Active immunisation for creating soluble a-beta antibodies
SUBSTANCE: methods according to the invention consist in introducing an Aβ 16-23 fragment having an amino acid sequence KLVFFAED of 16-23 residues SEQ ID NO:1. Besides the invention concerns the Aβ 16-23 fragment and a pharmaceutical composition containing it.
EFFECT: feasible prevention and treatment of Alzheimer's disease ensured by inhibition of amyloid deposition in cerebrum.
72 cl, 2 dwg, 1 ex
The technical FIELD
The invention relates to the field of immunology and medicine.
BACKGROUND of INVENTION
Alzheimer's disease (AD). Alzheimer's Disease) is a progressive disease leading to senile dementia. Cm. mainly Selkoe, TINS 16, 403-409 (1993); Hardy et al., WO 92/13069; Selkoe, J. Neuropathol. Exp. Neurol. 53, 438-447 (1994); Duffet al., Nature 373, 476-477 (1995); Games et al., Nature 373, 523 (1995). In General, there are two variants of this disease: from late debut, when the disease manifests itself in old age (65 + years), and with early debut, when the disease develops in presenilny period, i.e. between 35 and 60 years. In both variants of the disease pathologic basis of the same, but in the case of earlier changes are often more coarse and common. For the disease characterized by at least two types of brain lesions, senile plaques and neurofibrillary plexus. Senile plaques are areas of disorganized neuropile up to 150 microns in diameter with extracellular deposits of amyloid in the centre, which are visible on the slices of brain tissue under the microscope. Neurofibrillary plexus are intracellular deposits of microtubule-associated Tau protein, which consists of two threads, woven together in pairs.
The basis is the major component of plaques is a peptide, called β peptide or β-amadoda. β peptide is an internal fragment size 39-43 amino acids of the protein predecessor protein called amyloid precursor (APP). Amyloid Precursor Protein). Several mutations of the protein RDA, presumably associated with the development of Alzheimer's disease. See, for example, Goate et al., Nature 349, 704) (1991) (valine717for isoleucine); Chartier Harlan et al., Nature 353, 844 (1991) (valine717for glycine); Murrell et al., Science 254, 97 (1991) (valine717on phenylalanine); Mullan et al., Nature Genet. 1, 345 (1992) (double mutation leading to the replacement pair lysine595- methionine596for a couple of asparagine595- leucine596). It is believed that such mutations causing Alzheimer's disease, because they lead to increased or incorrect processing of APP in β, in particular the processing of APP with the formation of increased amounts of the long form β (i.e. β1-42 and β1-43). It is believed that mutations in other genes, for example genes presenilin, PS1 and PS2, directly affect the processing of APP with the formation of increased amounts of the long form β (see Hardy, TINS 20, 154 (1997)). These observations suggest that β, in particular its long form, is the cause of the development of Alzheimer's disease.
Immunization of transgenic mice models of AD immunogenum, saturated β-amyloid peptide (β), led to or antibody-based test response, is which inhibits the formation of amyloid plaques in the brain of mice or leads to their resorption (Schenk et al., (1999) Nature 400, 173-177; Janus et al., (2000) Nature 408, 979-982, Morgan et al. (2002) Nature 408, 982-985, Sigurdsson et al., (2001) Am. J. Pathol. 159, 439-447, 1-4)). Introduced passively ready antibody β led to the same effects. An assumption was made that the main mechanism of purification of brain tissue from existing amyloid plaques is mediated by antibodies, Fc-dependent phagocytosis by microglial cells and/or macrophages (Bard et al., (2000) Nat. Med., 6, 916-919)). This assumption is based on the fact that certain antibodies to β entered on the periphery, reaching the Central nervous system of transgenic mice, are associated with amyloid plaques and induce their resorption. There was also noted a strong correlation between the antibodies that were effective in vivo, and in studies of ex vivo held on brain slices PDAPP or people affected by Alzheimer's disease, to measure the resorbing activity of antibodies. In studies of ex vivo it was shown that resorption plaques involved Fc-receptors of microglial cells. However, it was also observed that the effectiveness of the antibodies can also be achieved in vivo through mechanisms that are independent Fc interactions (Bacskai et al., (2002) J. Neurosci. 22, 7873-7878). It was reported that the antibody directed against the middle section β, which cannot detect amyloid plaques, binds soluble β and reduces the deposition of plaques (eMattos et al., (2001) Proc. Natl. Acad. Sci. USA, 98, 8850-8855). It was reported that short-term treatment with this antibody resulted in improvement in patients ability to recognize, without affecting amyloid load (Dotart et al., (2002) Nat. Neurosci., 5, 452-457).
BRIEF DESCRIPTION of DRAWINGS
Figa-C. Antibodies obtained by immunization of mice with N-terminal fragments β, are associated with amyloid plaques. Figa. For immunization PDAPP mice used peptides comprising different domains Aβ1-42 (SEQ ID NO:1) (synthetic peptides related to T-cell epitope derived from ovalbumin). As a negative control were used reverseme β5-1 (SEQ ID NO:2). Figv. Titers in ELISA against the aggregated Aβ1-42 were significantly higher throughout the study in groups β5-11 and β15-24, compared with a group β1-5 (1:14, 457, p<0.01 and 1:12, 257, p<0.05 vs. 1:3, 647, respectively; ANOVA with subsequent post hoc test of Tukey). Figs. Flow kristalicheskie brain slices untreated PDAPP mice were treated with serum of mice immunized β5-1, β3-9, β5-11 or β15-24 (titers were normalized to 1:1000 for painting). Antibodies to β15-24 was not associated with amyloid plaques. The unit of the scale corresponded to 500 microns.
Figa-C. Capture of soluble Aβ1-42 antibodies was not associated with reduced amyloid load or nevroticheskoi pathology. Figa. Estimated production ability is here sera of mice immunized with Aβ fragments, capture radioactively labeled soluble Aβ1-42 in radioimmunological analysis. The serum of all animals immunized β15-24, had the ability to capture soluble Aβ1-42 (one serum sample had a titer higher than 1:1,350, the exact title was not defined), in comparison with 27% capture in the group β1-5 and 3% of the capture group β3-9. Figw-N-Amyloid load (Pigv) and neuroticescoy pathology (Figs) was estimated using image analysis in microscopy dark-field. The results were expressed in average interest rates on the group Aβ5-1 (as a negative control was used reversely peptide). Group β5-11 was investigated in a separate content from other groups, but in close relationship with the same group of negative control as an internal standard for comparison (the second set of reversalof β5-1). Amyloid burden was significantly decreased in groups β1-5, β3-9 and β5-11 (p<0.001). The arrows correspond to average values and the dashed horizontal lines indicate the reference level. Nevraticheskaya load was significantly changed after immunization groups β3-9 and β5-11 (p<0.05). None of the endpoints was not significantly changed after immunization group β15-24. Statistical analysis was carried out at an average quadratic deviation (to normalize aparametric the ski variance) and were analyzed by ANOVA method. Test Dunnett then used to compare groups β1-5, β3-9, Aβ15-24 control β5-1, and the test Mann-Whitney - group β5-11 and the corresponding reversemortgage control β5-1.
A DETAILED DESCRIPTION of the INVENTION
This invention relates to a method of prevention, effective prevention or treatment of diseases associated with amyloid deposits, consisting in the use of fragments of the Central or C-terminal sites β. These fragments can induce the formation of a polyclonal mixture of antibodies that are specific bind soluble β without binding to amyloid plaques. Antibodies can inhibit the formation of amyloid deposits β of soluble β in the brain of the patient, thereby ensuring the prevention and treatment of disease. The preferred immunogenum 15-24 are fragments of A-beta and subfragment 5-10 their contiguous amino acids, because they have the ability to generate high titers of antibodies.
For the purpose of classifying amino acids substitutions as conservative and nonconservative, amino acids are grouped as follows: Group I (hydrophobic side chains): norleucine, met, Ala, Val, LEU, Ile; Group II (neutral hydrophilic side chains): Cys, ser, tra; Group III (acidic side chains): ASP, Glu; Group IV (osnovnaya chains): ASN, GLn, GIS, Lys, arg; Group V (residues influencing chain orientation): Glu, Pro, and Group VI (aromatic side chains): three, Tyr, Hairdryer. Conservative substitutions include substitutions between amino acids of the same class. Non-conservative substitutions are substitutions of amino acids of the same class of amino acids of another class.
The term "all-D" refers to peptides having≥75%, ≥80%, ≥90%, ≥95% and 100% of the amino acids in the D-configuration.
The term "agent" is used to describe a compound that has, or may have pharmacological activity. Agents are compounds known as medicines, compounds with proven pharmacological activity, but whose therapeutic activity is subject to additional analysis, and connections that are part of the collections and libraries, the pharmacological activity of which have yet to be investigated.
Therapeutic agents declared in accordance with the present invention, are usually totally free from undesirable impurities. This means that the agent is typically at least about 50% V/V (weight/weight) clean and totally free from impurity proteins and contaminants. Sometimes agents have at least 80% V/V and more preferably at least 90 or 95%/purified. However, using routine techniques, the sight of the possible proteins, it is possible to obtain a homogeneous peptides at least 99% in/in cleaning. therapeutic agents in accordance with the present invention can effectively prevent or treat diseases associated with amyloid deposits.
Specific binding between two compounds indicates that these two compounds have mutual affinity that is at least 10, 100 times higher than the affinity of each of these compounds in relation to control, for example, as an unrelated antigen or antibodies to other antigens. The mutual affinity of the two compounds in relation to each other is usually 107, 108, 109M-1or 1010M-1. The affinity of more than 108M-1are preferred. Specific binding of the polyclonal antibody with epitope β means that antibodies in the group of polyclonal antibodies specific contact one epitope β and does not bind to other epitopes β.
The term "antibody" or "immunoglobulin" is used to denote a complete antibody molecules and their binding fragments. Usually fragments compete with the complete molecule of the antibody from which they are derived for specific binding to antigenic fragment, including separate heavy chains, light chains. Fab, Fab'F(ab')2, Fabc, and Fv. Slices are using recombina is the shaft DNA technology or by chemical separation complete molecules of immunoglobulins. The term "antibody" also refers to one or more chains of immunoglobulins, which are chemically conjugated to, or expressed as fusion proteins with other proteins. The term "antibody" also refers to bispecific antibody. Bispecific or bifunctional antibody is an artificial hybrid antibody having two different pairs of heavy/light chains and two different binding site. Bispecific antibodies can be obtained using a variety of methods including fusion of hybridomas or linking of Fab fragments. See, for example, Songsivilai & Lachmainn, Clin. Exp. Immunol. 79: 315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992).
β, also known as β-amyloid peptide, or A4 peptide (see U.S. patent 4666829; Glenner &Wong, Biochem. Biophys. Res. Commun., 120, 1131 (1984)), is a peptide consisting of 39-43 amino acids and is the main component of the plaques characteristic of Alzheimer's disease. β has several natural forms. Natural form β person designated as β39, β40, Aβ41, β42 and Aβ43. The sequence of these peptides and their relation to the predecessor of the RDA figure 1 illustrates the Hardy et al., TINS 20, 155-158 (1997). For example, β42 has the following sequence:
N2H-ASP-Ala-Glu-Hairdryer-Arg-GIS-ASP-Ser-Glu-Tyr-Glu-Val-GIS-GIS-GLn-Lys-LEU-Val-dryer-Hairdryer-Ala-Glu-ASP-Val-Glu-ASP-Val-Glu-Ser-ASN-Lys-Glu-Ala-Ile-Ile-Glu-LEU-Met-In the l-Glu-Glu-Val-Val-Ile-Ala-HE (SEQ ID NO:1).
β41, β40 and β39 differ from β42 absence of Ala, Ala-Ile, and Ala-Ile-Val, respectively, at the C-end. β43 differs from β42 the presence of a threonine residue at the C-end.
ADR695, ADR751and ADR770refer to long polypeptides, including 695, 751 and 770 amino acid residues, respectively, and encoded by the genome of the RDA person. Cm. Kang et al., Nature, 325, 773 (1987); Ponte et al., Nature, 331, 525 (1988), and Kitaguchi et al., Nature, 331, 530 (1988). Amino acids of the protein amyloid precursor person (RDAs) are numbered according to the sequence isoforms ARR. Terms such as β39, Aβ40, β41, Aβ42 and Aβ43 refer to β the peptide comprising amino acid residues 1-39, 1-40, 1-41, 1-42, and 1-43, respectively.
Disaggregated β or fragments thereof is Monomeric peptide units. Disaggregated β or their fragments are usually soluble and have the ability to samearearule with the formation of soluble oligomers. Oligomers β and their fragments are usually soluble and exist mainly in the form of alpha-helical or random coils. One of the ways to obtain Monomeric β is dissolving lyophilized peptide in pure DMSO and subsequent destruction by ultrasound. The resulting solution was centrifuged to remove insoluble particles. Aggregated β or fragments thereof are oligomers β or it is unagency fragments, in which the Monomeric units are connected together by non-covalent bonds and associated in the insoluble beta-folded structure. Aggregated β or fragments thereof also apply to fibrillar polymers. Fibrils are usually insoluble. Some antibodies bind both soluble β and fragments thereof, and the aggregated β and their fragments. Some antibodies bind soluble β, but does not bind plaques.
"Antigen" is a connection to which a specific antibody binds.
The term "epitope" or "antigenic determinant" refers to the site of antigen interacts with b and/or T-cells. B-cell epitopes can be formed from contiguous or non-contiguous amino acid that comes into contact with Quaternary laying protein. Epitopes formed from contiguous amino acids, typically stable when exposed to denaturing agents, while the epitopes generated during the Quaternary laying protein, usually dissolved in the processing of denaturing agents. An epitope typically includes at least 3, more often at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Eptope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996). Antibodies that recognize the same epitope can be identified using conventional immunological analysis showing the ability of one antibody to block the binding of another antibody to a target antigen. T-cells recognize continuous epitopes of about 9 amino acids for CD8 cells and from 13-15 amino acids for CD4 cells. T cells that recognize the epitope can be identified using in vitro assays, which allows us to measure antigen-dependent proliferation, as determined by the inclusion of3H-thymidine premirovanii T-cells responsible for the epitope (Burke et al., J. Inf. Dis., 170, 1110-19 (1994)), by antigen-dependent killing (method of cytotoxic T-lymphocytes, Tigges et al., J. Immunol., 156, 3901-3910) or secretion of cytokines.
N-terminal epitope β refers to the epitope formed by residues 1-11. Epitope at the C-end refers to the epitope comprising residues 29-43, and the epitope located in the Central area, formed by residues 12-28.
The term "immunological" or "immune" response is the development of the preferred humoral (mediated by antibodies) and/or a cellular (mediated by antigen-specific T cells or their secretion peptides) response against amyloid plaques in the patient's body. This response can be active, induced in the introduction of the immunogen, or passive, induced by the introduction of antibodies or premirovany T cells. Cellular immune response is called a presentation of the polypeptide epitopes associated with molecules of I or Class II major Histocompatibility Complex, to activate antigen-specific CD4+ T helper and/or CD8+ cytotoxic T cells. The response may also include the activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia, eosinophils, other components of the immune system. The presence of a cellular immune response can be confirmed through research of proliferation (CD4+ cells) or CTL (cytotoxic T lymphocytes). cytotoxic T lymphocyte) (see Burke, supra; Tigges, supra). The relative contribution of humoral and cellular responses in protective therapeutic effect of immunogen can be distinguished by separate selection of antibodies and T-cells from immunized syngeneic animal and measuring their protective or therapeutic effect in relation to the second entity.
"Immunological agent" or "immunogen" has the ability to induce an immune response directed against himself, when introduced into the body of a mammal, possibly in combination with adjuvant.
The term "naked" polynucleotide refers to polynucleotide not associated with colloidal material. "Naked" nucleotides is sometimes clone in plasmid vector.
The term "adjuvant" refers to a compound that, when introduced together with an antigen, enhances the immune response to this antigen, and with self-introduction does not lead to immune response to this antigen. Adjuvants can enhance the immune response through several mechanisms, including recruitment (permanent updating of the pool of lymphocytes, stimulation and/or T-cells and stimulation of macrophages.
The term "patient" refers to a person or other mammal receiving prophylactic or therapeutic treatment.
Competition between antibodies were determined using an analysis in which the test immunoglobulin inhibited specific binding of the control antibody with normal antigen, such as β. Currently there are many methods of competitive binding, for example, solid phase direct or indirect radioimmunoassay analysis (RIA). Radioimmunoassay), solid phase direct or indirect enzyme immune assay (EIA). Enzyme Immunoassay), the sandwich method of analysis of competitive binding (see Stahli et al., Methods in Enzymology, 9: 242-253 (1983)); solid phase direct EIA with Biotin-Avidya (see Kirklaud et al., Immunol. 137: 3614-3619 (1986)); solid phase direct analysis of labeled, solid phase direct sandwich assay with labeled (see Harlow and Lane, "Antibodies, A Laboratory Manual", Cold Spring Harbor Press (1988)); solid phase direct RIA labeled with I-125 (with whom. Morel et al., Molec. Immunol. 25 (1): 7-15 (1988)); solid phase direct EIA with Biotin-Avidya (Cheung et al., Virology, 176: 546-552 (1990)) and direct RIA labeled (Moldenhauer et al., Scand. J. Immunol., 32: 77-82 (1990)). Typically, these methods consist in the use of purified antigen bound to a solid surface or cells bearing as unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition is measured by determining the number of labels, contacting a solid surface or cells in the presence of the test antibody. Usually the test immunoglobulin is present in excess. Antibodies determined using the methods of competitive binding (competing antibodies include antibodies that bind to the same epitope as the reference antibody, and antibodies that bind with an adjacent epitope located proximally relative to the epitope is associated with a control antibody for maintenance of a steric barrier. Usually in the case when a competing antibody is present in excess, it will inhibit the binding of the control antibody with normal antigen by at least 50 or 75%.
The antibody that is specific binds soluble β, it is the antibody that binds to β with affinato constituting at least 107M-1. Some antic the La bind soluble β with an affinity of 10 8M-1up to 1011M-1.
The antibody that is specific associated with soluble β without specific binding to plaques, is an antibody that is specific binds soluble β, as described above, and has at least ten, and usually at least 100 times lower affinity specific binding relative to the plaques (i.e. in relation to β in the form of aggregated β-folded structures), obtained from the bodies of patients suffering from Alzheimer's disease, or from transgenic animal models. For example, such an antibody may be contacted with soluble β with an affinity of 109M-1and plaques with affinato less than 107M-1. The affinity of these antibodies against platelets are usually less than 107or 106M-1. Such antibodies additionally or alternatively determined by fluorescence intensity relative to the irrelevant control antibody (for example, the antibody or polyclonal mixture of antibodies to reversearray β the peptide), when antibodies are in contact with the plaques, and binding is monitored using fluorescent labels (as described in the examples). The intensity of fluorescence of antibodies that bind to soluble β peptide without binding to plaques, is within a factor of five, sometimes within a factor of two and sometimes indistinguishable within experimental error from that of the control antibody.
Compositions or methods "includes" one or more repetitive elements may include other elements that are specific not repeated. For example, the composition containing β peptide includes both stand-alone β peptide and β peptide as a component of a larger polypeptide sequence.
β peptides for active immunization
β peptides used in the methods in accordance with the present invention are immunogenic peptides, which when introduced into the human or animal contribute to the formation of antibodies that are specific associated with one or more epitope within 12 to 43 balance β, and do not lead to the formation of antibodies that are specific associated with one or more epitope within residues 1-11 β. The specific antibodies bind to epitopes located between 12 and 43 residues, specific contact with soluble β and are not associated with β in the plaques. Antibodies of this type can be specific to bind soluble β when circulating in the body of the patient or animal model without specific binding to plaques represented by deposits β in the brain of the patient or animal model. Specific antibody binding to soluble β inhibits the inclusion β in plaques, thus leading also the cessation of development of plaques in the brain of the patient or the termination of further increasing their size and number, in that case, if plaques were formed prior to the treatment.
Preferably introduced fragment β has no epitope, which will generate a T-cell response directed against the same fragment. In General, the epitopes of T cells include more than 10 contiguous amino acids. In addition, preferred fragments β consist of 5-10 or preferably from 7-10 contiguous amino acids, i.e. have sufficient length in order to generate or antibody-based test response, but not be involved in the immune response of T-cells. The absence of epitopes for T-cells is preferred because these epitopes are not required for immunogenic fragments, but can cause unwanted inflammatory response in many patients (Anderson et al., (2002) J. Immunol. 168, 3697-3701; Senior (2002) Lancet Neurol. 1, 3). In some methods is a fragment represents a fragment other than β 13-28, 17-28, 25-35, 35-40, 33-42 or 35-42. The majority of T-cell epitopes are within 14-30 amino acids β.
Fragment Aβ15-24 and subfragment 7-9 and their associated amino acids are preferred, since these peptides are equally generate high immune response to β peptide. These fragments include β15-21, β16-22, β17-23, β18-24, β19-25, β15-22, β16-23, β17-24, β18-25, β15-23, β16-24, β17-25, β18-26, β15-24, β16-25 and β15-25. Designation β15-21, for example, refers to a fragment, on the expectation residues 15-21 β and does not include other residues β. Also preferred are C-terminal fragments β42 or 43 of 5-10, preferably 7-10 adjacent amino acids. These fragments can generate or antibody-based test response, which involves end-specific antibodies. These antibodies have the advantage of the specific binding β42 and β43 without specific binding to β39-41. These antibodies bind soluble β without binding to plaques.
In some methods, the fragment of the Central or C-terminal site β entered mode, which also includes the introduction of a fragment from the N-terminal segment. In General, these fragments contribute to the formation of antibodies that are specific associated with plaques and induce their destruction faguoqitirute cells. This immune response is the most appropriate for the destruction of existing sediments β. However, after the destruction of these deposits is preferred to continue the introduction of a fragment from the Central or C-terminal site β to stimulate the formation of antibodies to soluble β to prevent further deposition β without the risk of unwanted inflammatory reactions in certain patients. N-terminal fragments, beginning with 1-3 residues β and ending at residues 7-11 β are the most preferred. Examples of N-terminal fragments is s are β1-5, 1-6, 1-7, 1-10, 3-7, 1-3, and 1-4.
If not stated otherwise, references to the fragments β include fragments of the natural amino acid sequence of a person, as defined above, and their analogues, including alellie, species and induced variants. Analogues β fragments induce the formation of antibodies that are specific contact natural β peptide (for example, β42). Analogues β fragments typically differ from the natural peptide fragments by approximately 30% 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid positions. Each deletion or replacement of natural amino acid residue is considered for position changes, like the inclusion of amino acid residue substitutions. Amino acid substitutions are typically conservative substitutions.
If not stated otherwise, references to β fragments include fragments of the natural amino acid sequence of a person, as defined above, and their analogues, including allelic, species and induced variants. Analogues β induce the formation of antibodies that are specific contact natural β peptide (for example, β42). Analogues β fragments typically differ from the natural peptide fragments by 30%. For example, similar β 15-21 may differ by 1, 2, 3, 4, or 10 amino acid positions. Each deletion or replacement naturallooking balance considered for the position change, as the inclusion of amino acid residue substitutions. Amino acid substitutions are typically conservative substitutions.
Some analogues β or β fragments also include non-natural amino acids or modifications of N or terminal amino acids in one, two, five, ten, or eleven, or even in all positions. For example, the natural aspartic acid residue at position 1 and/or 7 β may be substituted itsprevious acid. Examples of synthetic amino acids are D, alpha, alpha-duhsasana amino acids, N-alkylaminocarbonyl, lactic acid, 4-hydroxyproline, gamma-carboxyglutamate, Upsilon-N-acetylized, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, omega-N-methylarginine, β-alanine, ornithine, norleucine, Norvaline, hydroxyproline, thyroxine, gamma-aminobutyric acid, homoserine, citrulline and itaporanga acid. Some therapeutic agents, notified in accordance with the present invention, are all-D peptides, such as all-D-β or all-D β fragments, or all-D peptide analogues. The effectiveness of fragments or analogues in the prevention and treatment can be studied on a model of transgenic animals when compared with control groups not receiving treatment or placebo, as described below.
β, his piece is s and their analogues can be synthesized using solid phase peptide synthesis or recombinant expression, or can be obtained from natural sources. Automatic system for peptide synthesis are commercially available and provided by different suppliers, such as Applied Biosystems, Foster City, California. Recombinant expression can be carried out in bacteria such as E. coli, yeast cells, insect cells or mammalian cells. Methods recombinant expression are described in Sambrook et al., Molecular Cloning: A Laboratory Manual (C.S.H.P. Press, NY 2d ed., 1989). Some forms β peptide are also commercially available (for example, American Peptides Company, Inc., Sunnyvale, CA and California Peptide Research, Inc. Napa, CA).
Therapeutic agents also include longer polypeptides, including, for example, immunogenic fragment β peptide with one or more other amino acids flanking β peptide with one or both sides. For example, a preferred therapeutic agents include fusion proteins containing the segment β, fused with a heterologous amino acid sequence, which induces T-cell response directed against it, and, thus, promotes the development of b-cell immune response to β segment. For kupirovaniya β peptide to protect it from degradation during production, storage or use, you can use one or more flanking the heterologous amino acids. The effectiveness of these peptides against Professor the prevention and treatment can be studied on a model of transgenic animals when compared with control groups, not receiving treatment or placebo, as described below. Therapeutic agents declared in accordance with the present invention include immunogenic fragment β, flanked polylysine sequences. Polilizinov sequence can be fused to the N-end C-end or with both N - and C-terminal parts immunogenic fragment β. β peptide, its analog, active fragment or other polypeptide can be administered in an associate or a polymeric form or in dissociated form. Therapeutic agents also include polymeric or Monomeric immunogenic agents.
Another option is exhibiting monogenea fragment β with a virus or bacteria as part of an immunogenic composition. Nucleic acid encoding the immunogenic peptide, including the genome or the formation of the episome virus or bacteria. Optionally, nucleic acid include so that immunogenic peptide is expressed as a protein secretion or as a protein fusion with the outer protein surface of the virus or transmembrane protein of the bacteria, so that the peptide exhibited. Viruses or bacteria used in such techniques must be non-pathogenic or attenuated. Suitable viruses include adenovirus, HSV, the virus Venezuelan equine encephalitis and other alpha-viruses, VIR is with vesicular stomatitis and other rhabdovirus, vaccinia virus and avian pox. Suitable bacteria are bacteria of the genus Salmonella and Shigella. Merge monogenea peptide with HBsAg HBV is especially preferred.
Therapeutic agents also include peptides and other compounds, whose amino acid sequence is not necessarily similar to those β, but which can mimic the action β and cause the same immune response. For example, any of the peptides and polypeptides that form β-folded structure can be suitable. Anti-idiotypical antibodies against monoclonal antibodies to β or other amyloidogenic peptides can also be used. Such anti-Id antibodies mimic antigen and generate an immune response directed at him (see Essential Immunology (Roit ed., Blackwell Scientific Publications, Palo Alto, 6thed.), p.181). Agents other than β peptide must induce an immune response directed against one or more preferred segments β listed above (e.g., 15-24). Preferably, these agents cause an immune response, which focuses on one of these segments and is not directed to other segments β.
Also suitable for these purposes may be different libraries of peptides and other compounds. Combinatorial libraries can be created for connections of different types that m is able to be synthesized in stages. These compounds include polypeptides, mimetics of beta-turn, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycine and oligocarbonate. Large combinatorial libraries of compounds can be designed using the method of the encoded synthetic libraries (ESL). The Encoded Synthetic Libraries)that are described in Affymax, WO 95/12608, Affymax, WO 93/06121, Columbia University, WO 94/08051, Pharmacopeia, WO 95/3503 and Scripps, WO 95/30642 (all sources presented here as a reference). Peptide libraries can also be produced using phage display methods. See, for example, Devlin, WO 91/18980.
First of all establish whether combinatorial libraries and other suitable connection, by definition of their specific abilities to communicate with antibodies or lymphocytes (b or T), about which we know that they are specific to β or other amyloidogenic peptides. For example, initial studies can be carried out in any polyclonal serum or monoclonal antibody to β or its fragments. You can then explore how the connection-specific contact with a specific epitope β (e.g., 15-24). Compounds can be tested using technologies that are used to determine the population specificity of the epitope. Compounds identified by such studies, then subjected to analysis to determine their ability to induce the production of antibodies or re-activated lymphocytes to β or its fragments. For example, multiple dilution of sera can be tested on tablets for micrometrology, which are pre-coated β or its fragments, and to determine its ability to induce antibody production to β or its fragment, you can make a standard ELISA. It is then possible to investigate the preventive and therapeutic efficacy of these compounds in transgenic animals predisposed to Alzheimer's disease, as described in the examples. Such animals include, for example, mice having 717 mutation of APP described by Games et al., above, and mouse with 670/671 Swedish mutation of APP, as described McConglogue et al., US 5612486, Hsisao et al., Science, 274, 99 (1996); Staufenbiel et al., Proc. Natl. Acad. Sci. USA, 94: 13287-13292 (1997); Struchler-Pierrat et al., Proc. Natl. Acad. Sci. USA, 94: 13287-13292 (1997); Borchelt et al., Neuron, 19: 939-945 (1997)). The same screening approach can be applied to other potential agents, analogs β long and polypeptides, including fragments β described above.
Some of the agents for stimulating the immune response, are suitable epitope to induce an immune response against LBs, but have too mA is Enki size, to be immunogenic. In this situation, the peptide immunogen can be connected with a suitable media molecule for the formation of the conjugate, which will induce an immune response. One agent can be connected with a single carrier, multiple copies of the agent can be linked to multiple copies of the media, which, in turn, linked, multiple copies of the agent can be connected to one media or one agent can be connected with multiple copies of media or different media. Suitable carriers include serum albumin, hemocyanin mollusk fissurella, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxin or toxins other pathogenic bacteria, such as diphtheria bacteria, E. coli, Vibrio cholerae or .pylori, or attenuated derivative of the toxin. The epitopes for T-cells are also suitable carriers. Some conjugates can be formed by connecting agents declared in accordance with the present invention, with boosting polymer molecules (for example, dipalmitoyl-S-glycerine (Pam3Cys), mannan (polymer of mannose), or glucan (beta 1→2 polymer)), cytokines (eg, IL-1, IL-1 alpha and beta peptides, IL-2, gamma-interferon, IL-10, GM-CSF) and chemokines (e.g., MIP1 alpha and beta, and RANTES). Immunogen the major agents can also be combined with peptides, to improve the transport of substances through the tissue, as described by O Manony, WO 97/17613 and WO 97/17614. Immunogen can also be connected to the media as a means of separating amino acids (e.g., gli-gli), and without them.
Some conjugates can be formed by connecting agents declared in accordance with the present invention, at least one T-cell epitope. Some T-cell epitopes are universal, while others are not. Non-universal T-cell epitopes can enhance the induction of T-cell immunity in a large number of people with different HLA types. On the contrary, a universal T-cell epitopes can enhance the induction of T-cell immunity in a larger percentage of cases, at least 75% of people who have found various HLA molecules encoded by different HLA-DR alleles.
There are a large number of natural T-cell epitopes, such as, for example, tetanus toxin (e.g., epitopes P2 and RO), the surface antigen of hepatitis b virus, pertussis toxin, F protein of measles virus, the major outer membrane protein of Chlamydia trachomatis, diphtheria toxin, circumsporozoite T Plasmodium falciparum CS antigen of Plasmodium falciparum, triazolopyrimidine Schistosoma mansoni, TraT gene, the hemagglutinin of influenza virus (AT). Immunogenic peptides, declared with whom accordance with the present invention, can also be connected to T-cell epitopes described in Sinigaglia et al., Nature, 336: 778-780 (1988); Chicz R. et al., J. Exp. Med., 178: 27-47 (1993), Hammer, J. et al., Cell 74: 197-203 (1993); K. Falk et al., Immunogenetics, 39: 230-242 (1994); WO 98/23635; and S. Southwood et al., J. Immunology, 160: 3363-3373 (1998).
Other examples are the following:
The hemagglutinin of influenza virus:307-319
Malaria CS: T3 epitope EKKIAKMEKASSVFNV (SEQ ID NO:4)
The surface antigen of hepatitis b: HbsAg19-28FFLLTRILTI (SEQ ID NO:5)
Protein toxic shock 65: hsp65153-171DQSIGDLIAEAMDKVGNEG (SEQ ID NO:6)
Bacillus Calmette-guérin: QVHFQPLPPAVVKL (SEQ ID NO:7)
Tetanus toxin: TT830-844QYIKANSKFIGITEL (SEQ ID NO:8)
Tetanus toxin: TT947-967FNNFTVSFWLRVPKVSASHLE (SEQ ID NO:9)
HIV gpl20 T1: KQIINMWQEVGKAMYA (SEQ ID NO:10)
Alternatively, the conjugates can be formed by connecting agents declared in accordance with the present invention, at least one artificial T-cell epitope capable of contact with a large number of molecules HLA Class II, such as pan DR epitope (PADRE). PADRE described in U.S. patent US 5736141, the patent application WO 95/07707 and Alexander J. et al., Immunity, 1: 751-761 (1994) (each source is given here as a reference). Preferred PADRE peptide is AKXVAAWTLKAAA (SEQ ID NO:11) (total residues in bold), where X preferably represents cyclohexylamine, tyrosine or phenylalanine, while cyclohexylaniline most preferred.
Immunogenic agents can be connected to the media using chemical cross-linking. The method of connection of the immunogen to a carrier includes the creation of disulfide linkages using N-Succinimidyl-3-(2-pyridylthio)propionate (SPDP) and Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (if the peptide does not have a sulfhydryl group, this may be provided by including a cysteine residue). These reagents create a disulfide linkage between the peptide bond between cysteine residues of one protein and an amide linkage through the Upsilon-amino group of lysine, or other free amino group in other amino acids. Different variants of such agents that form disulfide/amide linkages described in Immun. Rev. 62, 185 (1982). Other bifunctional linking agents to form thioethers along with the formation of disulfide bonds. Many of these compounds, forming thioethers, are commercially available and include reactive esters of 6-multimediaphoto acid, 2-bromoxynil acid and 2-todokanai acid, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid. Carboxyl groups can be activated by combining them with suktinimida or 1-hydroxyl-2-nitro-4-sulfonic acid, its sodium salt.
Immunogenicity compounds can be us the Lena by including separating residues (for example, Gli-Gli) between Txepitope and peptide immunogen declared in accordance with the present invention. For additional physical separation of Txepitope and b cell epitope (i.e. peptide immunogen), glycine residues can disrupt any secondary structure created by the connection of the Txepitope and the peptide immunogen, and, consequently, to eliminate the interaction between T and/or b-cell responses. Conformational separation between helper epitope and an antibody comprising a domain that provides efficient communication between the exposed immunogen and appropriate Thepitope and b-cells.
To enhance the induction of T-cell immunity in a large number of people with different HLA types, in relation to the agent, notified in accordance with the present invention, can be prepared mixture of conjugates with different Tx-cell epitopes. The mixture can contain a mixture of at least two conjugates with different Tx-cell epitopes, a mixture of at least three conjugates with different Tx-cell epitopes or a mixture of at least four conjugates with different Tx-cell epitopes. The mixture may be injected with adjuvant.
Immunogenic peptides can also be expressed as fusion proteins with nositelyami (i.e. as heterologous peptides). The carrier may be attached to aminobenzo immunogenic peptide, to its carboxyl end or to both ends. Choose fusion protein can include multiple copies of the immunogenic peptide. It is also possible to immunogenic peptide was connected with many copies of a heterologous peptide, for example, at its C - and N-ends. It is also possible that multiple copies of the immunogenic peptide were associated with multiple copies of the heterologous peptide, which, in turn, are connected. Some peptide carriers can serve as stimulants of the immune Tx-cell response against this peptide carrier. Induced T-cells, in turn, induce b-cell immune response directed against the immunogenic peptide linked to a carrier.
Some examples of fusion proteins that are appropriate in the framework of the present invention below. Some of these fusion proteins contain segments β associated with epitopes of tetanus toxin, as described in U.S. patent US 5196512, European patent EP 378881 and European patent EP 427347. Some proteins contain merge segments β associated with at least one peptide PADRE, as described in U.S. patent US 5736142. Some heterologous peptides are not universal T-cell e is Itami, while other heterologous peptides represent a universal T-cell epitopes. In some methods, the agent for injection is a simple protein fusion with β segment associated with the heterologous segment in a linear configuration. Therapeutic agents declared in accordance with the present invention, can be represented using the formula. For example, in some methods, the agent is a polymer fusion proteins, corresponding to the formula 2xin which x is an integer from 1 to 5. Preferably x is 1, 2 or 3, while 2 is more preferable. In the case when x is equal to two, such a polymer has four protein fusions, United in the most preferred configuration, denoted as MAR (see U.S. patent US 5229490).
MAP4 configuration shown below, a branched structure which is obtained by the initial peptide synthesis on N-end, and amines have had the side chain of lysine. In the sequence include lysine required number of times and its branch, the final structure will have several N-ends. In this example, four identical N-end received on a branched nucleus containing lysine. Such a complex structure greatly enhances the reactivity of related b-cells. In the examples below, Z denotes the IMM is nagency fragment β, a Z1-4 mean immunogene fragments β. The fragments may be identical or different.
Other examples of fusion proteins include the following:
Z-Tetanus toxin 830-844 in MAP4 configuration:
Z-QYIKANSKFIGITEL (SEQ ID NO:12)
Z-Tetanus toxin 947-967 in MAP4 configuration
Z-FNNFTVSFWLRVPKVSASHLE (SEQ ID NO:13)
Z-Tetanus toxin 830-844 in MAR configuration:
Z-QYIKANSKFIGITEL (SEQ ID NO:14)
Z-Tetanus toxin 830-844 + 947-967 in a linear configuration:
Z-QYIKANSKFIGITELFNNFTVSFWLRVPKVSASHLE (SEQ ID NO:15)
PADRE peptide (all in a linear configuration), in which X preferably represents cyclohexylamine, tyrosine or phenylalanine, cyclohexylamin is the most preferred -Z:
AKXVAAWTLKAAA-Z(SEQ ID NO:16)
Zx 3-PADRE peptide:
Z-Z-Z-AKXVAAWTLKAAA (SEQ ID NO:17)
Z-ovalbumin 323-339 in a linear configuration:
Z-ISQAVHAAHAEINEAGR (SEQ ID NO:20)
Other examples of fusion proteins include:
AKXVAAWTLKAAA-Z-Z-Z-Z(SEQ ID NO:18)
Z-AKXVAAWTLKAAA (SEQ ID NO:19)
PKYVKQNTLKLAT-Z-Z-Z(SEQ ID NO:21)
Z-PKYVKQNTLKLAT-Z(SEQ ID NO:22)
Z-Z-Z-PKYVKQNTLKLAT (SEQ ID NO:23)
Z-Z-PKYVKQNTLKLAT (SEQ ID NO:24)
Z-Z-Z-QYIKANSKFIGITEL-FNNFTVSFWLRVPKVSASHLE (SEQ ID NO:25)
Z-QYIKANSKFIGITELCFNNFTVSFWLRVPKVSASHLE-Z-QYIKANSKFIGITELCFNNFTVSFWLRVPKVSASHLE-Z(SEQ ID NO:26)
Z-QYIKANSKFIGITEL (SEQ I NO:27) durisotti resin fragments may be identical or different.
To create immunogens, which stimulate the production of antibodies to β or immunogenic fragments β, can be used the same or different carrier proteins and methods of their connection. For example, β or immunogenic fragment β connected with the carrier, there may be a laboratory animal to obtain monoclonal antibodies to β or immunogenic fragment.
V. Nucleic acid encoding a therapeutic agent
The immune response directed against amyloid deposits can also be caused by introduction of a nucleic acid that encodes the segments β peptide fragments, or other peptide immunogen, or antibodies and their component chains used for passive immunization. Such nucleic acids can be DNA or RNA. The segment of the nucleic acid encoding the immunogen, is usually connected with regulatory elements such as promoter and enhancer, which allows the segment of DNA to be expressed in the target cells of the patient. For expression in blood cells, which is desirable to stimulate an immune response, are suitable promoter and enhancer elements from the genes of heavy and long chains of immunoglobulin or major intermediate early promoter and CMV enhancer. Associated regulatory elements and coding is the following sequence usually clone in the vector. For the introduction of double-stranded antibodies two chains can be cloned in the same or different vectors. Nucleic acids encoding therapeutic agents in accordance with the present invention can also encode at least one T-cell epitope. The conclusions presented here and related to the use of adjuvants and carriers, with certain changes, imply their use together with nucleic acids encoding therapeutic agents in accordance with the present invention.
Currently, there are many viral vector systems, including retroviral system (see, for example, Lawrie and Tumin, Cur. Opin. Genet. Develop. 3, 102-109 (1993)); adenoviral vectors (see, for example, Bett et al., J. Virol. 67, 5911 (1993)); adeno-associated viral vectors (see, for example, Zhou et al., J. Exp. Med. 179, 1867 (1994)), viral vectors from the family of poxviruses including vaccinia virus and avian pox, viral vectors of the genus alpha virus, including derived from Sindbis virus and Semliki Forest (see, for example, Dubensky et al., J. Virol. 70, 508-519 (1996)), the virus Venezuelan equine encephalitis virus (see U.S. patent US 5643576) and rhabdoviruses, such as vesicular stomatitis virus (see patent application WO 96/34625), and papillomaviruses (Ohe et al., Human Gene Therapy 6, 325-333 (1995); Woo et al., patent application WO 94/12629, and Xiao &Brandsma, Nucleic Acids. Res. 24, 2630-2622 (1996)).
The DNA code is the first immunogen, or a vector containing the same, can be enclosed in liposomes. Suitable lipids and related analogs are described in U.S. patent US 5208036, 5264618, 5279833 and 5283185. Vectors and DNA encoding the immunogen may also be associated with certain media or absorbed by them, examples are polymers of polymethylmethacrylate and polyactide and poly(lactide-co-glycolide), see, e.g., McGee et al., J. Micro. Encap. (1996).
Vectors for gene therapy or naked DNA can be delivered in vivo by introducing them to a specific patient, mainly by systemic administration (e.g., by intravenous, intraperitoneal, nasal, gastric, intradermal, intramuscular, subcutaneous and intracranial injection or by topical application (see, for example, U.S. patent US 5399346). Such vectors may also include potentiating agents such as bupivacaine (see U.S. patent US 5593970). DNA can also be introduced using a gene gun (see Xiao &Brandsma, supra). DNA encoding the immunogen, are precipitated on the surface of microscopic metal beads. Microsystem give the acceleration using shock waves or expanding flow of helium gas, after which they penetrate tissue to a depth of several cell layers. For example, is appropriate device for gene delivery Accel™, manufactured by the company Agacetus, Inc. Middleton WI. Al is ernative, "naked" DNA can pass through the skin into the bloodstream just after application to the skin, which is accompanied by chemical or mechanical irritation (see application WO 95/05853).
In another embodiment, vectors, encoding immunogenic, can be introduced into cells ex vivo, such as cells isolated from an organism of a particular patient (e.g., lymphocytes, bone marrow aspirate, tissue biopsy) or hematopoietic stem cells universal donor, after which these cells are again administered to the patient, usually after selection of those cells that contain the vector.
Immunogenic agents in accordance with the present invention, such as peptides, sometimes administered in combination with an adjuvant. Adjuvant increases the titer induced antibodies and/or binding affinity of induced related antibodies, if you used one peptide. For stimulation of the immune response can be used in a variety of adjuvants in combination with immunogenic fragments β. Preferred adjuvants enhance the immune response to the immunogen and do not cause changes its conformation, which can affect the quality of the immune response. Preferred adjuvants include aluminum hydroxide and aluminum phosphate, 3-De-O-acylated monophosphoryl lipid A (MPL™) (see UK patent GB 2220211) (RIBI ImmunoChem Research Inc., Hamilton, ontana, now part of Corixa). Stimulon™ QS-21 is a triterpene glycoside or saponin isolated from the bark of the tree Typically Saponaria Molina, growing in South America (see Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell &Newman, Plenum Press, NY, 1995); U.S. patent US No. 5057540), (Aquila BioPharmaceuticals, Framingham, MA). Other adjuvants are oil emulsion in water (such as squalene or peanut oil), possibly in combination with Immunostimulants, such as monophosphoryl lipid a (see Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)), pleurosicya polymers and killed mycobacteria. Another adjuvant is CpG (see application WO 98/40100). Adjuvants may be introduced as components of pharmaceutical compositions together with an active agent or separately, before, during or after administration of therapeutic agent.
A preferred class of adjuvants are aluminum salts such as aluminum hydroxide, aluminum phosphate, aluminum sulfate. Such adjuvants can be used both separately and together with other immunostimulating agents such as MPL or 3-DMP, QS-21, polymeric or Monomeric amino acids, such as polyglutamine acid or polylysine. Another class of adjuvants are emulsion compositions are oil-in-water. Such adjuvants can be used both separately and in conjunction with other immunostimulating agents such as morelove peptides (in the example, N-acetylmuramyl-L-threonyl-D-isoglutamine) (thr-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyrisperidone)ethylamine (MTP-PE), N-acetylglucosaminyl-N-acetylmuramyl-L-Al-D-asoglu-L-Ala-duplicateable (DTP-DPP) terameds) or other components of the cell wall of bacteria. Emulsion oil-in-water include (a) MF59 (see patent application WO 90/14837), containing 5% squalene, 0.5% tween 80, and 0.5% span 85 (option containing various amounts of MTP-PE), turned into submicron particles using microfluidizer, such as microfluidizer Model 110Y (Microfluidics, Newton MA), (b) SAP, containing 10% squalene, 0.4% tween 80, 5% pleuroscopy blocked polymer LI 21 and thr-MDP, also microfluidizer in submicron emulsion or in an emulsion of particles of larger size (obtained using a funnel), (C) Ribi™ adjuvant system (RAS), (Ribi ImmunoChem, Hamilton, MT)containing 2% squalene, 0.2% tween 80, and one or more components of the cell wall of bacteria from the group comprising monophosphorylated peptide A (MPL), trehalose of dimycolate (TDM) and cell wall skeleton (CWS), preferably MPL+CWS (Detox™).
Another class of adjuvants is class sapojnikova adjuvants, such as Stimulon™ (QS-21, Aquila, Framingham, MA), or particles obtained based on it, such as ISCOMs (immunostimulating idea what si) and ISCOMARTIX. Other adjuvants are RC-529, GM-CSF and complete adjuvant's adjuvant (CFA). Complete Freund''s Adjuvant and incomplete adjuvant's adjuvant (IFA). Also adjuvants include cytokines, such as interleukins (such as IL-1 α and β peptides, IL-2, IL-4, IL-6, IL-12, IL-13 and IL-15), colony stimulating factor, macrophage (M-CSF), colony stimulating factor granulocyte-macrophage (GM-CSF), tumor necrosis factor (TNF), chemokines, such as MIP1 α and β and RANTES. Another class of adjuvants are glycolipid analogues including N-glycosylated, N-glycosylation, N-glycosylceramide, each of which charigny residue substituted amino acid representing immunomodulators adjuvants (see U.S. patent US No. 4855283). Proteins toxic shock, such as HSP70 and HSP90, can also be used as adjuvants.
The adjuvant may be administered with the immunogen in a single composition or may be administered before, during or after administration of the immunogen. The immunogen and adjuvant can be Packed in one bubble or each of them may be contained in a separate vial, then they are mixed before introduction. In that case, if the immunogen and adjuvant Packed separately, the package usually includes instructions for proper mixing before use. The choice of adjuvant and/or carrier depends on the stability of immunogenic compositions containing and juvant, the way it was conducted, dosage, effectiveness of adjuvant in relation to the animal, which is the subject of vaccination, and, in the case of use in humans, pharmaceutically available adjuvant is one that has already been tested or have been tested on the possibility of introducing a person accordingly. For example, the full beta-blockers is not suitable for administration to humans. Preferred are salts of aluminum, MPL and QS-21. You can use two or more adjuvants at the same time. Preferred combinations are aluminium salts and MPL, aluminium salts and QS-21, MPL and QS-21, MPL or RC-529 together with GM-CSF, and aluminum salts, QS-21 and MPL together. Can also be used incomplete beta-blockers (Chang et al., Advanced Drug Delivery Reviews 32, 173-186 (1998)), possibly in combination with aluminum salts, and QS-21 or MPL, and simultaneously with all of these adjuvants.
Active immunization fragments β can be combined with passive antibodies. Antibodies used for passive injection may represent antibodies against N-terminal epitopes β in order to stimulate the phagocytic response, aimed at the destruction of amyloid plaques, and can also be an antibody to the Central or C-terminal region β to bind soluble β. In some method is Kah passive antibodies to N-terminal site is used primarily for the destruction of existing amyloid deposits. After that enter a fragment of the Central or C-terminal site β to prevent further fat free β in plaques. According to other methods of active immunization fragments to the Central or C-terminal region β is primarily for the generation of antibodies that bind soluble β. Then, when the level of antibodies in the blood begins to decline, introduce additional dose of antibodies that are specific link Central or C-terminal epitope β.
Antibodies that are suitable for passive introduction, described in applications WO 00/72880 and WO 02/46237. Preferred antibodies bind to the N-terminal epitope β, join, since residues 1-3 and ending at residues 7-11 β. Certain preferred antibodies specifically bind to epitopes within 1-3, 1-4, 1-5, 1-6, 1-7 or 3-7 amino acid residues. Certain preferred antibodies specifically bind to the epitope, starting with 1-3 amino acid residues and ending at residues 7-11 β. Such antibodies are usually specifically associated with amyloid deposits, and how to associate or not to associate soluble β. Certain preferred antibodies specifically bind to the C-terminal epitope β specifically bind natural long form β (i.e. β42 and β43) without specification of the specific binding with natural short form β (i.e. β39 or β41). Antibodies to C-terminal and Central epitopes β usually specifically associated with soluble β, but are not associated with amyloid plaques. In the case where the antibody specifically binds to an epitope on its specific residues, such as, for example, residues 1-5 β, this means that the antibody specifically binds with a polypeptide having a specific amino acid residues (i.e β 1-5, as in this example). This antibody does not need to contact each of these residues (1-5). Also a single amino acid substitution or division within residues 1-5 β did not significantly affect binding affinity. Epitope specificity of antibodies can be determined, for example, as described in the application WO 00/72880.
Antibodies may be monoclonal or polyclonal. Polyclonal serum usually contains a mixed population of antibodies that specifically bind to multiple epitopes located throughout the molecule β. However, polyclonal serum may be specific to a particular segment β, such as β 1-10. Preferred antibodies are chimeric, humanitarianism (see Queen et al., Proc. Natl. Acad. Sci. USA 86: 10029-10033 (1989) and the patent application WO 90/07861, U.S. patent US 5693762, US 5693761, US 5585089, US 5530101 and Winter, U.S. patent US 5225539) or human (Lonberg et al., patent application WO 93/12227 (1993); atenti USA US 5877397, US 5874299, US 5814318, US 5789650, US 5770429, US 5661016, US 5633425, US 5625126, US 5569825, US 5545806, Nature 148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, patent application WO 91/10741 (1991)). Some mouse antibodies with different binding specificity are acceptable for use as starting material for the production of humanized antibodies. The isotype IgG1 person is preferred to generate antibodies to the N-terminal site, because it has the highest affinity among all isotypes person to FcRI receptor located on the faguoqitirute cells. Some antibodies specific contact β with a binding affinity of 107, 108, 109, 1010M-1above.
VIII. Patients under treatment
Treatment of subject patients with individual risk of developing the disease, but no symptoms, and patients with a comprehensive clinical picture of having symptoms. In the case of Alzheimer's disease to those who have risk include men or women who live long enough. In addition, the methods in accordance with the present invention can be used as a preventive in relation to the entire population, without the need of specific people with risk. Methods in accordance with the present invention is especially recommended to people kotoryi.kot a genetic predisposition to Alzheimer's disease. Such people include those who have relatives suffering from this disease and those who have this predisposition has been identified through the identification of genetic or biochemical markers. Genetic markers of Alzheimer's disease include mutations of the gene RDAs, particularly mutations at position 717 and positions 670 and 671, referred to as mutations hardy and Swedish mutations, respectively (see Hardy, TINS16 above). Other markers are mutations in the genes presenilin, PS1 and PS2, and ApoE4, in the presence of a family history of hypercholesterolemia or atherosclerosis in a patient. People suffering from the disease of Allgamer, it is possible to identify characteristic for this disease dementia, as well as the markers described above. In addition, currently there are a number of diagnostic tests to identify patients suffering from Alzheimer's disease. Such tests include the measurement of the levels of CSF-Tau and β42. Elevated levels of Tau-protein and reduced β42 indicate the presence of Alzheimer's disease. Patients suffering from Alzheimer's disease, can also be detected using ADRDA criteria, as described in patent application WO 00/72880.
In patients without symptoms, treatment can be started at any age (for example, 10, 20, 30 years). However, usually does not make sense to start treatment until the patient has not reached the child's age 40, 50, 60 or 70 years. Treatment usually includes the use of multiple doses within a certain period of time. Treatment can be monitored by determining or antibody-based test, T-cell (side effect) or b-cell responses to therapeutic agent (e.g., β peptide) during any period of time. If the answer is reduced, the required reinforcing dose. In the case of potential patients with down syndrome treatment can be initiated antenatal and consist in the introduction of therapeutic agent to the mother or immediately after birth.
IX. Modes of therapy
In General, treatment regimens include administration to a patient a therapeutic agent that is effective in stimulating the immune response to β, preferably immunogenic fragment β. In the case of prophylactic treatment, the pharmaceutical compositions or drugs are typed in patients susceptible to or having a risk of developing Alzheimer's disease, in quantities sufficient to reduce the risk, lessen the severity, or delay onset of the disease, including psychological, biochemical, histological and/or behavioral symptoms, and complications and intermediate pathological manifestations that occur in the process of development of the disease. In the case of a therapeutic agent is administered to the patient, Powergen the mu or already suffering from this disease, in the regime, including the introduction of a certain number of the agent with a certain frequency to cure or at least reduce the symptoms (psychological, biochemical, histological and/or behavioral) or its complications and intermediate pathological manifestations that occur in the process of development of the disease. According to some methods of introduction of the agent reduces or stops the development biocognitive disorders in patients who have not yet revealed all the typical symptoms of Alzheimer's disease. The amount of agent sufficient to achieve a therapeutic or prophylactic effect, referred to as therapeutic or prophylactic effective dose. The combination of quantity and frequency of dose sufficient to achieve a therapeutic or prophylactic effect, referred to as therapeutic or prophylactic is an effective mode. When using both a preventive and a therapeutic mode, the agents are usually entered in several dosages until you reach the desired immune response. The dosage and frequency of administration, sufficient for achieving a therapeutic or prophylactic effect, are referred to as therapeutic or prophylactic is an effective mode. Usually, the immune response of the patient carefully controlrooms is, so if he starts to fall, re-introduces the agent. The immune response can be monitored by detection of antibodies to β in the blood of a patient, determining the level β or the number of plaques in the brain or by identifying symptoms using psychometric measurements, such as MMSE or ADAS, which allow you to fully evaluate the status and functional status of patients with Alzheimer's disease.
Effective doses of the agents and compositions in accordance with the present invention used for the treatment of the above conditions vary depending on various factors, including route of administration, the target tissue, the physiological condition of the patient, the patient is human or an animal, if he gets any other medicines whether the introduction of therapeutic or prophylactic. Usually the patient is a man, but treatment and other mammals, including transgenic animals. Therapeutic doses should be titrated to ensure the safety and effectiveness of treatment. The amount of an agent depends on whether jointly with him adjuvant, while in its absence, require higher doses of the agent. The amount of immunogen for the introduction sometimes varies from 1-500 μg for one patient before, more often, 5-500 μg per ml is in the shot man. Sometimes used a higher dose of about 1-2 mg per one injection. Usually, one injection human use 10, 20, 50 or 100 mg quantity of the injected immunogen also depends on the mass ratio of the immunogenic epitope to the total weight of the immunogen. Typically used from 10-3up to 10-5the micromol monogenea epitope on micrograms of immunogen. The time of introduction of the agent may also vary considerably: from once a day, once a year to once in ten years. Every day dose of immunogen is more than 1 mg for one patient and is usually more than 10 µg / if adjuvant is used, and is more than 10 µg and usually more than 100 μg, if adjuvant is not used. The normal mode includes immunization followed by the introduction of reinforcing doses at regular intervals, components, for example, 6 weeks. Another mode involves immunization with the subsequent introduction of a booster dose of 1, 2 and 12 months later. Another mode involves the introduction of the drug every two months throughout life. Alternatively, the reinforcing injections may not have regular injection mode, depending on the determined level of the immune response of the patient.
The doses of nucleic acids encoding immunogenic range from 10 ng to 1 g, 100 ng to 100 mg, 1 μg to 10 mg is the same from 30 to 300 μg DNA per patient. Dosage viral vectors vary from 10-100 or more virions per dose.
For passive immunization with an antibody (in the case of combination therapy, the dose varies from 0.0001 to 100 mg/kg, usually from 0.01 to 5 μg/kg weight of the patient. For example, the dose may be 1 μg/kg or 10 mg/kg, or to be in the range from 1 to 10 mcg/kg, in other words, 70 μg or 700 μg, or in the range from 70 to 700 μg, respectively, for a patient weighing 70 kg Approximate treatment consists of the administration of the agent once in two weeks once a month or once every 3 or 6 months. According to some methods introduced simultaneously with two or more monoclonal antibodies with different binding specificity in each case, the dosage of each antibody correspond to the above. The antibody is typically administered in a variety of ways. The interval between the introduction of a single dose may be one week, one month or even one year. The frequency of injection may also be irregular and decide measured in the patient's blood levels of antibodies to β. According to some methods, the dose is calculated so that the concentration of antibodies in the plasma was 1-1000 μg/ml, according to another 25-300 μg/ml Alternatively, the antibody may be administered in the form of a special composition with delayed release, which requires a more rare introduction. Dosirak and frequency of introduction vary depending on the half-life of antibodies in the patient's body. In General, the greatest half-life are human antibodies, then humanized antibodies, chimeric antibodies and antibodies are not human. Dosage and frequency of injection also depend on whether treatment is prophylactic or therapeutic. In the case of prophylactic treatment, a relatively small doses with relatively infrequent intervals over a long period of time. Some patients receive treatment throughout their life. In the case of therapeutic treatment is administered fairly large doses at short intervals until until you see the signs of the regression of the disease, preferably before until the patient is partially or completely disappears symptoms. After this, the patient will receive antibodies in prevention mode.
Agents that cause an immune response and is used for prevention and treatment can be administered parenterally, topically, intravenously, orally, subcutaneously, intraarterially, intracranial, administered intraperitoneally, intranasally or intramuscularly. The most frequently immunogenic agent is injected subcutaneously, although other routes of administration are also effective. The other most frequently used method of administration is intramuscular. Usually, the injection is carried out in the muscles of the shoulder or hip. In some methods, agents centuries is changed directly to the fabric, in which there are deposits of amyloid, for example, using intracranial (intracranial) introduction. Preferred routes of administration of antibodies is intramuscular and intravenous (in combination therapy). According to some methods specific therapeutic antibodies are injected directly into the skull. In some methods, the antibody is administered in the form of special compositions with delayed release, such as, for example, Madipad™.
Agents in accordance with the present invention are usually used in the form of pharmaceutical compositions containing a therapeutically active agent and a variety of other pharmaceutically available components. Cm. Remington''s Pharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pennsylvania, 1980). The preferred form depends on the assigned route of administration and therapeutic application. The composition may also contain, depending on the desired formulation, pharmaceutically available, non-toxic carriers or diluents, which are typically used as a binder compounds in the pharmaceutical compositions used in humans and animals. The diluent is selected so that it does not affect the biological activity of the composition. Examples of such diluents are distilled water, physiological phosphate buffer saline, ringer's solution, actor dextrose, the solution Khanka. In addition, the pharmaceutical composition or formulation may also include other carriers and adjuvants, or nontoxic, non-immunogenic non-drug stabilizers or similar connection.
Pharmaceutical compositions may also contain large, slowly metabolisable macromolecules, such as proteins, polysaccharides, for example chitosan, polylactic acid, polyglycolic acids and copolymers (such as latex functionalized sepharose (TM), agarose, cellulose and similar compounds), polymeric amino acids, copolymers of amino acids, lipid aggregates (such as oil droplets or liposomes). In addition, these carriers can act as immunostimulating agents (i.e. adjuvants).
For parenteral administration, the agents in accordance with the present invention can be administered in injectable form comprising a solution or suspension of the active agent in a physiologically acceptable solvent together with a pharmaceutical carrier which can be a sterile liquid such as water oils, saline, glycerol, or ethanol. In addition, the composition may contain auxiliary compounds such as moisturizing or emulsifying agents, surfactants, pH bothersome connections and similar substances. Other com is hentai composition can be compounds of the animal, vegetable, synthetic or petroleum origin, such as peanut oil, oil from soybean seeds, mineral oil. In General, the preferred liquid carriers are glycols, such as propylene glycol or polyethylene glycol, particularly for injectable solutions. Antibodies can be administered in the form of a depot injection or in the form of compositions for implantation, which are designed to provide a slow and gradual release of the active agent. An exemplary composition comprises monoclonal antibody at a dose of 5 mg/ml in aqueous buffer containing 50 mm L-histidine, 150 mm NaCl, brought to pH 6.0 with HCl. Compositions for parenteral administration are generally completely sterile, isotonic, prepared in accordance with GMP-conditions of the PDA or in a similar manner.
Typically, the compositions are prepared in a format suitable for injection, in the form of solutions or suspensions, and solid forms for dissolution or suspension in liquid prior to introduction. The composition also can be emulsified or encapsulated in liposomes or microparticles, such as polylactide, polyglycolides or copolymers to enhance the adjuvant effect, as discussed above (see Langer, Science 249, 1527 (1990), and Hanes, Advanced Drug Delivery Reviews 28, 97-119 (1997). Agents, notified in accordance with n is a worthwhile invention, can be entered in the form of a depot injection or compositions for implantation, which are created in such a way as to ensure a gradual and slow release of the active ingredient.
Additional compositions include compositions for oral, intranasal, pulmonary applications, compositions for application to the skin, as well as suppositories.
To create suppositories as forming compounds and carriers used, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in an amount of from 0.5% to 10%, preferably 1%-2%. Compositions for oral administration include the so-called receptive medium (medium for drugs), such as pharmaceutical mannitol, lactose, starch, magnesium stearate, saccharine sodium, cellulose and magnesium carbonate. These compositions can take the form of solutions, supense, tablets, pills, capsules, drugs with a slow release of the active ingredient or powders and contain 10% to 95%, preferably from 25% to 70% of the active connection.
A local application is cutaneous or intradermal administration. Local injection can be enhanced by combining the agent and the cholera toxin or its detoxificating derivatives is whether subunits, or by joint application of the active agent and another identical bacterial toxin (see Glenn. et al., Nature 391, 851 (1998)). Co-administration can be achieved using the specified components in the mixture, as well as associated with the chemical synthesis of molecules or expression as fusion proteins.
Alternatively, the percutaneous introduction can be carried out using the skin path, or when using transversum (Paul et al., Eur. J. Immunol. 25, 3521-24 (1995); Cevc et al., Biochem. Biophys. Acta 1368, 201-15 (1998)).
The invention relates also to methods for determining or antibody-based test response against β peptide in patients suffering from diseases associated with amyloid deposits, or predisposed to it. These methods are especially suitable for monitoring treatment of patients. These methods are suitable for monitoring treatment in patients with symptoms of the disease, and for monitoring preventive treatment in patients who do not have symptoms. Some ways are to determine the baseline level of antibodies in the blood of the patient before treatment and compared with the level or antibody-based test response after treatment. A significant increase (more than usual boundary of experimental error in repeat measurements of the same sample, expressed as one standard the deviation from the mean of such measurements) in value or antibody-based test response indicates a positive treatment outcome (i.e., the introduction of the agent resulted in the achievement or increase the immune response). If the level or antibody-based test response was not significantly increased or even decreased, this indicates a negative treatment outcome. In General, patients who received initial treatment immunogenic agents, in effective doses, are expected to increase the immune response, which eventually reaches a plateau phase. The introduction of the active agent continue to until there is a growth of the immune response. Reaching the plateau phase is a signal to stop treatment or reduce the dose and frequency of the conduct of the agent.
Other ways are to determine the control value (i.e. the average and standard deviations) or antibody-based test response in the control population. Usually patients from control populations do not receive prior treatment. Measured values or antibody-based test response in patients after administration of therapeutic agent compared with control values. A significant increase in comparison to the control value (for example, more than one standard deviation from the average) indicates a positive treatment outcome. No significant increase or decrease in immune response indicates a negative treatment outcome. Introduction agent usually continue until or antibody-based test response increases compared to the control value. Ka is in the previous method, reaching the plateau phase is a signal to stop treatment or reduce the dose and frequency of administration of the agent.
Other ways are to determine the control value (i.e. the average and standard deviations) or antibody-based test response in a control population of patients who have already undergone treatment therapeutic agent and have or antibody-based test response reached a plateau phase. Measured values or antibody-based test response in these patients compared with the control values. If measured at the patient level does not differ significantly (i.e. more than one standard deviation from the control, this means that treatment can be discontinued. If the level or antibody-based test response is significantly lower than the control, it is necessary to continue the introduction of therapeutic agent. If the value or antibody-based test response remains at the same level, you must change the mode of treatment, for example to use a different adjuvant, fragment, or go to the passive introduction.
Another way is to monitor or antibody-based test response in a patient who is not currently receiving treatment, but has already undergone pre-treatment, in order to determine whether this patient continued treatment. Measured value or antibody-based test response in a patient can be compared with the value obtained from E. the CSOs same patient immediately after the preliminary course of treatment. A significant reduction relative to the first dimension (i.e. more than the usual minimum of errors in repeated measurements of the same sample) suggests that treatment should be resumed. Alternatively, the value measured in the patient compared with the control value (mean plus standard deviation)measured in patients already treated. Alternatively, the value measured in the patient compared with the control value obtained in patients receiving prophylactic treatment and have no symptoms of the disease, or in patients who have received therapeutic treatment and have decreased manifestations of the disease. In all these cases, a significant decrease compared to control (i.e. more than one standard deviation) indicates that the treatment of this patient must be renewed.
Usually for the analysis to take samples of blood, plasma, serum, mucus, or cerebrospinal fluid. The sample is examined for the presence of an immune response to any form β peptide, usually to Aβ42 or peptide used for immunization. The immune response is determined by the presence of antibodies that are specific contact β peptide. Antibodies can be identified through analysis of binding with a ligand which is specific associated with them Usually ligand immobilized. Binding may be determined using a labeled antiidiotypic antibodies.
When using combined modes, including both active and passive introduction, for monitoring the level of antibodies resulting from passive introduction, you can apply the same approaches described, for example, in the patent application WO 00/72880.
Materials and methods
β fragments. Peptides corresponding to β1-5, β3-9, β5-11, β15-24 and reverse sequence Aβ5-1, is able to interact with T-cell epitope of 17 amino acids, derived from ovalbumin, were synthesized on a branched peptide skeleton (the core of the three lysine residues with four peptide branches) to obtain multiantigenic peptide, as described by Tam, J.P. (1988) Proc. Natl. Acad. Sci. USA 85, 5409-5413. Got polyclonal antibodies (Pab) β1-42 and the immunoglobulin fraction was isolated as described by Bard, F. et al., (2000) Nat. Med. 6, 916-919. Polyclonal antibody Pab-EL16, Pab-EL17 and Pab-EL20 were obtained from serum PDAPP mice immunized with peptides corresponding to β1-7, Aβ15-24 and β3-9, which were synthesized on an extensive frame, as described above. Pab-EL26 was obtained from the serum of a mouse immunized β(7-1)42. Peptides were synthesized by AnaSpec, San Jose, CA, USA.
The immunization procedure. 100 mcg β fragment in complete Freund's adjuvant was injected by NutriBar the bus injections, then in 2 weeks, 4 weeks and 1 month introduced an additional 100 μg peptide in incomplete Freund's adjuvant.
Antibody binding to aggregated and soluble Aβ1-42. Titers of serum (obtained by multiple dilution) and monoclonal antibody to bind to the aggregated synthetic β1-42 were determined using ELISA as described by Schenk D. et al., (1999) Nature 400, 173-177. Soluble Aβ1-42 corresponds to the synthetic Aβ1-42 peptide, destroyed by ultrasound in dimethyl sulfoxide. Several dilutions of antibody were incubated with 50,000 cmp125I Aβ1-42 over night at room temperature. 50 μl of liquid clay, containing 75 mg/ml protein And sepharose (Amersham Biosciences, Uppsala, Sweden)/200 mcg artemisinin IgG (H+L) rabbit (Jackson ImmunoResearch, West Grove, PA, USA) were incubated with diluted antibody for 1 hour at room temperature, washed twice, and counted on a gamma counter Wallac (PerkinElmer Life Sciense, Grove, IL, USA). All steps were carried out in buffer for radioimmunological studies containing 10 mm Tris, 0.5 M NaCl, 1 mg/ml gelatin and 0.5% Nonidet P-40, pH 8.0.
Compared the ability of several series of peptides to induce effective or antibody-based test response in vivo. PDAPP mice aged twelve to thirteen months were immunized with one of three N-terminal peptide fragments (Aβ1-5, Aβ3-9 or β5-11) or a fragment derived from the internal teaching the TKA peptide (β15-24) (figa). Internal peptide Aβ15-24 with the epitope of antibody 266, which has a high affinity to soluble Aβ (Seubert et al. (1992) Nature 359, 325-327), does not recognize plaques in the flow sections of the brain affected by Alzheimer's disease, or in PDAPP tissue. Thus, it was interesting to determine whether the polyclonal response directed against this peptide may lead to the formation of antibodies having the ability to recognize plaques, or the introduction of a single soluble β is quite effective. In these studies, the peptide with the reverse sequence of Aβ5-1 was used as negative control. Synthesized peptides capable of interacting with T-cell epitope of 17 amino acids, derived from ovalbumin, and represented them in identical multivalent configurations (see Materials and methods). All peptides (with the exception of reversely β5-1) resulted in the formation of serum, which recognizes the aggregated synthetic Aβ1-42 ELISA, although β5-11 and Aβ15-24 caused the formation of higher titers of antibodies than AβB1-5 (p<0.05 and p<0.05, respectively) (pigv). On the contrary, only serum against the N-terminal peptides was able to recognize β in plaques; anticavity against β15-24 not connected plaques, despite the strong reactivity against synthetic agrege is consistent with the peptide (figs). There are also differences in the ability of different groups of sera to capture soluble β (figa). Less than 30% of sera of mice immunized β1-5 or β3-9, took soluble peptide (27% and 5% respectively). In contrast, serum approximately half of the animals immunized Aβ5-11, and all animals immunized β15-24, was captured soluble Aβ1-42.
Because the level of deposits β varies considerably depending on the age of PDAPP mice in vivo was carried out on groups that include at least 30 animals. Data on the effectiveness of immunization are shown for one mouse and expressed as a percentage of the amyloid or nevroticheskoi load to the average control value (100%). Immunization with each of the three N-terminal peptides significantly reduced amyloid load (46-61%, p<0.002) (pigv). In addition, β3-9 and Aβ5-11 significantly reduced neuroticescoy pathology (34% and 41% respectively, p<0.05) (figs). Immunization β15-24 did not lead to the reduction of amyloid or nevroticheskoi load. These results indicate that binding to plaques is one of the mechanisms of the effectiveness of antibodies. They also show that the capture of soluble β not required to reduce nevroticheskoi pathology and up until or antibody-based test response against β3-9 provides a strong re aktivnosti against plaque and a high level of protection against neural degeneration, even with a weak ability to bind soluble peptide. Antibodies that bind soluble β without binding to plaques may also have such activity, if you write them in high titers, or within a longer period of time. Antibodies that bind soluble β without binding to plaques may also be useful to prevent the formation of plaque and/or sediments β. High titers of antibodies obtained by immunization β15-24 shows that this fragment or subfragment are most suitable for generating high titers of soluble antibodies.
1. Method for the treatment or prevention of Alzheimer's disease, which consists in the introduction of the fragment β16-23 having an amino acid sequence KLVFFAED from 16-23 residues of SEQ ID NO:1, or a fragment having the amino acid sequence KLVFFAED from 16-23 residues of SEQ ID NO:1 and is related to media molecule for the formation of the conjugate.
2. The method according to claim 1, in which β fragment induces antibodies that bind soluble β the patient.
3. Method for the treatment or prevention of Alzheimer's disease, which consists in the introduction of polynucleotide encoding: fragment β16-23 having an amino acid sequence KLVFFAED from 16-23 residues of SEQ ID NO:1, or a fragment having the amino acid sequence KLVFFAED from 16-23, astatke is SEQ ID NO:1 and is related to media molecule for the formation of the conjugate.
4. The method according to claim 3, in which polynucleotide is expressed in a patient with production β fragment that generates antibodies that bind soluble β the patient.
5. The method according to claims 1 to 4, which further added fragment β, causing the formation of antibodies, specific binding β through one or more epitope β1-11.
6. The method according to claim 5, in which the fragment β, which induces the formation of antibodies, specific binding β through one or more epitopes β1-11, introduced before the introduction of the fragment β16-23.
7. The method according to claims 1 to 4, which further added antibody that is specific links β through epitope β1-11.
8. The method according to claim 7, in which the antibody that is specific links β through epitope of Aβ1-11, introduced before the introduction of the fragment β16-23.
9. The method according to any one of claims 1 to 8, where the disease is cognitive impairment.
10. The method according to any one of claims 1 to 8, which are assessed in relation to the patient, who is a man.
11. The method according to any one of claims 1 to 8, further comprising monitoring induced antibodies in a patient.
12. The method according to any one of claims 1 to 8, which are assessed in relation to the patient without symptoms of Alzheimer's disease.
13. The method according to any one of claims 1 to 8, which are assessed in relation to the patient having symptoms of Alzheimer's disease, the method leads to the decrease of et the x symptoms.
14. The method according to any one of claims 1 to 8, used in relation to a patient under the age of 50.
15. The method according to any one of claims 1 to 8, which are assessed in relation to the patient with an inherited risk factor, indicating a predisposition to Alzheimer's disease.
16. The method according to clause 15, which are assessed in relation to the patient, which during the five years after the start of treatment was not observed progression of the symptoms.
17. The method according to any one of claims 1 to 14 and 16, which are assessed in relation to the patient, with no known risk factors for Alzheimer's disease.
18. The method according to any one of claims 1 to 8, which is the introduction of a fragment β dose, component, at least 50 μg, for many days.
19. The method according to any one of claims 1 to 8, in which the fragment is administered in multiple doses over a period of time comprising at least three months.
20. The method according to claim 19, in which the dose is at least 50 μg.
21. The method according to claim 1 or 3, which impose additional adjuvant that increases the level of antibodies produced by the fragment β.
22. The method according to item 21, in which the adjuvant is pharmaceutically acceptable.
23. The method according to claim 1 or 3, wherein the fragment is administered intraperitoneally, orally, intranasally, subcutaneously, intramuscularly, topically or intravenously.
24. The method according to claim 1 or 3, further comprising monitoring at the anti-shudder performance of induced antibodies in the patient's blood.
25. The method according to claims 1 to 4, in which the fragment is associated with media molecule for the formation of the conjugate.
26. The method according A.25, in which multiple copies of the fragment associated with media molecule for the formation of the conjugate.
27. The method according A.25, in which multiple copies of a fragment is associated with multiple copies of the molecules of the medium.
28. The method according to p, in which the carrier is a heterologous polypeptide comprising the amino acid sequence QYIKANSKFIGITEL (SEQ ID NO:8).
29. The method according to p, in which the carrier is a heterologous polypeptide comprising the amino acid sequence AKXVAAWTLKAAA (SEQ ID NO: 11).
30. The method according to p or 29, in which the polypeptide induces T-cell response directed against the heterologous peptide and, therefore, b-cell response directed against a fragment.
31. The method according to claims 1 to 4, which impose additional adjuvant that increases the titer and/or the binding affinity of induced antibodies compared with the introduction of only one fragment.
32. The method according to p, in which the adjuvant is pharmaceutically acceptable.
33. The method according to p, in which the fragment β or polynucleotide encoding fragment β injected in the form of a composition with adjuvant.
34. The method according to p, in which the adjuvant is administered prior to administration of the fragment or polynucleotide encoding fragm the NT.
35. The method according to p, in which the adjuvant is administered after administration of the fragment or polynucleotide encoding the fragment.
36. The method according to p, in which the adjuvant is an aluminium salt, or MPL, or QS-21, or the incomplete beta-blockers.
37. The method according to claim 1, in which the dose of the fragment is more than 10 µg per injection.
38. Fragment β having the amino acid sequence KLVFFAED from 16-23 residues of SEQ ID NO:1.
39. Fragment by § 38 associated with media molecule for the formation of the conjugate.
40. Fragment by § 39, in which multiple copies associated with a single molecule of the media.
41. Fragment by § 39, of which one copy is associated with one molecule of the media.
42. Fragment by § 39, associated with the molecule of the carrier via a spacer.
43. Fragment by § 39, associated with the molecule of the carrier by a chemical bond.
44. Fragment by § 39, in which the carrier is a heterologous polypeptide.
45. The fragment in item 44, in which the heterologous polypeptide contains the sequence QYIKANSKFIGITEL (SEQ ID NO:8).
46. The fragment in item 44, in which the heterologous polypeptide contains the sequence AKXVAAWTLKAAA (SEQ ID NO:11).
47. Fragment by § 39, in which the carrier is a diphtheria toxin.
48. The fragment in § 38, further containing a pharmaceutically acceptable carrier.
49. The fragment in § 38, which is compatible with the STN with the N-terminal section of the fragment β.
50. The fragment in § 49, in which the N-terminal site is a β1-5.
51. The fragment in § 49, in which the N-terminal site is a β1-6.
52. The fragment in § 49, in which the N-terminal site is a β1-7.
53. A pharmaceutical composition comprising a fragment β comprising the amino acid sequence KLVFFAED from 16-23 residues of SEQ ID NO:1, and a pharmaceutically acceptable carrier.
54. The composition according to item 53, in which the fragment is associated with media molecule for the formation of the conjugate.
55. The composition according to item 54, in which multiple copies of the fragment associated with one molecule of the media.
56. The composition according to item 54, in which one copy of a fragment is associated with one molecule of the media.
57. The composition according to item 54, in which the fragment is associated with a molecule of the carrier via a spacer.
58. The composition according to item 54, in which the fragment is associated with a molecule of the carrier by a chemical bond.
59. The composition according to item 54, in which the carrier is a heterologous polypeptide.
60. The composition according to p, in which the heterologous polypeptide contains the sequence QYIKANSKFIGITEL (SEQ ID NO:8).
61. The composition according to p, in which the heterologous polypeptide contains the sequence AKXVAAWTLKAAA (SEQ ID NO:11).
62. The composition according to item 54, in which the carrier is a diphtheria toxin.
63. The composition according to item 53, additionally aderasa the N-terminal β fragment.
64. The composition according to p, whose N-terminal fragment is a β1-5.
65. The composition according to p, whose N-terminal fragment is a β1-6.
66. The composition according to p, whose N-terminal fragment is a β1-7.
67. The composition according to p, optionally containing adjuvant.
68. The composition according to p, in which the adjuvant is an aluminium salt.
69. The composition according to p, in which the adjuvant is an MPL.
70. The composition according to p, in which the adjuvant is a QS-21.
71. The composition according to p, in which the adjuvant is an RC-529.
72. The composition according to item 53, further containing a surfactant.
SUBSTANCE: invention refers to medicine, namely to neurology, and can be used in treating the patients with multiple sclerosis. That is ensured by infusion of rituximab in a dose 1 g followed by introduction of mitoxantrone in a dose 20 mg and another infusion of rituximab in a dose 1 g 13-15 days later. A common preanaesthetic medication precedes infusion of rituximab.
EFFECT: method ensures fast therapeutic effect and prolonged remission in all forms of multiple sclerosis without a concomitant therapy, including chemotherapy.
1 ex, 11 dwg
SUBSTANCE: present invention relates to new compounds of formulae and , in which radicals and symbols assume values defined in the formula of invention, e.g. to 1H-indazoles, 1,2-benzisoxazoles and 1,2-benzisothiazoles. Said compounds are receptor ligands of the α-7 nAChR subtype. The invention also relates to a pharmaceutical composition containing the said compounds.
EFFECT: possibility of using the said compounds to make medicinal agents for treating diseases associated with impaired functioning of nicotinic acetylcholine receptors and their abnormal functioning, primarily in brain cells.
46 cl, 85 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: according to the invention, a pharmaceutical composition contains aripiprazole metabolite combined with serotonin reuptake inhibitor. Aripiprazole metabolite is chosen from dehyoaripiprazole DM-1458, DM-1451, DM-1452, DM-1454 or DCPP. Aripiprazole metabolite is a stabiliser of dopamine-serotonin system. Serotonin reuptake inhibitor can be fluoxetine, duloxetine, venlafaxine, milnaciprane, citalopram, fluvoxamine, paroxetin, sertraline or escitalopram and their salts. The pharmaceutical composition contains one weight part of said aripiprazole metabolite and 0.01 to 50 weight parts of said serotonin reuptake inhibitor. The pharmaceutical composition under the invention can be used for treating the patients with phrenopathies, particularly with depression or major depressive disorder.
EFFECT: pharmaceutical composition is effective and does not cause by-effects.
22 cl, 8 dwg, 3 tbl, 9 ex
SUBSTANCE: invention relates to medical agents and concerns a remedy in the form of an injection precursor product for obtaining liquid injectable composition, containing as agent for prevention and/or treatment of neurodegenerative diseases, nervous disorders or diseases requiring nerves regeneration the effective quantity of (2R)-2-propyloctane acid or salt thereof, and 1-5 equivalents of the basic ion of metal as per 1 equivalent of (2R)-2-propyloctane acid or salt thereof, metal salt selected from phosphoric acid metal salt, carbonic acid metal salt, and sulfurous acid metal salt and may optionally contain metal hydroxide as the source(s) of the basic metal ion.
EFFECT: development of the remedy which is resistant to pH fluctuations and does not grow turbid at solution.
21 cl, 3 dwg, 21 tbl, 22 ex
SUBSTANCE: invention relates to novel naphthalene derivatives of general formula I , as well as to their pharmaceutically acceptable salts, which can be used for treating and/or preventing diseases associated with H-3 receptor modulation. In formula I, R1 is selected from hydrogen, lower alkyl, phenyl, phenyl-lower alkyl and lower alkoxyalkyl; R2 is selected from hydrogen, lower alkyl, C3-C7-cycloalkyl, lower alkoxyalkyl or lower alkylsuphanylalkyl (all values of R1 and R2 are given in the formula of inventions); or R1 and R2 together with the nitrogen atom to which they are bonded form a 4-7-member saturated or partially unsaturated heterocyclic ring which can contain one more heteroatom selected from nitrogen, oxygen and sulphur atoms, where the said heterocyclic ring can be unsubstituted or substituted with 1-2 groups, or can be condensed with an unsubstituted phenyl ring; A is selected from (values of R3-R7, R9, R10, X, m, n, t, p, q and s are given in the formula of invention). Invention also pertains to a pharmaceutical composition containing formula I compounds.
EFFECT: increased effectiveness of application.
30 cl, 2 tbl, 188 ex
SUBSTANCE: therapeutically effective amount of a proteosome composition containing a preparation of outer membrane protein of gram-negative bacteria.
EFFECT: reduced amyloid deposit ensured by activated microglial phagocytosis without induction of neurotoxic effects.
17 cl, 9 dwg, 4 tbl, 1 ex
SUBSTANCE: invention relates to medicine, namely to neonatology, and can be used in treatment of newborns who are on ILV in critical state, conditioned by perinatal affection of central nervous system. For this purpose preliminarily rate of blood flow in vein of Galen is determined by dopplerography. If rate of blood flow decreases lower than 4 cm/s, inhalation with nitrogen oxide in concentration 15 ppm during 24 hours is carried out.
EFFECT: method allows to considerably reduce lethality of newborns in case of said pathology due to respiratory therapy with nitrogen oxide which is endothelial relaxing factor, and in case of introduction through inhalation - selective pulmonary vasodilatator.
SUBSTANCE: invention relates to compounds of formula (I) , where A, B represent C; D is selected from N; X is selected from -CRaRb-, -O-; Y is selected from O; Z1 and Z2 are independently selected from hydrogen, substituted or unsubstituted alkyl having 1-8 atoms, R1-R4 are independently selected from hydrogen or halogen; R5-R16 independently represent hydrogen; where Ra and Rb independently represent hydrogen; n equals 1-3; m equals 0 or 1, k equals 2; or to pharmaceutically acceptable salts or solvates of the said compounds. The invention also relates to a pharmaceutical composition based on the said compounds which inhibits butyrylcholin esterase (BuChE).
EFFECT: obtaining novel compounds and a pharmaceutical composition based on the said compounds, which can be used for treating or preventing cognitive disorders and/or neurodegenerative disorders.
10 cl, 1 tbl, 2 ex
SUBSTANCE: invention relates to novel pyrrolopyrimidine and pyrrolopyridine of general formula (I), substituted with a cyclic amino group (II), or their pharmaceutically acceptable salts having CRF antagonist properties. In general formula the cyclic amino group has formula , in which the cyclic amino group is a 6-member saturated cyclic amine, the said cyclic amine is substituted with a group of formula -(CH2)mX; in which X is -CO2H, -CONH2,-P(=O)(OH)2 or -S(=O)2OH; Y is N or CH; m is an integer selected from 1, 2 and 3; R4 is hydrogen; R5 is hydrogen; R6 is C1-5alkyl; R7 and R8 are identical or different and independently represent hydrogen, C1-5alkyl, Ar is phenyl which is unsubstituted or substituted with one or more substitutes which are identical or different and are selected from a group consisting of halogen, C1-5alkyl, C1-5alkoxy, C1-5alkylthio, trifluoromethyl and trifluoromethoxy.
EFFECT: compounds can be used for therapeutic or preventive treatment of diseases where CRF is considered to be involved, such as depression, anxiety, Alzheimer's disease, Parkinson's disease, Huntington's chorea, eating disorder, hypertension etc.
12 cl, 6 dwg, 1 tbl, 10 ex
SUBSTANCE: invention relates to novel antagonists of serotonin 5-HT6 receptors - substituted 2-amino-3-sulfonyl-6,7,8,9-tetrahydro-pyrazolo[1,5-a]pyrido[3,4-e]pyrimidines of general formula 1 and substituted 2-amino-3-sulfonyl-5,6,7,8-tetrahydro-pyrazolo[1,5-a]pyrido[4,3-d]pyrimidines of general formula 2 or their pharmaceutically acceptable salts and/or hydrates, method of producing said compounds and pharmaceutical compositions, medicinal agents and treatment method. In compounds of formula 1 and general formula 2 , Ar is phenyl which is possibly substituted with halogen atoms, or a 6-member nitrogen-containing heteroaryl; R1 is a hydrogen atom, C1-C3alkyl which is possibly substituted with phenyl, C1-C5alkoxycarbonyl; R2 is a hydrogen atom, halogen or C1-C3alkyl; R1 3 and R2 3 are optionally identical substitutes selected from a hydrogen atom, optionally substituted C1-C3alkyl or R1 3 and R2 3 together with the nitrogen atom with which they are bonded form a nitrogen-containing 6-member saturated heteroaryl optionally substituted with C1-C5alkoxycarbonyl, where the said heteroaryl has 1-2 heteroatoms selected from nitrogen.
EFFECT: compounds can be used to prevent and treat diseases of the central nervous system, pathogenesis of which is associated with 5-HT6 receptors for enhancing mental capacity.
14 cl, 3 tbl, 19 dwg, 16 ex
SUBSTANCE: group of the inventions refers to medicine, namely to oncology, and can be used in bone cancer related pain management. The method involves administration to the patient of effective amount of a nerve growth factor (NGF) antagonist representing a NGF antibody. A set includes the NGF antagonist in the form of the NGF antibody and application instructions.
EFFECT: inventions allow for effective pain management, both constant, and motion related, due to high affinity of the NGF antibody without affecting the initial thresholds of thermal and mechanical sensitivity.
20 cl, 4 tbl, 14 dwg, 4 ex
SUBSTANCE: invention refers to medicine, namely to oncology, and can be used in treating the patients with Non Hodgkin Lymphoma. That is ensured by administration of a combination containing an effective amount of CCI-779 and rituximab in the form of a dosage form with one or more neutral components added. The combination is introduced simultaneously, separately or consistently with the other agents.
EFFECT: method allows improving clinical effectiveness for the given pathology, including in the patients resistant to rituximab due to synergetic interactions of these preparations.
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to pharmaceutical industry, particularly to new drugs and preparations containing effective anticancer agent with anti-Hsp90 antibody.
EFFECT: invention improves clinical effectiveness in treating cancer and leukemia.
48 cl, 25 tbl
SUBSTANCE: there are offered versions of human IL-13 antibodies, including based on CDR antibody BAK278D6. There is described a based composition, and also isolated nucleic acid, a host cell for preparing antibodies and versions of the method for preparing antibodies. There is disclosed application of antibodies for preparing a drug and a composition for treating various diseases mediated by IL-13 activity. Application of the invention provides antibodies neutralising IL-13.
EFFECT: applicable in medicine for preparing a vaccine.
52 cl, 32 dwg, 7 tbl, 29 ex
SUBSTANCE: invention relates to immunology and biotechnology. Described are versions of the humanised antibody CD45RO/RB which carry a light and a heavy strand. Versions of the following are disclosed: isolated polynucleotide, coding antibody, expression vector containing a polynucleotide and host cells containing the expression vector. Described also is use of the antibody to treat and/or prevent various diseases, including as a component of a pharmaceutical composition.
EFFECT: invention provides antibodies identified as CD45RO and CD45RB, which can find use in medicine.
9 cl, 14 dwg, 2 tbl, 13 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to medical technologies, immunology, pharmacology and particularly to drugs for treating the patients with rheumatoid arthritis (RA) and to methods of treating thereof to be used in rheumatology. Substance of the invention includes application of Fc-fragments of immunoglobulin class G as an antigen for treating RA, a remedy for treating RA, produced by mixing the antigen and an adjuvant, and the related method of treating RA.
EFFECT: improved effect to suppress autoimmune response.
6 cl, 3 ex, 4 dwg
SUBSTANCE: invention is related medicine and concerns applications of antibodies specifically recognising any prevailing variants of beta-amyloid peptide, Aβ40 and Aβ42, in preparation of a drug applied for prevention and-or treatment of Alzheimer's disease.
EFFECT: invention provides prevention of progression or reduction of symptoms, and/or decrease in amyloid deposition in an individual when administering an immunostimulating dose of peptide or specific antibody.
7 cl, 3 ex, 2 dwg
SUBSTANCE: anti-Cd20 antibodies are administered in amount of 0.5-4.0 g. 16-60 weeks later administration is repeated. To 4 and more antibody effects are performed. Each effect comprises one or two dosages of antibody. Antibody can be introduced intravenously, subcutaneously or subtunically. The second medicine can be used therewith. A product containing the instructions and a container with 0.5-4.0 g of antibody. In addition, the product can contain the container with the second medicine and the application instructions.
EFFECT: invention allows for prolonged period before stated progression of disease, reduced recurrence rate.
38 cl, 6 dwg, 4 ex
SUBSTANCE: there is offered a monoclonal antibody specific to human interleukine-4 (hIL-4) containing two domains with the related CDR1-3 region. There are described versions thereof that contain specified CDR, polynucleotide coding said antibody. There are described an expression vector and a host-cell for preparing the antibody to human interleukine-4 (hIL-4). There are opened: application of the antibody for preparing a pharmaceutical agent for treating the diseases mediated by interleukine-4 and/or IgE. There is discovered the pharmaceutical composition for treating the diseases mediated by interleukine-4 and/or IgE is opened.
EFFECT: application of the invention ensured the high-affinity neutralised monoclonal antibodies to human interleukine-4.
14 cl, 1 tbl, 6 ex
SUBSTANCE: substance of the invention involves sterile liquid or dry specifically active F(ab')2-fragments of anti-anthrax antibodies containing (35±5) mg·cm3 of protein and at least 96% of F(ab')2-fragments of antibodies recovered from liquid equine anti-anthrax immunoglobulin prepared of blood serum of horses preliminary immunised with strains B anthracis "СТИ"-1 and Ichtiman, and also a anthrax toxin produced by the Kohn's spirit deposition method.
EFFECT: lower reactogenicity and improved immunogenicity.
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to glucagon-like peptide 1 secretion stimulation and to foodstuffs and beverages stimulating glucagon-like peptide 1 secretion and containing k-casein as the active component. declaration is made for a medicinal glucagon-like peptide-1 secretagogue containing k-casein as the active component.
EFFECT: invention ensures glucagon-like peptide 1 secretion stimulation and postprandial hyperglycemia inhibition.
2 cl, 2 ex, 4 tbl, 2 dwg