G, Moritz E, and Nitsch RM, 2002. Generation of antibodies specific for beta - amyloid by vaccination of patients with Alzheimer's disease. Nature Med. 8, 1270-1275.

Hutchinson G. E., Sessions, R. B, Thornton J. M., D. N. Woolfson, 1998. Determinants of strand register in antiparallel beta-sheets of proteins. Protein Sci. 7, 2287-2300.

Klein WL, Stine WB, and Teplow DB, 2004. Small assemblies of unmodified amyloid β-protein are the proximate neurotoxin in Alzheimer's disease. Neurobiol. Aging 25, 569-580.

Lambert M. P., Velasco P. T., L. Chang, K. L. Viola, S. Fernandez, P. N. Lacor, Khuon D., Gong Y., Bigio E. H., Shaw P., De Felice F. G., Krafft G. A., Klein W. L., 2007. Monoclonal antibodies that target pathological assemblies of Abeta. J. Neurochem. 100, 23-35.

Lee M., Bard, F., Johnson-Wood K, Lee C, Hu K., Griffith, S. G., Black, R. S., Schenk D., Seubert P., 2005. Abeta42 immunization in Alzheimer's disease generates Abeta N-terminal antibodies. Ann.Neurol. 58, 430-435.

Lemere CA, Maier M, Jiang L, Peng Y, and Seabrook TJ, 2006. Amyloid-beta immunotherapy for the prevention and treatment of Alzheimer's disease: lessons from mice, monkeys, and humans. Rejuvenation Res. 9, 77-84. Lemere C. A., M. Maier, Y. Peng, L. Jiang, J. J. Seabrook, 2007. Novel Abeta immunogens: is shorter better? Curr.Alzheimer Res. 4, 427-436.

Maier M, Seabrook TJ, Lazo ND, Jiang L, Das P, Janus C, and Lemere CA, 2006. Short amyloid β (Aβ) immunogens reduce cerebral Aβ load and learning deficits in an Alzheimer's disease mouse model in the absence of an Aβ-specif[iota]c cellular immune response. J. Neurosci. 26, 4717-4728.

McDonnell J, Redekop WK, Van der Roer N, E Goes, Ruitenberg A, Busschbach JJ, Breteler MM, and Rutten FF, 2001. The cost of treatment of Alzheimer's disease in The Netherlands: a regression-based simulation model. Pharmacoeconomics 19, 379-390.

McLaurin J, Cecal R, Kierstead ME, Tian X, Phinney AL, Manea M, French JE, Lambermon MHL, Darabie AA, Brown ME, Janus C, Chishti MA, Home P, Westaway D, Fraser PE, Mount HTJ, Przybylski M, and St George-Hyslop P, 2002. Therapeutically effective antibodies against amyloid-beta peptide target amyloid-beta residues 4-10 and inhibit cytotoxicity and fibrillogenesis. Nature Med. 1263-1269.

Meyer-Luehmann m, Spires-Jones T. L., Prada C, Garcia-Alloza M. de C. A., Rozkalne A., Koenigsknecht-Talboo j, Holtzman D. M., Bacskai BJ, Hyman B. T., 2008. Rapid appearance and local toxicity of amyloid-beta plaques in a mouse model of Alzheimer's disease. Nature 451, 720-724.

<> Movsesyan N., Ghochikyan A., Mkrtichyan M., Petrushina L, Davtyan H., Olkhanud P. B., E. Head, A. Biragyn, Cribbs D. H., Agadjanyan M. G., 2008. Reducing AD-like pathology in 3xTg-AD mouse model by DNA epitope vaccine - a novel immunotherapeutic strategy. PLoS. ONE. 3, e2124.

O'brien J. T., 2007. Role of imaging techniques in the diagnosis of dementia. Br. J. Radiol. 80 Spec No 2, S71-S77.

Olofsson A, Sauer-Eriksson AE, and Ohman A, 2006. The solvent protection of Alzheimer amyloid-[beta]-(l-42) fibrils as determined by solution NMR spectroscopy. J. Biol Chem. 281, 477-483.

Oomen, C. J., Hoogerhout P., Bonvin, A. M. and B. Kuipers, Brugghe, H., Timmermans H., S. R. Haseley van A. L., Gros P., 2003. Immunogenicity of peptide - vaccine candidates predicted by molecular dynamics simulations. J. Mol. Biol. 328, 1083-1089.

Oomen CJ, Hoogerhout P, Kuipers B, Vidarsson G, van Alphen L, and Gros P, 2005. Crystal structure of an anti-meningococcal subtype P 1.4 PorA antibody provides basis for peptide-vaccine design. J. MoI. Biol. 351, 1070-1080.

M Sadowski and Wisniewski T, 2004. Vaccines for conformational disorders. Expert Rev. Vaccines 3, 279-290.

Sawaya MR, Sambashivan S, Nelson R, Ivanova MI, Sievers SA, Apostol M, Thompson MJ, Balbirnie M, Wiltzius JJW, McFarlane HT, Madsen A0, Riekel C, and Eisenberg D, 2007. Atomic structures of amyloid cross-beta spines reveal varied steric zippers. Nature 447, 453-457.

Schenk, Barbour R, Dunn W, Gordon G, Grajeda H, Guido T, Hu K, Huang J, Johnson - Wood K, Khan K, Kholodenko D, Lee M, Liao Z, Lieberburg I, Motter R, Mutter L, Soriano F, Shopp G, Vasquez N, Vandevert C, Walker S, Wogulis M, Yednock T, Games D, Seubert P, 1999. Immunization with amyloid-beta attenuates Alzheimer - disease-like pathology in the PDAPP mouse. Nature 400, 173-177.

Sigurdsson EM, Knudsen E, Asuni A, Fitzer-Attas C, sage D, Quartermain D, Goni F, Frangione B, Wisniewski T, 2004. An attenuated immune response is sufficient to enhance cognition in an Alzheimer's disease mouse model immunized with amyloid-[beta] derivatives. J. Neurosci. 24, 6277-6282.

Stine WB Jr, Dahlgren KN, Rrafft GA, LaDu MJ, 2003. J. Biol. Chem., 278:11612.

Tomiyama, T., Nagata T., Shimada H., R. Teraoka, A. Fukushima, H. Kanemitsu, H. Takuma, Kuwano R., Imagawa M., S. Ataka, Y. Wada, E. Yoshioka, Nishizki T., Watanabe Y., Mori H., 2008. A new amyloid beta variant favoring oligomerization in Alzheimer's-type dementia. Ann.Neurol. 63, 377-387.

Vas CJ, Rajkumar S, Tanyakitpisal P, and Chandra V, 2001. Alzheimer's disease: the brain killer. Report of the World Health Organization.

Walsh D. M., Selkoe D. J., 2007. A beta oligomers - a decade of discovery. J. Neurochem. 101, 1172-1184.

Westdijk j, Van den Ijssel, J., M. Thalen, Beuvery C, W. collected constantly, 1997. Quantification of cell-associated and free antigens in Bordetella pertussis suspensions by antigen binding ELISA. J. Immunoassay 18, 267-284.

Woolfson D. N., Evans P. A., E. G. Hutchinson, J. M. Thornton, 1993. Topological and stereochemical restrictions in beta-sandwich protein structures. Protein Eng 6, 461-470.

Zeng H, Zhang Y, Peng L-J, Shao H, Menon NK, Yang J, Salomon AR, Freidland RP, and Zagorski MG, 2001. Nicotine and amyloid formation. Biol. Psychiatry 49, 248-257.

1. Cyclic peptide consisting of the formula X2X3VGSNK-Z or X3VGSNKG-Z, where X2is an E, G, Q or K, X3represents D or N, and Z is an agent stabilizing the bend present within the sequence of peptide X2X3VGSNK or X3VGSNKG, wherein the peptide cycletour by covalent binding of N-terminal amino acid of the peptide sequence Z, where Z is a peptide fragment consisting of YNGK, wherein said cyclic peptide is able to induce antibody response.

2. Conjugate containing cyclic peptide according to claim 1, conjugated to immunogenic carrier molecule, wherein said conjugate is capable of inducing an immune response to the peptide according to claim 1.

3. The conjugate according to claim 2, where the peptide con�Ugarova with the immunogenic carrier molecule by selective covalent bond Z with the immunogenic carrier molecule.

4. The conjugate according to claim 2, where the immunogenic carrier molecule is a tetanus toxoid.

5. The use of the peptide according to claim 1 and/or conjugate according to any of claims.2-4 as a vaccine against Alzheimer's disease.

6. The use of the peptide according to claim 1 and/or conjugate according to any of claims.2-4 for the production of medicinal products for the treatment or prevention of Alzheimer's disease.

7. A method of producing a cyclic peptide as defined in claim 1, wherein the method comprises the steps:
a) synthesis of a linear peptide consisting of a sequence X2X3VGSNK-Z or X3VGSNKG-Z, where X2is an E, G, Q or K, X3represents D or N, and Z is an agent stabilizing the bend present within the sequence of peptide X2X3VGSNK or X3VGSNKG, and where Z is a peptide fragment consisting of YNGK; and
(b) covalent binding of N-terminal amino acid of the linear peptide obtained in step (a), with Z with obtaining thus a cyclic peptide.

8. The method of producing a conjugate as defined in any one of claims.2-4, where the method includes the stage of conjugation of immunogenic carrier molecule with a cyclic peptide obtained according to claim 7 communication through Z with the immunogenic carrier molecule.



 

Same patents:

Peptides // 2496789

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a conformationally restricted cyclic oligopeptide producing two stable and specific epitopes each of which is able to react with a target ligand, to a pharmaceutical composition comprising the above oligopeptide, and to a method for producing the oligopeptide.

EFFECT: producing the conformationally restricted cyclic oligopeptide.

19 cl, 8 tbl, 8 ex

Peptide compounds // 2415149

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to new peptide compounds, pharmaceutical compositions containing them, application of said compound for making a drug exhibiting activity with respect to hV1a receptor for treating related conditions, particularly depressed cases. The compounds are presented by general formula (I) which is specified in the description of the invention.

EFFECT: preparation of new peptide compounds.

12 cl, 1 tbl, 1 ex

The invention relates to a new compound of chemical formula I,

where R= H, -COCH3or their salts; the way they are received by cultivation of the strain SANK 13899 (FERM BP-6851) a microorganism of the genus Phoma; strain SANK 13899 (FERM BP-6851) a microorganism of the genus Phoma; therapeutic or preventive agent against fungal infections; method of treating or preventing fungal infectious diseases

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to new compounds of formula I, wherein R1 and R2 are identical or different and specified in an alkyl or alkenyl hydrocarbon chain; the R3 group values split by lipase are specified in the patient claim. R4 and R5 are independently hydrogen or C1-C7alkyl; R6 represents hydrogen or C1-C7alkyl; and R7 and R8 are independently hydrogen or C1-C7alkyl. The invention also refers to using compounds of formulas ,

which are introduced into the mammalian biological system and increase the cell concentrations of specific sn-2 substituted ethanolamine-plasmalogens.

EFFECT: compounds are applicable in treating or preventing the age-related disorders associated with high membrane cholesterol, high amyloids and low plasmalogens, such as neurodegeneration, cognitive disorder, dementia, cancer, osteoporosis, bipolar disorder and vascular diseases.

11 cl, 18 dwg, 7 ex

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine. What is described is a transdermally absorbable formulation prepared by dissolving donepezil in an adherent plaster base, which contains a hydrophobic polymer and an absorption enhancer. The absorption enhancer represents one substance, or two or more substances specified in lauryl alcohol, triethyl citrate, isopropylmyristate, cetyl lactate, oleyl alcohol, sorbitan monooleate, polyethyleneglycol monostearate, lauromacrogol, N-methyl-2-pyrroldone and triacetin.

EFFECT: transdermally absorbable formulation can administer donepezil stably for a relatively long period of time and can provide both blood donepezil increase, and the properties of sustained release of donepezil.

4 cl, 4 dwg, 6 tbl, 26 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to compounds of formula (I) and their pharmaceutically acceptable salts, wherein A is thiazolyl, oxazolyl, thienyl, furyl, imidazolyl, pyrazolyl or oxadiazolyl (structures of which are presented in cl.1 of the patent claim), R1 represents C1-6alkyl; R2 represents (i) phenyl substituted by halogen; C1-6alkyl optionally substituted by morpholine or C1-6dialkylamino; C1-6alkoxy optionally substituted by halogen; or heterocyclyl, wherein a heterocyclyl substitute is specified in morpholine; pyrazolyl optionally substituted by C1-6alkyl; piperidinyl; pyrrolidinyl; oxadiazolyl substituted by C1-6alkyl; furyl substituted by C1-6alkyl; dioxydoisothiazolidinyl; triazolyl; tetrazolyl substituted by C1-6alkyl, tridiazolyl substituted by C1-6alkyl; thiazolyl substituted by C1-6alkyl; pyridyl; or pyrazinyl; (ii) substituted or unsubstituted heterocyclyl specified in quinolinyl; pyridyl substituted by C1-6alkoxy or morpholinyl; or benzo [d] [1, 2, 3] triazolyl substituted by C1-6alkyl; R3 represents phenyl substituted by 2 or 3 substitutes specified in halogen; C1-6alkyl; C1-6alkoxy optionally substituted by halogen; hydroxy group; cyano; or -C(=O)ORa, wherein Ra represents phenyl; R4 represents hydrogen, C1-6alkyl or C1-6halogenalkyl. The invention also refers to a pharmaceutical composition containing the compounds of formula (I), a method for PDE10 inhibition, a method of treating neurological disorders, and to intermediate compounds: 2-(4-chlor-3,5-dimethoxyphenyl)furan and 4-(5-methyl-1,3,4-thiadiazol-2-yl)benzaldehyde.

EFFECT: compounds of formula (I) as PDE10 inhibitors.

39 cl, 13 ex, 2 tbl, 77 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: given invention refers to new 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole derivatives, which can be used for modulating histamine receptors in individuals or for treating neurodegenerative diseases. The above derivatives have formula , wherein R1 is specified in an alkyl, substituted alkoxy or phenyl, and aralalkyl; R2 is specified in an alkyl, C6-C14-aryl optionally substituted by 1 to 5 substitutes specified in alkoxy and alkyl; R3 is an alkyl; and R4 is an alkyl.

EFFECT: presented are the new compounds effective as histamine receptor modulators, and a based pharmaceutical composition.

20 cl, 1 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to organic chemistry, namely to new quinolin-4-one derivatives of formula (1) or to its pharmaceutically acceptable salt, wherein R1 represents: (1) hydrogen, (2) C1-C6 alkyl, (35) carbamoyl-C1-C6 alkyl optionally containing morpholinyl-C1-C6 alkyl, or (36) phosphonoxy-C1-C6 alkyl optionally containing one or two C1-C6 alkyl groups on a phosphonoxy group; R2 represents: (1) hydrogen or (2) C1-C6 alkyl; R3 represents phenyl, thienyl or furyl, wherein a phenyl ring presented by R3, can be substituted by one C1-C6 alkoxy group; R4 and R5 are bound to form a group presented by any of the following formulas: , , , , , , or a group presented by the following formula: a group optionally containing one or more substitutes specified in a group consisting of C1-C6 alkyl groups and oxogroups; R6 represents hydrogen; and R7 represents C1-C6 alkoxy group. The invention also refers to a pharmaceutical composition based on the compound of formula , to a preventive and/or therapeutic agent based on the compound of formula (1), using the compound of formula (1), a method for preparing the compound of formula , as well as to specific compounds.

EFFECT: there are prepared new quinolin-4-one derivatives effective in treating neurodegenerative diseases, diseases caused by neurological dysfunction, or diseases caused by mitochondrial dysfunction.

18 cl, 1 tbl, 257 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a compound of structural formula (I), which possesses phosphodiesterase 10 inhibitory activity. In formula (I), R1 represents hydrogen, halogen or lower alkyl; the ring A represents optionally substituted 6-10-merous monocyclic or bicyclic heteroaryl containing 1 to 3 nitrogen atoms as heteroatoms, or a group containing a cycloaliphatic 6-merous ring condensed with the above heteroaryl, which is specified in 6-merous cycloalkane and aliphatic 6-merous heterocyclic ring containing an oxygen atom; the ring B represents optionally substituted 4-6-merous monocyclic nitrogen-containing group, which can additionally contain an oxygen atom or a 3-6-merous monocyclic hydrocarbonic group, which can be optionally saturated; R3 represents hydrogen; lower alkyl optionally substituted by a substitute specified in lower alkoxy; or lower cycloalky. The R2,Y radicals, as well as substitutes of the rings A and B are presented in the patent claim.

EFFECT: invention refers to the pharmaceutical composition containing the above compound, to a method of treating or preventing schizophrenia, anxiety disorders, drug addiction, disorders with a symptom of cognition deficiency, affective disorder or mood episode, each of which is mediated by phosphodiesterase 10 activity.

20 cl, 3 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: present group of inventions refers to medicine, namely to neurology, and concerns treating vegetative-vascular dystonia, dizziness syndrome of various origins, and kinetosis. That is ensured by administering a therapeutic agent containing an activated-potentiated form of brain-specific protein S-100 antibody and using the activated-potentiated form of endothelial NO-synthase antibodies as an additional exalting agent.

EFFECT: invention provides the effective treatment of the above pathological conditions by the synergetic effect of the ingredients of the therapeutic agent.

9 cl, 16 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: claimed group of inventions relates to medicine, namely to neurology, and deals with treatment of Alzheimer's disease. For this purpose pharmaceutical composition, which contains activated potentiated form of antibodies to brain-specific protein S-100 and activated potentiated form of antibodies to endothelial NO-synthase, is introduced.

EFFECT: introduction of claimed composition provides efficient treatment of Alzheimer's disease due to synergic neuroprotective, anti-ischemic and anxiolytic action of composition components.

9 cl, 5 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention represents a liquid dosage form of a hopantenic acid calcium salt possessing nootropic activity, containing an effective amount of the hopantenic acid calcium salt and additive agents. It represents drops, and as additive agents, it contains benzoic acid, sodium saccharinate, an orange flavour, hydrochloric 1M, Trilon B and water.

EFFECT: higher active substance content by improving its organoleptic properties and reducing a viscosity of the liquid dosage form by reducing the content of viscosity-enhancing agents and increasing the water content.

2 cl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention can be used in chemical-pharmaceutical industry for preparing effective tranquilising salamide preparations and analgesics. What is presented is using salicyloyl morpholine sodium salt of formula (I) as a tranquilising, nootropic and analgesic agent possessing low gastric toxicity.

EFFECT: implementing the declared application with the therapeutic index of salicyloyl morpholine of formula (I) is 3 times higher than that of mexidole, and 9,5 times higher than that of aspirin.

5 tbl

FIELD: biotechnology.

SUBSTANCE: invention is a method of producing an adjuvant for vaccines, comprising dissolving a mixture of triterpenoids of bark in tetrahydrofuran, addition of oleic acid, removal of tetrahydrofuran, addition of cryoprotectant, and lyophilisation, at that a mixture of triterpenoids of bark in tetrahydrofuran at a concentration of 5-10 g/l is obtained, followed by dissolving the resulting mixture in oleic acid in an amount of 5-10% by weight of triterpenoids of bark, sterilising filtration of the mixture is carried out, a homogeneous dispersion of spherical amorphous nanoparticles is formed by adding a 25-fold excess of 0.01 M tris buffer, pH - 9.0±0.2 with stirring, followed by ultrasonic treatment for 5-10 minutes, tetrogidrofuran is removed by ultrafiltration at a rate of 1.0-1.2 l/min at a pressure of 0.6-0.8 atm, adding the cryoprotectant the resulting concentrated mixture containing a mixture of terpenoids of 1 mg/ml with the temperature below minus 35°C is frozen, kept at this temperature for 4-6 hours, and lyophilised at a temperature of minus 35°C for 15 hours, followed by subsequent drying at 20-25°C for 15 hours.

EFFECT: invention enables to increase the immunogenic activity of viral vaccines and provides their stability while storage.

3 cl, 2 dwg, 6 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: invention is implemented by single vaccination of the non-acute COPD patients against pneumococcal disease with the preparation Prevenar-13.

EFFECT: method is sage for the COPD patients; it is available for health care facilities, increases the clinical effectiveness in the patients suffering chronic obstructive pulmonary disease.

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, namely to pharmacology and cardiology, and concerns endothelial dysfunction correction. That is ensured by administering a therapeutic agent containing an activated-potentiated form of antibodies to the endothelial synthase of nitrogen oxide in a complex of an activated-potentiated form of antibodies to the C-terminal fragment of the AT1 angiotensin II receptor.

EFFECT: administering this agent provides the effective endothelial dysfunction correction by the synergistic effect of its ingredients.

6 cl, 1 ex, 1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: present group of inventions refers to medicine, namely to neurology, and concerns treating vegetative-vascular dystonia, dizziness syndrome of various origins, and kinetosis. That is ensured by administering a therapeutic agent containing an activated-potentiated form of brain-specific protein S-100 antibody and using the activated-potentiated form of endothelial NO-synthase antibodies as an additional exalting agent.

EFFECT: invention provides the effective treatment of the above pathological conditions by the synergetic effect of the ingredients of the therapeutic agent.

9 cl, 16 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: claimed group of inventions relates to medicine, namely to neurology, and deals with treatment of Alzheimer's disease. For this purpose pharmaceutical composition, which contains activated potentiated form of antibodies to brain-specific protein S-100 and activated potentiated form of antibodies to endothelial NO-synthase, is introduced.

EFFECT: introduction of claimed composition provides efficient treatment of Alzheimer's disease due to synergic neuroprotective, anti-ischemic and anxiolytic action of composition components.

9 cl, 5 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to urology, and deals with treatment of erectile dysfunction. For this purpose pharmaceutical composition, containing activated potentiated form of antibodies to prostate-specific antigen and as additional enhancing component - activated potentiated form of antibodies to endothelial NO-synthase.

EFFECT: introduction of claimed composition provides efficient treatment of erectile dysfunction due to anti-inflammatory, antiproloferative and vascularisation-improving properties of its components.

9 cl, 1 ex

Up!