Enhancing immune response and target focus by antigens and/or drug

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to compounds providing the targeted drug delivery to CD1d receptor cells and inducing an immune response as compared to an antigen, to based compositions and to a method using them applicable in medicine, of formula

,

wherein R1, R2, R3, and R4 represents hydrogen; R6 represents -(CH2)xCH3, x represents an integer from 20 to 25, or -(CH2)xCH=CH(CH2)yCH3, x, y and z independently represent an integer from 1 to 14, R5 is described by formula

,

wherein R8 represents hydrogen, R7 represents C3-C15 alkyl; X represents N; X represents -(CH2)t, wherein t is an integer within 3-10; Z represents a peptide antigen.

EFFECT: prepared are the new biologically active compounds effective for simulating the immune response.

12 cl, 6 dwg, 5 ex

 

The technical field to which the invention relates

The present invention relates to glycolipids that activate NKT cells, covalently linked to antigens and/or medication.

The level of technology

The present invention relates to compounds consisting of glycolipids covalently associated with the antigen or drug via a linker. These compounds are able to induce a stronger immune response than composition comprising separately glycolipids and antigen. These compounds are also able to direct the drug to the cells bearing molecule CD1d.

Natural killer T-cells ("NKT-cells) are a population of cells, similar to that of natural cells of memory/effector cells that Express both receptors of natural killer cells (natural killer, NK) and a conservative seven-invariant receptors T-cells (TCR). NKT cells are involved in the suppression of autoimmune reactions and rejection of implants, stimulation of resistance to pathogens and stimulate the immune response against tumors.

NKT cells are responsible for the massive production of cytokines within a few hours after TCR activation, releasing cytokines TH1-type, including IFN-γ and TNF and cytokines TH2-type, including IL-4 and IL-13. Modulation of these lymphokines answers and is mainly intended�of education adjuvants, used in immunogenic compositions.

NKT cells recognize foreign and self lipid antigens presented by the molecules of the members of the family CD1d associated with β2-microglobuline. It was found that a large number of lipids with different structure binds CD1d molecules specific way, which is accompanied by the localization of the fatty acid chains in each of the two hydrophobic binding pockets (A') and (F) of the molecule CD1d. The types of lipids that are able to communicate with the CD1d molecules include microvia acid, diacylglycerols, sphingolipids, and polyisoprenoids, lipopeptides, phosphomimetic and small hydrophobic compounds.

A large number of molecules of natural and synthetic lipids are converted antigen-presenting cells and is presented by CD1 molecules to NKT cells. The prototype compound used to study the activation of NKT cells in vitro and in vivo, is KRN7000, as well as α-galactosylceramide ("αGalCer"), wiseley from the marine sponge Agelas mauritianus. Other recently identified compounds include isoprothiolane ("iGB3"), which is an endogenous glycolipid described in the Patent application PCT WO 2006/029010, and PBS-57, modified 6"amino-6"desoxycorticosterone, which has been described in PCT Patent application PCT/US 2007/066250, which is included in the present invented�e by reference. These compounds activate NKT cells and enhance cytokine responses in vitro and in vivo. Accordingly, it was suggested that the use of such compounds as adjuvants to improve vaccine efficacy in their joint introduction to the antigen (PCT Patent application WO 2006/083671).

In the context of vaccination, the antigen must be presented to antigen-presenting cells (antigen-presenting cell, APC), particularly dendritic cells (dendritic cells, DCs), conventional CD8+ and CD4+ T cells by MHC molecules of class I or class II, respectively, to induce a specific immune response against this antigen. For this purpose initiated reciprocal activation of NKT cells and dendritic cells by the presentation of α-galactosylceramide resting dendritic cells to NKT cells that induces NKT cells to increased regulation of cytokines CD40L and Th1 and Th2 and chemokines. After this "stitching" CD40 stimulates dendritic cells to increased regulation of CD40, B7.1 and V7.2 and IL-12, which in turn enhances the activation of NKT cells and cytokine production. The spread of this reaction involves activation of cytolysis NKT cells and production of IFN-γ and, most importantly, increased regulation of co-stimulatory properties of dendritic cells and presentation of antigens, mediated MHC class I and MHC class II

It was suggested that PR�to change glycolipids, directly linked to a reporter group, such as a fluorophore or other small molecule (e.g., Biotin), to using them as probes to observe the binding of glycolipids to CD1d and NKT cells (PCT Patent application WO 2004/094444). In particular, associated with the 6th amino-6"deoxy-galactosylceramide the fluorophore and Biotin were used to elucidate the role of the structure of the glycolipid-binding receptor CD1d and NKT-cells. This staining allowed us to observe the movement of glycolipids and quantify their receptor binding CD1d and NKT cells (Zhou et al, Org. Lett. 2000 4: 1267-1270). It was found that cancelroutine and prodanovi derivatives 6"-amino-6"-deoxy-galactosylceramide, as well as associated with Biotin 6"-amino-6"-deoxy-galactosylceramide stimulate NKT cells as the source glycolipid, which allowed to assume that these compounds are absorbed in the process of endocytosis, bind to CD1d, on the cell surface and provide a binding to the receptors of T-cells, causing stimulation of T-cells. However, Zhou et al. attached small molecules to the glycolipid for labeling NKT cells and/or cells having a receptor CD1d. It remains unknown whether the impact of the accession of larger molecules to the lipid chain for binding to CD1d. In addition, to date n� been reported glycolipids, associated with antigens or drugs.

Disclosure of the invention

The authors of the present invention unexpectedly found that a compound consisting of a glycolipid, covalently linked to the antigen by means of a linker that can cause a specific immune response to this antigen, and stronger than the response observed in the case when the glycolipid antigen and separately administered as a composition.

Without being bound by theory, the authors suggested that if the co-delivery of antigen and an agonist of NKT cells to the same cell APC, preferably to the same B-lymphocyte or the same cell DC, B-cell and/or DC-cell activated NKT-cell antigen and, thus, will be presented conventional T cells are fully activated B-cell and/or cell DC. The close location of activated T-cells may be useful when the cell APC presents antigen to the T cell, as that would contribute to its cytokine environment.

In addition, when the glycolipid is covalently bonded with the medication, the glycolipid is able to provide the specific delivery of the drug to NKT cells.

Thus, the present invention relates to compounds consisting of glycolipids covalently linked via a linker to an antigen or drug, and their applications.

Conn�use, consisting of a glycolipid, covalently linked through a linker to the antigen or medicine

The compound of the present invention has the Formula (I):

where,

R1, R2, R3and R4represent each independently from each other hydrogen, C1-C6alkyl, C6-C12aralkyl, or C1-C6acyl; and R1is either above or below the sugar ring.

R6is a

(a) -(CH2)xCH3where x is an integer selected from 1 to 100; or

(b) -(CH2)xCH=CH(CH2)ICH3or -(CH2)xCH=CH(CH2)yCH=CH(CH2)zCH3

where x, y and z are independently selected integers from 1 to 14.

R5represents one of the following formulae (II), (III) or (IV)

where

R8represents hydrogen, C1-C6alkyl, C6-C12aralkyl, or C1-C6acyl, and

R7represents a linear or branched C3-C100alkyl;

X is O, N or S;

Y is a degradable or not degradable linker group; and

Z is an antigen or drug and�and their pharmaceutically acceptable salt.

Used here meaning the term "alkyl" is a hydrocarbon chain which may be straight chain or branched chain, containing the indicated number of carbon atoms. For example, C1-C12alkyl indicates that the group may contain from 1 to 12 (inclusive) carbon atoms. The term "arylalkyl" or "aralkyl" refers to an alkyl residue in which the alkyl hydrogen atom replaced with an aryl group. Examples of "arylalkyl" or "aralkyl" include benzyl and 9-fluorophenyl group.

The term "acyl" refers to alkylcarboxylic, cycloalkylcarbonyl, arylcarboxylic, geterotsiklicheskie or heteroarylboronic Deputy, any of which may be optionally substituted by substituents.

The term "cycloalkyl" used here in the meaning includes saturated cyclic, bicyclic, tricyclic, or polycyclic hydrocarbon group containing from 3 to 12 carbon atoms, in which any ring atom available for substitution can be substituted by the Deputy. Examples cycloalkyl residues include, but are not limited to, cyclohexyl and adamantyl.

The term "aryl" refers to aromatic monocyclic, bicyclic or tricyclic hydrocarbon ring system, in which any ring atom that is available for Thames�tion, may be substituted by the Deputy. Examples of aryl residues include, but are not limited to, phenyl, naphthyl and anthracene.

The term "heterocyclyl" refers to non-aromatic 3-10-membered monocyclic, 8-12-membered bicyclic, or 11-14-membered tricyclic ring system containing 1-3 heteroatom in the case of monocyclic, 1-6 heteroatoms in the case of bicyclic, or 1-9 heteroatoms in the case of a tricyclic system where the named heteroatoms selected from O, N or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms selected from N, O or S, in the case of monocyclic, bicyclic or tricyclic system, respectively), where any ring atom available for substitution may be substituted by the Deputy.

The term "heteroaryl" refers to an aromatic 5-8-membered monocyclic, 8-12-membered bicyclic, or 11-14-membered tricyclic ring system containing 1-3 heteroatom in the case of monocyclic, 1-6 heteroatoms in the case of bicyclic, or 1-9 heteroatoms in the case of a tricyclic system where the named heteroatoms selected from O, N or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms selected from N, O or S, in the case of monocyclic, bicyclic or tricyclic system, respectively), where any ring atom available for substitution can be substituted by the Deputy./p>

The term "oxo" refers to an oxygen atom which forms a carbonyl when you join carbon, N-oxide, when attaching to the nitrogen, and a sulfoxide or sulfone when joining sulphur.

The term "substituents" refers to a group "substituted" alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl group at any atom of that group. Suitable substituents include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, halo, hydroxy, cyano, nitro, amino, - SO3H, sulfate, phosphate, perfluoroalkyl, perforamce, methylenedioxy, Ethylenedioxy, carboxyl, oxo, thioxo, imino (alkyl, aryl, aralkyl), S(O)nalkyl (where n is 0-2), S(O)naryl (where n is 0-2), S(O)nheteroaryl (where n is 0-2), S(O)nheterocyclyl (where n is 0-2), amine (mono-, di-, alkyl, cycloalkyl, aralkyl, heteroalkyl and combinations thereof), ester (alkyl, aralkyl, heteroaryl), amide (mono-, di-, alkyl, aralkyl, heteroalkyl and combinations thereof), sulfonamide (mono-, di-, alkyl, aralkyl, heteroalkyl and combinations thereof), unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heterocyclyl and the unsubstituted cycloalkyl.

R6may contain from 1 to 100 methylene (CH2) groups (e.g., R6represents (CH2)xCH3and x=1-100). In particular, R6can containing�ü 1-75 CH 2groups 1-50 of CH2groups, 1-25 CH2groups 1-20 CH2groups, 1-15 CH2groups 1-10 CH2groups or 1-5 CH2groups. Preferably, R6contains 15-25 CH2groups. More preferably, R6contains 20-25 CH2groups.

In certain embodiments, R6contains 22 or 24 CH2group (x=22 or x=24).

R7can represent, in particular, linear or branched C3-C75alkyl, C3-C50alkyl, C3-C25alkyl, C3-C20alkyl, C3-C15alkyl, C10-C15alkyl or C3-C10alkyl.

In certain embodiments, R7represents an unbranched alkyl group of 14 carbon atoms.

Preferably, R6represents C25H51and R7represents C14N29. More preferably, R6represents C23N45and R7represents C14N29. In another preferred embodiment R6represents C23N47and R7represents C14H29.

When R1-R3are not hydrogen, preferably each independently represent a methyl, benzyl or acetyl.

According to a particular embodiment (hereinafter referred to as PBS-6), R1, R2/sub> , R3, R4and R8represent hydrogen, R5represents (II), R6represents C25H51, R7represents C14N29and X represents N.

According to another embodiment (hereinafter referred to as PBS-57), R1, R2, R3, R4and R8represent hydrogen, R5represents (II), R6represents C23N45, R7represents C14H29and X represents N.

According to another embodiment (hereinafter referred to as PBS-14), R1, R2, R3, R4and R8represent hydrogen, R5represents (II), R6represents C25N51, R7represents C14N29and X represents N.

In another embodiment (hereinafter referred to as PBS-96), R1, R2, R3, R4and R8represent hydrogen, R5represents (II), R6represents C23N47, R7represents C14N29and X represents N.

Pharmaceutically acceptable salts of the compounds of the present invention include salts derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable salts of acids include acetate, adipate, and�iginal, aspartate, benzoate, benzolsulfonat, bisulfate, butyrate, citrate, comfort, camphorsulfonate, digluconate, dodecyl sulfate, aconsultant, forms(I)at, fumarate, glucoheptonate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonic, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate and undecanoate. Other acids such as oxalic acid, although in themselves and are not pharmaceutically acceptable may be used to obtain the salts are useful as intermediate components in obtaining compounds of the present invention and their pharmaceutically acceptable acid-additive salts (salts of accession acid). Salts derived from acceptable bases include alkali metal salts (e.g., sodium), alkaline earth metals (e.g. magnesium), ammonium and N-(alkyl)4+. The present invention also involves the quaternization of any alkaline nitrogen-containing group disclosed here compounds. Using this quaternization can be obtained products which are soluble or dispersible in water or oil. Salt forms of compounds that have indicated here� Formula can be salts of amino acids on the carboxyl groups (e.g. salts of L-arginine, -lysine, -histidine).

Glycolipid component compounds according to the present invention, described above, can be synthesized in such a manner as is disclosed in the International patent application WO 2004/094444, which is included in the present invention by reference.

The joining component linker-antigen or drug (Y-Z) to glycolipid component can be carried out according to the method described by Zhou et al. (Org. Lett. 2000 4:1267-1270).

Example conjugation of peptide antigen shown in the diagram below I:

To obtain such conjugation of peptide antigen peptide can be dissolved at a concentration of 50-100 µm in a suitable buffer at a pH of 7.0 to 7.5 at room temperature. The reduction of the disulfide bonds in the peptide can be performed by adding a 10-fold molar excess of a reducing agent such as DTT or TSAR. The glycolipid-containing reactive group may be added dropwise to the peptide solution while stirring to a final ratio of 10-20 moles of glycolipid per mole of peptide. The reaction mixture can be left for reaction for 2 hours at room temperature or at but�ü at 4°C. The conjugate can eventually be extracted using gel-chromatography on a column.

The linker group Y can be any carbon chain or ring. For example, the linker can be -(CH2)t- in which chain optionally includes one or more terminal heteroatoms (e.g., N, O, S), and/or one or more heteroatoms, rings, double bonds, triple bonds that inserted in this circuit. The value "t" can be 1-20, preferably 3-10.

In a preferred embodiment of the present invention, the linker comprises 6 carbon atoms.

Preferably, the linker contains proteoliticeski split plot, in particular, the site of cleavage of endo-lysosomal proteases. Alternative to this, the linker group contains the site of cleavage by lipases. In particular, the linker group may contain a cleavage site by the protease and/or a site of cleavage by lipases.

Preferably, the compound according to the present invention is capable of contacting the monomer or tetramer CD1d. More preferably, this compound is able to activate NKT cells.

Used here meaning the term "antigen" refers to any substance or material, which is specifically recognized by the binding component of the immune system, such as an antibody or antibody fragment, VK�uchumi Pratap or receptor of T cells (TCR).

Accordingly, the antigens of a compound of formula (I) originate from weakened or killed infectious agents. Can be used whole organisms or their parts (for example, "shadows" membranes, crude membrane preparations, lysates and other preparations of microorganisms). Suitable infectious agents, of which there can be obtained an antigen, include, but are not limited to, pathogens and microorganisms, such as bacteria, parasites and viruses. In certain cases, suitable antigens or get isolated from viral pathogen associated with human disease, including, but not limited to, HIV/AIDS (retroviridae are, for example, molecules of gp120 for HIV-1 and HIV-2 isolates, HTLV-I, HTLV-11), influenza viruses (Orthomyxoviridae, e.g., types A, b and C), herpes (e.g., simplex herpes viruses, HSV-1 and HSV-2 glycoproteins gB, gD and gH), rotavirus (Reoviridae), respiratory infections (parainfluenza and respiratory syncytial viruses), poliomyelitis (Picornaviridae, e.g., polioviruses, rhinoviruses), measles and mumps (Paramyxoviridae), rubella (Togaviridae, e.g., rubella virus), hepatitis (e.g., hepatitis viruses type A, b, C, D, E and/or G), cytomegalovirus (such as gB and gH), gastroenteritis (Caliciviridae), Yellow fever and West Nile virus (Flaviviridae), rabies {Rhabdoviridae), Korean hemorrhagic fever (Bunyaviridae), Venezuelan fever (Arenaviridae), warts (Papilomavirus), immunodeficiency virus monkeys, encephalitis virus, the virus of chickenpox and shingles, Epstein-Barr and other families of viruses, including Coronaviridae, Birnaviridae and Filoviridae.

Suitable bacterial and parasitic antigens can be obtained or isolated from known agents that cause disease, including, but not limited to, diphtheria, pertussis, tetanus, tuberculosis, bacterial or fungal pneumonia, otitis media, gonorrhea, cholera, typhoid, meningitis, mononucleosis, plague, shigellosis (bacterial dysentery) or Salmonella, Legionnaires ' disease, Lyme disease, leprosy, malaria, hookworm disease, Onchocerciasis, Schistosomiasis (bilharziasis), Trypanosomiasis, Leishmaniasis, ginasios (girlasian), amoebiasis (amoebic dysentery), filariasis, Borrelii and trichinosis. Other antigens can be obtained or isolated from unusual pathogens such as the causative agents of Kuru, a disease of Creutzfeld-Jakob disease (CJD), scrapie (pocesuhi), transmissible mink encephalopathy and chronic debilitating diseases, or from proteinaceous infectious particles such as prions associated with mad cow disease (spongiform encephalopathy).

Additional specific pathogens, which can be selected antigens include Mycobacterium tuberculosis, Chlamydia, Neisseria gonorrhoeae, Shigella, Salmonella, Vibrio, Treponema pallidum, Pseudomonas, Bordetella pertusss, Brucella, Francisella tularensis, Helicobacter pylori, Leptospira interrogans, Legionella pneumophila, Yersinia pestis, Streptococcus (types A and b), pneumococcus, meningococcus, Haemophilus influenzae (type b), Toxoplasma gondii, Moraxella catarrhalis, donovanosis, and actinomycosis, fungi pathogens including candidiasis and Aspergillus, pathogens parasites, including Taenia, flukes, roundworms, amebiasis (amoebic dysentery), ginasios (girlasian), Cryptosporidium, Schistosoma, Pneumocystis carinii, trichomoniasis and trichinosis. The present invention can also be applied to ensure an appropriate immune response against a variety of veterinary diseases, such as foot and mouth disease, coronavirus infection, Pasteurella multocida, Helicobacter, Strongylus vulgaris, Actinobacillus pleuropneumonia, virus diarrhea of cattle (BVDV), Klebsiella pneumoniae, Escherichia coli, and Bordetella pertussis, parapertussis and brochiseptica.

In other embodiments, the antigens for binding to glycolipids that can be applied, are antigens obtained from tumors or autologous or allogeneic whole tumor cells. Accordingly, the tumor antigen is an antigen specific to the tumor (tumor specific antigen, TSA) or an antigen associated with a tumor (tumor associated antigen, TAA). Some tumor antigens and their expression is known in the art and can be selected based on the type of tumor, which will Podgoritsa treatment. Limitiruyuschie examples of tumor antigens include cdk4 (Meylan�mA), p-catenin (melanoma), caspase-8 (squamous cell carcinomas), MAGE-1 and MAGE-3 (melanoma, breast cancer, glioma), tyrosinase (melanoma), surface Ig idiotype (e.g., BCR) (lymphoma), Her-2/neu (breast cancer, ovarian cancer), MUC-1 (breast cancer, pancreatic cancer) and HPV E6 and E7 (cervical carcinoma). Additional suitable tumor antigens include prostate-specific antigen (PSA), sialyl Tn (STn), heat shock proteins and tumor associated peptides (e.g., gp96), molecules of gangliosides (e.g., GM2, GD2, and GD3), carcinoembryonic antigen (CEA) and MART-1.

According to another embodiment of the glycolipid joins the cure. Examples of suitable drugs include cyclosporine, FK 506 and rapamycin.

Compositions for vaccination and pharmaceutical compositions Another aspect of the present invention relates to compositions for vaccination, which includes the compound of the above formula (I) where Z represents the antigen.

The term "vaccine" refers to a composition which when administered to a subject induces cellular and/or humoral immune responses, as described here.

In the context of the present invention, the term "subject" refers to an animal, preferably a mammal, which no man or person. Examples of mammalian, non-human, include rodents, etc�mats. Most preferably the subject is a person.

The present invention also provides a method of inducing, especially stimulation of the immune response in a subject, which comprises administering to a subject named compounds or compositions for vaccination according to the present invention.

In the context of the present invention, the term "stimulation of an immune response" means increasing the immune response that is induced by the presence of the antigen.

In a preferred embodiment, the immune response is a humoral immune response. Used here meaning the term "humorally immune response" is a production of antibodies In the cells and support processes that accompany these products, including, but not limited to, for example activation and Th2 cytokine production, formation of germinal center (center of reproduction), and isotype switching, the process of affinity maturation and the formation of memory cells (T lymphocytes). To determine whether stimulated humoral immune response, can be carried out a quantitative comparison of the signal in a sample obtained from a subject who received any vaccinations compound or a composition for vaccination, as defined above, with the signal in the sample obtained from the subject who received any vaccinations only antigen�m in itself. Humoral immune response may be assessed by measuring the effector functions of antibodies, including neutralizing the pathogen or toxin, classical activation of complement and the stimulation of opsonins in phagocytosis and elimination of the pathogen. Antibodies produced in response to the introduction of compounds or compositions for vaccination, as defined above, and the antigen may belong to any type, for example IgM, IgA or IgG. Humoral immune response may be measured by any quantitative method known in the art, e.g., ELISA, a simple method of radial immunodiffusion (SRID), enzyme immunoassay analysis (EIA) or in the test of inhibition of hemagglutination (HAI).

In another preferred embodiment of the stimulation of the immune response corresponds to the activation of CD4+ T lymphocytes. As should be known to experts in the art, CD4+ T cells or cells T helper cells" are cells that recognize antigens presented by a marker of major histocompatibility complex class II on the surface of antigen-presenting cells and secrete lymphokines to stimulate as it is mediated by cells and of antibody-mediated branches of the immune system. Activation of CD4+ T cells stimulates the secretion of lymphokines, isotype switching of immunoglobulins, affinity maturation of the response of the antibodies�, activation of macrophages and enhancement of the activity of natural killer (NK) and cytotoxic T cells (CTL). Lymphokines are proteins synthesized by lymphocytes, which have an impact on their own activity and/or activity of other cells. Lymphokines include, but are not limited to, interleukins and cytokines, e.g., IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 and INFγ. To clarify, are activated whether CD4+ T-lymphocytes, may be performed a quantitative comparison of the signal in the sample taken from the subject who received any vaccinations connection or vaccine composition, as defined above, and the signal in the sample taken from the subject who received any vaccinations only antigen. Methods of determining the activation of CD4+ T cells known in the art.

In another preferred embodiment of the stimulation of the immune response corresponds to the activation of CD8+ T lymphocytes. CD8+ T lymphocytes recognize antigens presented by molecules of the MHC Class I (present on all containing core cells). The involvement of the peptide MHC complex class I provides delivery of lytic granules to the target cell, causing lysis of the target cells. The methods used to determine the activation of CD8+ T cells, known in the art and include, but are not limited to, ELISPOT, ELISA, and analysis of cytotoxicity. An alternative for monitoring �Stivali CD8+ T cells can be used the mouse model using the definition of fluorescence to measure mediated cell cytotoxicity, as described in the work of Hermans et al. (2004) J. Immunol. Meth. 285:25-40.

Suitable effective dosage of a compound of formula (I) in the compositions for vaccination may be determined by the person skilled in the technical field, but they usually are in the range from about 1 micrograms to about 10,000 micrograms per kilogram of body weight, although, as a rule, they are approximately 1000 micrograms or less per kilogram of body weight. In some embodiments, effective dosages are in the range of from about 10 to about 5000 micrograms per kilogram of body weight. In another embodiment an effective dosage is in the range from about 50 to about 1000 micrograms per kilogram of body weight. In another embodiment, an effective dosage is in the range from about 75 to about 500 micrograms per kilogram of body weight. The composition may be administered in a single dosage or may be divided into multiple dosages that are found in within weeks or months. You must understand that the dosage of the antigen will depend on the specific antigen and the age and immune status of the subject, as well as from other important factors that can determine a person skilled in the technical field.

The vaccine of the present invention can provide therapeutic or profilakticheskaya infectious disease or disease associated with the infectious agent. The term "treat" or "treatment" of an infectious disease includes one or more of the following values: (1) suppression of infection, i.e. the prevention of infection under the action of an infectious agent, (2) preventing the spread of an infectious agent that is spread to other areas (organs) of the subject or from one subject to another, (3) reducing the severity of the disease, (4) prevention of recurrent infections, i.e., prevention of reactivation of latent or chronic infections, and (5) reducing symptoms of infectious diseases.

Another aspect of the present invention relates to pharmaceutical compositions containing the compound of formula (I) in which Z represents a cure.

The present invention also provides a method of treatment of a subject who needs the treatment, which includes the introduction of named entity the named compounds or pharmaceutical compositions.

The term "effective amount" means an amount of compound that provides a therapeutic effect in treatment of the subject. therapeutic effect may be objective (i.e., as measured by some test or marker) or subjective (i.e. the subject is showing signs of effect or feeling Dunn�th effect). The effective amount of the compounds described above can be varied in the range from about 0.01 μg/kg to about 500 μg/kg, this alternative from about 0.1 μg/kg to about 100 μg/kg, an alternative to this, from about 1 μg/kg to about 50 μg/kg. Effective dosage will also vary depending on the method of administration, and also on the possibility of joint use with other agents.

As should be understood specialists in this field of technology, vaccines are prepared in accordance with the method of administration is assumed. Examples of suitable routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, intramuscular, oral (e.g., by inhalation), transdermal (topical), transmucosal and rectal administration.

Preferably, the vaccine according to the present invention can be administered intramuscularly, intravenously, subcutaneously, intradermally, intraperitoneally, nasally, parenterally or by inhalation. Most preferably, the vaccine according to the present invention administered intramuscularly or subcutaneously.

The vaccine may also include a physiologically acceptable excipient. "Physiologically acceptable" excipient is any filler that is suitable for administration in vivo (e.g., by TRANS�General, transdermal or parenteral administration) or application of in vitro, i.e., in cell culture. Suitable physiologically acceptable excipients for administration in vivo include, among others, water, buffer solutions and glucose solutions. Additional components of the compositions in addition to a physiologically acceptable excipient may respectively include excipients, such as stabilizers, preservatives, solvents, emulsifiers or lubricants. In particular, suitable excipients include, but are not limited to, tween 20, DMSO, sucrose, L-histidine, Polysorbate 20 and whey.

Compounds of formulas described herein, can, for example, be administered by injection, intravenously, intraarterially, subcutaneously, intraperitoneally, intramuscularly or subcutaneously, or oral, buccal, nasal, transmucosally, topically, in the form of ophthalmic preparations, or by inhalation at doses in the range of from about 0.5 to about 100 μg/kg of body weight, this alternative at doses in the range from about 1 mg to about 100 mg/dose, every 4-120 hours, or in accordance with the requirements for a particular drug. The methods described here assume the introduction of an effective amount of compound or compound composition to achieve the desired or necessary effect. Typically, pharmaceutical �oppozitsii of the present invention should be administered from about 1 to about 6 times per day or, alternative to this, in the form of a continuous infusion. Such administration can be used as a chronic or acute therapy. Amount of active ingredient that may be combined with the materials of the carrier to obtain a single dosage form will vary depending on the treatment facility and the specific method of administration. A typical preparation will contain from about 5% to about 95% active compound (by weight). Alternative to this, such preparations contain from about 20% to about 80% active compound.

May require lower or higher doses than those indicated above. Specific dosage and treatment regimen for any particular patient will depend upon a variety of factors including the activity of the specific compound, the age, body weight, General health, sex, diet, time of administration, rate of excretion, combination of drugs, the severity and nature of the disease, condition or symptoms, the patient's relationship to the disease, condition or symptoms, and the decision of the treating physician.

To improve the condition of the patient, if necessary, may be given a maintenance dose of a compound, composition or combination of the present invention. After that the dosage or frequency of administration, or dosage, and frequency, mo�ut to be reduced depending on your symptoms to a level on which the improved condition is retained when the symptoms been alleviated to the desired level. However, patients may require intermittent treatment on a long-term basis as the appearance of recurrence of symptoms.

Described herein compositions comprise compounds of formulas described herein, as well as additional therapeutic agents if present, in amounts effective for achieving a modulation of disease or disease symptoms, including those described here.

The term "pharmaceutically acceptable carrier" means a carrier that may be administered to the patient together with the compound of the present invention and which does not violate the pharmacological activity of this compound is non-toxic when administered in doses sufficient to deliver a therapeutic quantity of the connection.

Pharmaceutically acceptable carriers and excipients that may be used in pharmaceutical compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, offering self-emulsifying delivery system of drugs (self-emulsifying drug delivery systems SEDDS) such as d-α-tocopherol polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms, such as Twins or other similar p�likernye matrices for drug delivery, plasma proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes, such as protaminsulphate, disodium phosphate, sodium, odnozameshchenny potassium phosphate, sodium chloride, zinc salts, colloidal silicon oxide, magnesium trisilicate, polyvinylpyrrolidone, substances based on cellulose, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and lanolin. To improve the delivery of compounds with the formula described here also can be applied mainly cyclodextrin such as α-, β-, and γ-cyclodextrin, or chemically modified derivatives, such as hydroxyacylglutathione, including 2 - and 3-hydroxypropyl-β-cyclodextrins, or other solubilized derivatives.

Pharmaceutical compositions of the present invention can be administered orally, parenterally in the form of a spray for ingalicia, topically, rectal, nasal, buccal, vaginally or via implanted reservoir, preferably by oral administration or administration by injection. Pharmaceutical compositions of the present invention may contain any conventional� non-toxic pharmaceutically acceptable carriers or excipients. In some cases, the pH of the composition may be brought to the desired value by using pharmaceutically acceptable acids, bases or buffers to enhance the stability of the cooking compounds or formulations. Used here meaning the term "parenteral" includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, nutricentials, intramammary, intrathecal (intrathecal) injections, administered in the affected tissues, and intracranial injection or introduction using the infusion.

Pharmaceutical compositions may be prepared in the form of a sterile injection preparations, for example, in the form of a sterile aqueous or oily suspension for injection.

This suspension can be prepared using methods known in the art, using suitable dispersing or wetting agents (e.g., tween 80) and suspendida agents. A sterile preparation for injection may also be a sterile solution or suspension for injection in a non-toxic acceptable for injecting a solvent or diluent, such as, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be used are mannitol, water, a solution of the Ring�RA and isotonic sodium chloride solution. In addition, typically as a solvent or medium for suspending apply a sterile non-volatile oil. For this purpose you may use a soft, non-volatile oils, including synthetic mono - or diglycerides. For the preparation of injectable forms are applicable fatty acids such as oleic acid and its glyceride derivatives, as well as natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated forms. Solutions or suspensions of these oils may also contain as solvents long-chain alcohols or dispersing agents, or carboxymethyl cellulose or similar dispersing agents that are commonly used in the preparation of pharmaceutically acceptable dosage forms such as emulsions and/or suspensions. Other commonly used surfactants, such as Twins or Spiny and/or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for production.

Pharmaceutical compositions of the present invention can be administered orally in any orally available dosage form, including, but not limited�ü, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. In the case of tablets for oral use media that are commonly used include lactose and corn starch.

Usually also added lubricating agents such as magnesium stearate. Suitable solvents for oral administration in the form of capsules include lactose and corn starch.

When aqueous suspensions and/or emulsions are introduced orally, the active ingredient may be suspended or dissolved in the oil phase and is combined with emulsifying and/or suspendresume agents. If desired, can be added, certain sweetening and/or flavoring and/or coloring agents.

Pharmaceutical compositions of the present invention can also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing the compounds of the present invention with a suitable non-irritation excipient which is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.

Topical pharmaceutical compositions of the present�status (active or inactive of the invention is applicable, when the desired treatment involves areas or organs accessible for topical application. For topical application on the skin of the pharmaceutical composition should be prepared in the form of a suitable ointment containing the active components suspended or dissolved in the carrier. Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oil, liquid oil, white petrolatum, propylene glycol, a mixture of polyoxyethylene and polyoxypropylene, emulsified wax and water. Alternative to this, the pharmaceutical composition may be prepared in the form of a suitable lotion or cream containing the active compound suspended or dissolved in a carrier with suitable emulsifying agents.

Suitable carrier materials include, but are not limited to, mineral oil, servicemonitor, Polysorbate 60, wax cetylated esters, Cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Pharmaceutical compositions of the present invention can also be applied topically in the lower intestine in the form of rectal suppositories or suitable dosage forms for enemas. Topical transdermal patches are also included in the present invention.

Pharmaceutical compositions of the present invention can�titsa as a nasal aerosol or inhalation. Such compositions are prepared according to methods well known in the art for the preparation of pharmaceutical compositions and can be prepared as solutions in saline with the use of benzyl alcohol or other suitable preservatives, stimulants suction to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.

A composition comprising a compound of the formula given here may be introduced with the use of implantable devices.

Implantable devices and similar technologies known in the art and are useful as delivery systems in the case when the desirable is continuous or timed delivery of the compounds described herein or compositions. In addition, implantable system for drug delivery is suitable for the targeted delivery of compounds or compositions in certain points (for example, in localized areas, water bodies) (see Negrin et al., (2001) Biomaterials, 22 (6): 563).

Timed delivery technology, including alternative delivery methods, can also be used in the present invention. For example, for delivery of the compounds described herein and compositions can also be applied timed kom�osili, based on polymer technologies, sustained release technologies and methods with the use of capsule (e.g., polymeric, liposomal).

In the scope of the present invention also includes a plaster (bandage) for delivery described herein chemotherapeutic compounds. The patch includes a layer of material (e.g., polymer, cloth, gauze, bandage) and the compound of the above formula. One side of the layer of material may have a protective layer to prevent penetration of the compounds or compositions. The patch may further comprise a sticky component to hold it on the right place on the subject. Sticky component is a composition comprising a sticky substance of natural or synthetic origin which, in contact with skin of the subject at the time stick to the skin. Sticky component can be water-resistant. Sticky component can be deposited on the adhesive to hold it in contact with the skin of a subject over a long period of time. Sticky component can have a certain stickiness or adhesive force, so that he kept the patch on the right place the subject in case of accidental contact, however, with a targeted action (e.g., tearing, removal or other method of intentional removal) of the adhesive layer should provide opportunities� for application of external force to the entire patch or to the sticky layer, to break the adhesive contact. Sticky component can be pressure sensitive, that is, it should allow to place the adhesive layer (and the device that must be attached to the skin) on a certain area of the skin by application of pressure (e.g., pressing, grinding) in adhesive component or device in General.

Brief description of the drawings

Figure 1 shows the definition of SIINFEKL-specific cytolytic activity in the blood of immunized mice. All samples were selected on the next day after injection of target cells. Mice were immunized by intravenous injection of 100 μg of one of the Ova peptide (group 1), 1 µg of one of the Ova peptide (group 2), the combination of 100 μg of Ova peptide and 1 μg of glycolipid (group 3), the combination of 1 μg of Ova peptide and 1 μg of glycolipid (group 4), 1 μg of Ova peptide covalently linked to glycolipid (group 5), 100 ng of Ova peptide covalently linked to glycolipid (group 6) or 10 ng of Ova peptide covalently linked to glycolipid (group 7).

Figure 2 shows histograms representing the percentage SIINFEKL-H2Kb- specific CD8+ cells (CD8+ SIINFEKL Pentamer + (%), abscissa) in the blood of mice immunized with PBS (PBS), ovalbumin peptide (OVA), ovalbumin peptide covalently linked to PBS-6 (PBS6-OVA), ovalbumin peptide covalently linked to PBS-1 (PBS14-OVA), or ovalbumin peptide covalently linked to PBS-57 (PBS57-OVA).

Figure 3 shows histograms representing expressed as a percentage) SIINFEKL-specific cytolytic activity (% specific lysis, abscissa) in the blood of mice immunized with PBS (PBS), a short peptide of ovalbumin (50 ág short OVA), long peptide of ovalbumin (50 μg long OVA), ovalbumin peptide covalently linked to PBS-6 (1 μg PBS6-OVA), ovalbumin peptide covalently linked to PBS-14 (1 µg PBS14-OVA), or ovalbumin peptide, covalently linked to PBS-57 (1 µg PBS57-OVA).

Figure 4 shows histograms representing the titer of OVA-specific IgG1 antibodies in the blood of mice immunized with ovalbumin in CFA/IFA (positive control, CTRL), a short peptide of ovalbumin (50 ág short OVA), long peptide of ovalbumin (50 μg long OVA), ovalbumin peptide covalently linked to PBS-6 (1 μg PBS6-OVA), ovalbumin peptide covalently linked to PBS-14 (1 µg PBS14-OVA), or ovalbumin peptide, covalently linked to PBS-57 (1 µg PBS57-OVA).

Figure 5 shows histograms representing the titer of OVA-specific IgG2a antibodies in the blood of mice immunized with ovalbumin in CFA/IFA (positive control, CTRL), a short peptide of ovalbumin (50 ág short OVA), long peptide of ovalbumin (50 μg long OVA), ovalbumin peptide covalently linked � PBS-6 (1 μg PBS6-OVA), the ovalbumin peptide covalently linked to PBS-14 (1 µg PBS14-OVA), or ovalbumin peptide covalently linked to PBS-57 (1 µg PBS57-OVA).

Figure 6 shows histograms representing expressed as a percentage Trp2-specific cytolytic activity (% specific lysis, abscissa) in the blood of mice immunized with PBS (PBS), short Trp2 peptide (50 µg short Trp2), long Trp2 peptide (50 μg long Trp2), the Trp2 peptide covalently linked to PBS-6 (1 μg PBS6-Trp2), the Trp2 peptide covalently linked to PBS-14 (1 µg PBS14-Trp2) or Trp2 peptide, covalently linked to PBS-57 (1 µg PBS57-Trp2).

The implementation of the invention

Example 1: Analysis of immunization induced antigen-related PBS6 injected intravenously in the test VITAL.

Material

For immunization were used thirty-three female mice of the C57B1/6J CD45.2 the age of 8 weeks. They were divided into 8 groups: group 1, 2, 4, 6 and 8 consisted of 3 animals and groups 3, 5 and 7 consisted of 6 animals.

Twenty female mice of the C57B1/6J CD45.2 the age of 8 weeks were used to obtain the target cells.

Target cells to measure cytotoxicity in vivo

Cytotoxicity in vivo ovalbumin peptide sequence SIINFEKL (SEQ ID NO: 1) (peptide or Ova) induced response of CD8+ T cells was assessed using VITAL test, as described by Hermans et al (2004, J. Immunol. Methods, 285:25-40). Briefly, congenia ass�acii of splenocytes (splenic macrophages) were labeled with a fluorescent dye CFSE, taken either in low concentration (0.6 μm for 10 min at 37°C) or high concentration (6 μm for 10 min at 37°C). Population, marked by a high concentration of CFSE, was previously loaded with SIINFEKL peptide (5 μm for 60 min at 37°C) whereas the population labeled with a low concentration of CFSE, was previously loaded neutral (control) LCMV peptide gp33-41 (5 μm for 60 min at 37°C).

Equal amounts of both populations were mixed and were injected by intravenous immunized mice. 10.106cells obtained in different conditions (total 20.106cells) in a volume of 100 μl were injected into an orbit (orbital) sinus or the lateral tail vein of each immunized mice 10 days after immunization.

Vaccination

The ovalbumin peptide includes the sequence SIINFEKL (SEQ ID NO: 1) (i.e. amino acids of ovalbumin 257-264) and used the glycolipid is PBS-6.

Mice were immunized by intravenous injection on day "0" of 50 μl of the following solutions prepared respectively for different groups:

1 - 100 μg of Ova peptide in 50 μl PBS

2 - 1 μg of Ova peptide in 50 μl PBS

3 - 100 μg of Ova peptide in combination with 1 μg of PBS-6 in 50 µl PBS

4 - 1 μg of Ova peptide in combination with 1 μg of PBS-6 in 50 µl PBS

5 - 1 μg of Ova peptide covalently svyazannogo PBS-6, in 50 µl PBS

6 - 100 ng of Ova peptide covalently linked to PBS-6, in 50 µl PBS

7 - 10 ng of Ova peptide covalently linked to PBS-6, in 50 µl PBS

Get data

Monitoring of specific lysis of target cells loaded SINFEKL, FACS was performed in peripheral blood cells or splenocytes. Blood samples from the orbital sinus and spleen were collected from mice after their killing 11 days after immunization. The average survival of targets containing the peptide and a fluorescent dye, in percent, was calculated relative to similar values obtained for the control population, and the cytotoxic activity was expressed as the percentage of specific lysis (100 minus the average survival of targets containing the peptide and a fluorescent dye, in percent.

Results

The aim of this study was to evaluate the effect of peptide covalent associated with glycolipids an agonist of NKT-cells when it is administered intravenously, at a response in the form of specific lysis compared with the same peptide that was introduced in a mixture with the same glycolipid. Mice, which were injected with 1 μg - 100 μg of one of the Ova peptide did not show antigen-specific lysis. It was determined that the baseline natural lysis of the placebo control animals is values between -6% and +3% (�of figure 1). The results obtained for mice that received 100 μg of Ova peptide in combination with 1 μg PBS6 by intravenous injection showed strong specific lysis in all mice, as expected. Mice, which were injected with 1 μg of Ova peptide in combination with 1 μg of PBS-6 did not show any specific lysis after immunization by intravenous injection. However, the results obtained for mice that received 1 μg of Ova peptide covalently linked to PBS-6, showed the presence of strong specific lysis of all mice. This activity was still preserved with the introduction of 100 and 10 ng. This result indicates that immunization by intravenous covalently linked anaganaga peptide gives better results than injection of a mixture.

Finally, the results of this experiment indicate that the linkage between the antigen and adjuvant, towards CD1d, increases the strength of the response.

Example 2: Evaluation of stimulation of the immune response induced glycolipid adjuvant, using the model antigen OVA.

Material

For immunization were used forty-eight female mice of the C57B1/6J CD45.2 the age of 9 weeks. They were divided into 9 groups: group 1 and 2 consisted of 3 animals and groups 3 through 9 consisted of 6 animals.

Vaccination.

The ovalbumin peptide includes a follower�spine VSGLEQLESIINFEKLTEWTS (SEQ ID NO:2) and used the glycolipids are PBS-6, PBS-14 and PBS-57. Mice were immunized by intravenous injection on day 0 and day 14 following solutions prepared respectively for different groups:

- Group 1: 100 μl PBS;

- Group 2: 50 μg of Ova in 100 μl PBS;

- Group 3: 1 µg Ova-PBS-b in 100 μl PBS;

- Group 4: 1 µg Ova-PBS-14 in 100 μl PBS;

- Group 5: 1 µg Ova-PBS-57 in 100 µl PBS.

Get data.

Detection of SIINFEKL-specific CD8+ cells FACS was performed after joint-penetration of pentamer H-2Kb SIINFEKL and CD8. Monitoring the response of cytokines in the spleen was performed using CBA analysis. Monitoring of secretion of cytokines was performed using a mouse Th1/Th2 cytokine CBA on a flow cytometer, and the concentration of cytokines was determined using the software FCAP Array software, BD.

Results

The aim of this study was to determine the effectiveness of the link between the antigen and a glycolipid with their intramuscular administration in relation to the induction of specific immune responses.

Mice, which were injected with 1 μg of OVA covalently linked or with PBS-6, PBS-14 or PBS-57, showed a higher percentage of SIINFEKL-H2Kb-specific CD8+in the blood than mice, which were injected with 50 μg of one OVA (figure 2).

Thus, this experiment demonstrates that the linkage between the antigen and glycolipid adjuvants increases the strength of the response.

Example 3: Analysis �manisali, induced antigen associated with a different glycolipid adjuvants, and intramuscular injection.

Material

For immunization were used sixty-nine female mice of the C57B1/6J CD45.2 the age of 9 weeks. They were divided into 14 groups: groups 1 through 5 consisted of 3 animals / group 6 to 14 included 6 animals.

Target cells to measure cytotoxicity in vivo

Cytotoxicity in vivo ovalbumin peptide sequence SIINFEKL (SEQ ID NO:1), induced response, CD8+ T cells was assessed using VITAL test as described in Example 1.

Vaccination

Short ovalbumin peptide includes the sequence SIINFEKL (SEQ ID NO:1; amino acids of ovalbumin 257-264), long ovalbumin peptide includes the sequence VSGLEQLESIINFEKLTEWTS (SEQ ID NO:2; amino acids of ovalbumin 250-269) and used the glycolipids are PBS-6, PBS-14 and PBS-57.

Mice were immunized by intravenous injection on day "0" of the following solutions prepared respectively for different groups:

Group 1: 50 µl PBS;

Group 2: 50 µg Short OVA in 50 μl PBS/DMSO;

Group 3: 50 mg Long OVA in 50 μl PBS/DMSO

Group 4: 1 μg of PBS-6-OVA in 50 μl PBS;

Group 5: 1 μg of PBS-57-OVA in 50 μl PBS;

Group 6: 1 μg of PBS-14-OVA in 50 μl PBS.

Get data.

Data were obtained as described in Example 1.

Results

The purpose of this ISS�of adowania was to evaluate the efficiency of covalent binding of the peptide with glycolipids an agonist of NKT-cells when administered intramuscularly in relation to induction of response in the form of specific lysis, in comparison with the same peptide in itself.

Mice, which were injected with 1 μg of OVA covalently linked or with PBS-6, PBS-14 or PBS-57, showed a higher percentage of SIINFEKL-specific cytolytic activity in the blood than mice, which were injected with 50 μg of one OVA (figure 3).

Thus, this experiment demonstrates that the linkage between the antigen and glycolipid adjuvants increases the strength of the response.

Example 4: Evaluation of specific antibody responses against ovalbumin peptide.

The aim of this study was to evaluate the response of antibodies IgG1 and IgG2a after immunization with ovalbumin in combination with various covalently bound adjuvants.

Material and methods

Were analyzed following serum:

Group 1; 6 mice immunized with 500 μg of ova protein with CFA/IFA (Positive control)

Group 2: 6 mice immunized with 50 µg of long ova peptide with sequence ISSAESLKISQAVHAAHAEINEA (SEQ ID NO: 3; amino acids of ovalbumin with 316 338)

Group 3: 6 mice immunized with 50 µg short ova peptide with sequence KISQAVHAAHA (SEQ ID NO: 4; amino acids of ovalbumin with 323 333)

Group 4: 6 mice immunized with 1 μg of PBS-6-OVA

Group 5: 6 mice immunized with 1 μg of PBS-14-OVA

Group 6: 6 mice immunized with 1 μg of PBS-57-OVA

All mice were immunized by intramuscular injection in den� 0 and day 14. All samples were taken 14 days after the second immunization and were used to verify the titer of IgGI and IgG2a specific against ovalbumin.

Results

Mice, which were injected with 1 μg of OVA covalently linked either with PBS-6, PBS-14 or PBS-57, showed a higher titer as IgG1 (figure 4) and IgG2a (figure 5) that are specific for ovalbumin than mice, which were injected with 50 μg OVA in itself.

Thus, this experiment demonstrates that the binding of the antigen with glycolipid adjuvants increases the strength of the response.

Example 5: Analysis of immunization induced antigen tyrosinase-like protein 2 (Trp2) associated with different glycolipid adjuvants, and intramuscular injection.

Material

For immunization were used sixty-nine female mice of the C57B1/6J CD45.2 the age of 9 weeks. They were divided into 14 groups: groups 1 through 5 consisted of 3 animals / group 6 to 14 included 6 animals.

Target cells to measure cytotoxicity in vivo

Cytotoxicity in vivo Trp2 181 to 188, induced response, CD8+ T cells was assessed using VITAL test as described in Example 1.

Vaccination

Short Trp2 peptide comprising the sequence of amino acids 181 to 188 of protein Trp2, long Trp2 peptide includes the amino acid sequence of 174-193 Trp2 and used the glycolipids are PBS-6, PBS-14 and PBS-57.

M�Shea were vaccinated by intramuscular injection at day "0" of the following solutions prepared respectively for different groups:

- Group 1: 50 µl PBS;

- Group 2: 50 µg short Trp2 peptide in 50 μl PBS/DMSO;

- Group 3: 50 mg long Trp2 peptide in 50 μl PBS/DMSO

- Group 4: 1 μg of PBS-6-Trp2 in 50 μl PBS;

- Group 5: 1 μg of PBS-57-Trp2 in 50 μl PBS;

- Group 6: 1 μg of PBS-14-Trp2 in 50 µl PBS.

Get data.

Data were obtained as described in Example 1.

Results

The aim of this study was to evaluate the efficiency of covalent binding different antigens with glycolipid agonists NKT cells in the induction of response in the form of specific lysis intramuscular injection, in comparison with the same antigen in itself.

Mice, which were injected with 1 μg Trp2, covalently linked or with PBS-6, PBS-14 or PBS-57, showed a higher percentage of Trp2-specific cytolytic activity in the blood than mice, which were injected with 50 μg of one Trp2 (figure 6).

Thus, this experiment demonstrates that the results obtained with the ovalbumin peptide covalently linked to glycolipid adjuvants can also be observed with other antigens.

1. The compound of formula (I)

where
R1, R2, R3and R4represent each independently from each other hydrogen; and R1is either above or below the sugar ring.
R6PR�dstanley a
(a) -(CH2)xCH3where x is an integer selected from 20 to 25; or
(b) -(CH2)xCH=CH(CH2)yCH3
where x, y and z are independently selected integers from 1 to 14. R5represents the following formula (II):

where
R8represents hydrogen, and
R7represents a linear or branched C3-C15alkyl;
X represents N;
Υ represents a biologically degradable by proteases and/or lipases of the linker group -(CH2)twhere t represents an integer selected from 3-10; and
Ζ is a peptide antigen.

2. The compound according to claim 1, where R6represents C25H51, R7represents C14H29.

3. The compound according to claim 1, where R6represents C23H45, R7represents C14H29.

4. The compound according to claim 1, where R6represents C23H47, R7represents C14H29.

5. The compound according to claim 1, where the linker contains a plot of degradable by proteases.

6. The compound according to claim 1, where the linker contains a plot split by lipase.

7. The compound according to claim 1, wherein the compound capable of activating NKT-cell.

8. Composition for vaccination to induce cellular and/or �amoralnih immune responses, comprising an effective dosage of a compound according to claim 1 and a physiologically acceptable excipient.

9. Pharmaceutical composition for the activation of NKT cells, comprising an effective amount of a compound according to claim 7 and a physiologically acceptable excipient.

10. Method of stimulating an immune response in a subject, comprising administering to the subject an effective amount of the composition for vaccination according to claim 8.

11. A method according to claim 10, wherein the immune response is a humoral immune response.

12. A method according to claim 10 for the activation of CD8+ T-lymphocytes.



 

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EFFECT: claimed novel compounds and pharmaceutical compositions based on them enhance immune response, increase production of antibodies in immunised animals, stimulate production of cytokines and activate macrophages.

7 cl, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutics, namely a method for preparing and purifying monosialoganglioside GM1. The method for preparing pure monosialoganglioside GM1 in the form of its sodium salt involves (a) GM1 separation from Fucosyl GM1 in a lipid mixture containing monosialoganglioside GM1 as a main ganglioside component by a column ion-exchange chromatography using an eluent comprising potassium or cesium ions, (b) recovery of the dissolved substance from the eluted solution, (c) diafiltration of the aqueous solution of the recovered dissolved substance of the stage (b), (d) addition of sodium salt, and diafiltration of the prepared aqueous solution (d) recovery of GM1 in the form of its sodium salt. The method for purifying monosialoganglioside GM1 from Fucosyl GM1 in the lipid mixture, the column ion-exchange chromatography using the eluent comprising potassium or cesium ions. The preparation of monosialoganglioside GM1 has a purity of 99.0% or more, and contains less than 0.1% of Fucosyl GM1. The method of treating disorders and diseases of the central nervous system and the peripheral nervous system, comprising administering the preparation of monosialoganglioside GM1 to the patient in its effective amount. The use of the preparation of monosialoganglioside GM1 in preparing a pharmaceutical composition.

EFFECT: use the above preparation of monosialoganglioside GM1 in treating has the considerable advantages due to reducing side effects.

17 cl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, more specifically to pharmacology, and concerts agents having an effect on the rheological properties of blood. What is presented is using the chemical modification product of hydroxyethylated starch O-(2-hydroxyethyl)-(1,4)-α-D-glucan, the hybrid macromolecular compound O-(4-hydroxy-3,5-di(1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl)benzyl)oxy)ethyl)-O-(2-hydroxyethyl)-(1,4)-α-D-glucan as an agent improving the rheological properties of blood. The invention can be used in a complex therapy of pathologies accompanied by blood hyperviscosity.

EFFECT: limiting an increase in blood viscosity.

4 ex

FIELD: medicine.

SUBSTANCE: invention represents an antibacterial pharmaceutical composition containing clotrimazole, propylene glycol, macrogol 400, macrogol 1,500, macrogol 4,000, poloxamer 338, cetostearyl alcohol, macrogol 20 cetostearyl alcohol, disodium edetate, purified water with the ingredients of the compositions taken in certain proportions, g/100 g.

EFFECT: higher antibacterial and antifungal action.

3 cl, 3 dwg, 5 tbl

FIELD: chemistry.

SUBSTANCE: conjugate represents nanodiamond particles with size 2-10 nm with pyrophosphorase, immobilised on them by means of linker, containing amino or amide groups. Content of pyrophosphatase constitutes 0.1-1 mg per 1 mg of nanodiamond, with specific activity of pyrophosphatase constituting to 95±5% of native pyrophosphatase activity. method of conjugate obtaining includes dissolution of nanodiamond with grafted hexamethylenediamine and/or nanodiamond aminated with ammonia in water, successive addition of water buffer solution HEPES with pH 7-8, magnesium chloride, sodium fluoride, sodium pyrophosphate, pyriphosphatase, and glutaraldehyde. After that, obtained mixture is exposed for 0.5-12 h, centrifuged, washed with water buffer solution Tris-HCl and dried.

EFFECT: obtaining conjugate of nanodiamond with pyrophosphatase, possessing increased stability.

3 cl, 1 tbl, 2 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: therapeutic agent contains carboxymethyl cellulose sodium salt as a base and a combination of antiseptic, 0.01% Myramistinum and metronidazole as therapeutic ingredients. The invention provides preparing the therapeutic agent possessing the antiseptic, wound-healing and sorption action on local pyoinflammatory processes in soft tissues and mucous membranes, used in surgery, dermatology, obstetrics and gynaecology, otorhinolaryngology.

EFFECT: agent possesses the higher efficacy.

2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: therapeutic agent contains an alloy of polyethylene oxide of molecular weight 400 and 1500 as a base and comprises a combination of antiseptic, benzalkonium chloride and metronidazole as therapeutic ingredients. The invention provides preparing the therapeutic agent possessing the antimicrobial, sorption and wound-healing action on local pyoinflammatory processes in soft tissues and mucous membranes, used in surgery, dermatology, obstetrics and gynaecology, otorhinolaryngology.

EFFECT: agent possesses the higher efficacy.

2 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention represents a composition for surgical area infiltration in liposuction or lymphedema elimination, containing normal saline, 0.5% Naropin and phosphatidylcholine in ratio 9:1:6 respectively per 10-15 cm3 fat.

EFFECT: reducing oedema, tissue injuries and blood loss volume, preventing microcirculation disorders in soft tissues and complications, reducing a risk of recurrences and promoting a faster rehabilitation of the patients.

2 cl

FIELD: chemistry.

SUBSTANCE: invention relates to a method of producing paramagnetic iron oxide nanoparticles. The disclosed method includes reacting an iron complex having iron as a central atom and a carboxylate group having 10 to 22 carbon atoms which is bonded to the central atom in a ligand form; a C10-C22 fatty acid and a C10-C22 aliphatic alcohol or C10-C22 aliphatic amine to obtain iron oxide nanoparticles. The iron oxide nanoparticles are obtained by raising the temperature from room temperature to 200 to 250°C with at a rate of 5°C/min or higher, and conducting the reaction at 200 to 250°C for 5 to 60 min.

EFFECT: invention enables to obtain iron oxide nanoparticles having a size of 4nm or less, which can be used as a MRI T1 contrast agent.

7 cl, 1 tbl, 25 dwg, 31 ex

FIELD: medicine.

SUBSTANCE: group of inventions refers to a kit for targeted medical imaging and/or clinical medicine, and to a method for using the kit. The declared kit comprises a pre-guide probe and an effector probe, wherein the pre-guide probe comprises a primary guide fragment and a first bio-orthogonal reactive group, and wherein the effector probe comprises an effector element, such as a label or a pharmaceutically active compound, and a second bio-orthogonal reactive group. Any of the first and second bio-orthogonal reactive groups represents dienophile in the form of stressed 8-merous cycle (cyclooctene or cyclooctine), while diene represents electron-deficient tetrazine or other applicable diene. A method for using the kit involves administering the pre-guide probe into the individual and enabling the probe circulating in the individual system for a period of time effective to achieve binding of the primary guide fragment with the primary target with the unbounded substance discharge from the body, with the effector probe to be inserted.

EFFECT: invention involves using the [4+2] Diels-Alder retrochemical reaction with electron reuptake to bind the pre-guide probe and the effector probe that enables fast in vivo reaction behaviour.

12 cl, 12 dwg, 1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to a method for active substance (AS) delivery through an epidermal barrier. The declared method involves using a matrix-type transdermal patch containing a substrate, a protective tape and a polymer layer, and characterised by the fact that 10% PEG-12 dimethicone niosomes are introduced into the polymer layer of the transdermal patch; then the substrate is coated with the polymer layer. The ACs are encapsulated into the niosomes with the use of hemogeniser APV, as well as 10 wt % propylene glycol and 5 wt % isopropyl myristate.

EFFECT: improving the active substance penetration with maintaining the biological activity and prolonging the active substance action.

2 tbl, 3 ex

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