Conjugates consisting of polymer and peptides deriving from gp41 hiv and their using in therapy

FIELD: medicine, polymers.

SUBSTANCE: invention relates to conjugates consisting of a water-soluble polymer of molecular mass from 200 to 20000 Da and representing polyethylene glycol or alkyl chain to which two molecules of synthetic peptides, not less, are bound by reactive functional group and wherein each peptide comprises amino acid sequence originating from region HR1 or HR2 of human immunodeficiency virus (HIV) gp41. Invention relates to methods for using these conjugates for delivery inhibition of to HIV target-cell by addition of indicated conjugates in the amount providing effective inhibition of cell infection with indicated virus. Also, invention relates to methods for preparing conjugates by functional adding of each molecule of synthetic peptide to polymer through reactive functional group.

EFFECT: valuable biological properties of conjugates.

27 cl, 2 dwg, 6 tbl, 6 ex

 

The scope to which the invention relates

The present invention relates to conjugates comprising a polymer and synthetic peptides derived from gp41 of human immunodeficiency virus (HIV). More specifically the present invention relates to a conjugate formed by a functional connection to a polymer of at least two molecules of synthetic peptide containing the amino acid sequence originating either from the region of HR1 or HR2 region gp41 of HIV-1.

Prior art

Currently, it is well known that cells can be infected with HIV in accordance with the mechanism in which there is fusion of the cell membrane and the viral membrane. The conventional model of such a mechanism is that glycoprotein complex viral envelope (gp120/gp41) interacts with cell surface receptors on the membranes of target cells. After binding of gp120 to cellular receptors (e.g., CD4 in combination with the chemokine co-receptor, such as CCR-5 or CXCR-4), is induced conformational change in the complex of gp120/gp41, which facilitates the introduction of gp41 into the membrane of target cells and mediates fusion of membranes.

Amino acid sequence of gp41 and its variants in different strains of HIV are well known. Figure 1 schematically presents about neizvestnye functional domains of gp41 (it should be noted, the number of amino acid sequences may slightly vary depending on the strain of HIV). It is obvious that hybrid peptide (fusogenic domain) is involved in the introduction of the virus into the membrane of target cells and its disruptive. Transmembrane domain-containing transmembrane anchor sequence is located at the end of this protein. Between hybrid peptide and transmembrane anchor are two distinct areas, known as areas heptadic repeats (HR), each of which has many of heptad. The HR1 region, which is closest to the N-end of the protein than the HR2 region, generally described as the area containing the amino acid sequence represented by SEQ ID NO:1. However, due to the natural polymorphism of the amino acid sequence and numbering of residues) region of gp41 HR1 HIV-1 can vary depending on the viral strain originates from this amino acid sequence. Another area HR2, also presented in figure 1 and in SEQ ID NO:2, can also vary due to polymorphism. Each amino acid sequence containing the HR1 region, and the amino acid sequence containing the HR2 region represents one of the most highly conserved regions in the protein shell of the HIV-1 (Shu et al., 1999, Biochemistry, 38:5378-5385; Hanna et al., 2002, AIDS 16:1603-8). HR have set the TWT sections, consisting of 7 amino acid residues, or "gated" (7 amino acids in each heptade marked "a"-"g"), where the amino acid in position "a" and the position "d" are mainly hydrophobic. In each area of HR is also present one or more motifs of type "latinboy lightning" (also known as repetition type "latinboy lightning"), containing 8 amino acid sequence beginning with isoleucine or leucine and ending them. In most cases, the HR2 region has only one motive-type latinboy lightning", whereas the HR1 region has five motifs of type "latinboy lightning". Heptade and motives of the type "latinboy lightning participate in education superspiritual structure of gp41 and superspiritual patterns of peptides derived from the HR areas. In General it is known that the supercoiling consist of two or more helices, which are twisted relative to each other, forming oligomers, and characteristic of such superspray is heptanal repeat consisting of amino acids with a predominance of hydrophobic residues in the first ("a") and fourth (d) positions, and charged residues, most often localized in the fifth (e) and the seventh (g) provisions, where the amino acid in position "a" and the position "d" represent the determinants affecting the status the oligomer and the orientation of the chain (see, for example the EP, Akey et al., 2001, Biochemistry, 40:6352-60).

It was found that synthetic peptides originating either from the HR1 region (HR1 peptides"), or from the HR2 region (HR2 peptides") gp41 of HIV, inhibit transmission of HIV to cells of the host in in vitro assays and clinical in vivo studies (see e.g., Wild et al., 1994, Proc. Natl. Acad. Sci. USA, 91:9770-9774; U.S. patent No. 5464933 and 5656480, and Kilby et al., 1998, Nature Med. 4:1302-1306). More specifically HR1 peptides presented DP107 (also known as T-21; SEQ ID NO:3), inhibit infection of T cells at 50%effective concentration (EC), part 1 µg/ml (see, for example, Lawless et al., 1996, Biochemistry, 35:13697-13708). The HR2 peptides presented DP178 (also known as T-20; SEQ ID NO:4), typically block the infection of T cells at 50%effective concentration (EC) scale ng/ml. the First mention of high-performance synthetic peptides that include one or more enhancer sequences, attached to the amino acid sequence of the crustal HIV gp41 and inhibit membrane fusion of HIV with the cell membrane, thus preventing transmission of the virus to the cell master, have already met in the literature (see for example, U.S. patent No. 6258782 and 6348568). However, synthetic peptides derived from HIV gp41, have a relatively low molecular weight. To achieve and maintain a blood level that is sufficient for producing terapeutiche is one effect, these synthetic peptides like other known peptides that are effective therapeutic agents require frequent administration (e.g. daily injections). To solve this problem, researchers have attempted to chemically modify therapeutic agent, such as a peptide or peptidomimetic, for example, by attaching a therapeutic agent to a water-soluble polymer, such as polyethylene glycol (PEG)to provide a longer presence of therapeutic agent in vivo (for example, increasing its half-life in the bloodstream and/or the inhibition of the degradation of therapeutic agent in the bloodstream). However, as known in the art (see, for example, U.S. patents 6258774 and 6113906), such modifications therapeutic agent have natural limitations, i.e. such modifications usually limit the bioavailability of therapeutic agent. More specifically attaching a water-soluble polymer and a therapeutic agent, and in particular to a small peptide, often leads to negative modulation of the biological activity of therapeutic agent. This loss of activity and therapeutic efficacy often occurs in peptides with lower molecular weight (e.g. less than 4000 daltons), have small the binding sites, not associated with bioactivity. Although previous works could be described conjugation of therapeutic agents with a water-soluble polymer, however, in these works there is no mention of the conjugate containing a polymer attached to two or more molecules of synthetic peptide and which would preserve significant bioactivity (e.g., retain significant biological activity compared to the activity of individual synthetic peptide) and increased action (significant biological activity against HIV-1 strain resistant synthetic peptide, taken separately, i.e. not in the form of a conjugate, in comparison with the biological activity of synthetic peptide).

Thus, it is necessary to obtain such conjugates, which could prevent the interaction of the viral gp41 domains involved in the fusion process of the virus, and more preferably to prevent conformational changes in gp41 required for such a merger, and that would be the inhibition of gp41 fusion of HIV with the membrane of target cells. In addition, you must obtain conjugates, which could inhibit HIV transmission in the target cell, and at the same time would preserve significant biological activity and would have a stronger effect. The present invented the e aims to meet these requirements.

Brief description of the invention

The present invention relates to a conjugate comprising a polymer attached to two or more synthetic peptides derived from the gp41 HR (region HR1, HR2 region, or combinations thereof, where the said conjugate has the advantage that it retains a significant level of biological activity (i.e. activity against HIV) and has enhanced action (compared with a synthetic peptide taken separately (for example, without the polymer portion of the conjugate)). Other distinctive signs, such as increased biological half-life synthetic peptide, which is part of the conjugate (e.g., contributing to increase the half-life of synthetic peptide in vivo prior to its decomposition in the bloodstream and/or removal from the bloodstream compared with individual synthetic peptide), will be obvious to experts from the following description of the invention. The conjugate according to the present invention, in addition, may contain a pharmaceutically acceptable carrier.

In addition, the present invention relates to a method of applying a conjugate according to the present invention for inhibiting transmission of HIV to the target cell, where the method involves the introduction of the virus into the cell a certain amount of the conjugate according to the of the present invention, effective to inhibit infection of the cell specified by a virus. This method can be used for the treatment of HIV-infected individuals. In a preferred embodiment of the invention, inhibition of HIV transmission to the target cell involves the inhibition of gp41-mediated fusion of HIV-1 cell-target.

The present invention also relates to methods of obtaining conjugates in accordance with the present invention. One of these methods described here involves the following stages: (a) the reaction of the first molecule of synthetic peptide to the polymer with the formation of the intermediate, which includes the first intermediate, where the first molecule of synthetic peptide is functionally attached to the first reactive functional group of the polymer; (b) the reaction of the specified intermediate comprising the first intermediate with a second molecule of synthetic peptide with the formation of the conjugate, where the second molecule of synthetic peptide is functionally attached to the intermediate comprising the first intermediate, through the second reactive functional groups of the polymer. For professionals it is obvious that this method may also allow for simultaneous connection of multiple molecules of synthetic peptide to the polymer, where more than one of alcula synthetic peptide functionally attached to the polymer with the formation of the conjugate, where each molecule of synthetic peptide which is functionally connected, functionally associated with the reactive functional group of the polymer.

The above and other objectives, features and advantages of the present invention will be apparent from the following detailed description of the invention with reference to the accompanying graphic material.

Brief description of the graphical material

Figure 1 schematically presents gp41 of HIV-1, which shows the region 1 heptanol repeat (HR1) and region 2 heptanol repeat (HR2) together with other functional areas of gp41. Examples of amino acid sequences corresponding to the HR1 and HR2, and the numbering of the positions of the amino acids are given only for purposes of illustration, and relate to gp160, HIV strainIIIB.

Figure 2 schematically illustrates a variant of the synthesis of the conjugate according to the present invention.

Detailed description of the invention

Definitions:

In the present description, the terms "first", "second", "third", etc. can be used for: (a) specify the order; or (b) to identify these molecules (e.g., synthetic peptides, intermediates or conjugates, which has a composition different from each other); or (C) for the two objectives (a) and (b)). However, the terms "first", "second", "third is th" etc. should not be construed as limiting the present invention.

The term "functionally connected" (and its grammatical forms)used in the description and the claims, refers to the hybrid, connection or Association with stability sufficient for the polymer regardless of the conditions found in vivo, remained attached to two or more molecules of synthetic peptide within a certain period of time sufficient to increase the biological time-life synthetic peptide, which is part of a conjugate according to the present invention (compared with individual synthetic peptide). As known in the art, the communication may include one or more covalent, ionic, hydrogen, van der Waals, electrostatic ties, etc. As known in the art and as will be more apparent from the following description of the variants of the invention, there are several methods and compositions in which two or more molecules can be functionally connected with the use of reactive functional groups. As will be described in detail in the present invention, such reactive groups include, but are not limited to, the available chemical groups (e.g., thio, carboxyl, hydroxyl, amine, sulfo and p) and reactive chemical groups (reactive with the free chemical groups).

The term "individual"used in the description and in the claims, refers to a mammal, preferably human.

The term "cell-target"used in the description and the claims, means a cell that can be exposed to HIV infection. Such a cell is preferably a cell or human cell, and more preferably human cells, which can undergo HIV infection by a particular mechanism, including membrane fusion.

The term "pharmaceutically acceptable carrier"used in the description and the claims, means the carrier medium that does not have a significant impact on the biological activity of the active ingredient (e.g., conjugate according to the invention or compounds obtained by the method according to the invention), when adding it to the environment. As known in the art, suitable pharmaceutically acceptable carrier can contain one or several substances, including, but not limited to, water, buffered water, saline, 0.3% glycine, aqueous alcohols, isotonic aqueous buffer; and, in addition, it may include one or more substances, such as glycerin, oils, salts such as salts of sodium, potassium, magnesium and ammonium phosphonates, esters of carboxylic acids, fatty acids, saccharides, polysaccharides, glycopro teeny (to improve stability), fillers, preservatives and/or stabilizers (designed to increase shelf life, when necessary and appropriate for the manufacture and commercial distribution of this composition). Preferably, the medium was suitable for intravenous, intramuscular, subcutaneous or parenteral administration (e.g. by injection).

The term "amino acid"used in the description and in the claims relating to synthetic peptides according to the invention means a molecule that has at least one free amino group and at least one free carboxyl group. This amino acid may have more than one free amino group or more than one free carboxyl group or, alternatively, it may contain one or more chemical free reactive groups that are not amino group or a carboxyl group (for example, hydroxyl, sulfhydryl etc). This amino acid can be a natural amino acid (for example, L-amino acid), unnatural amino acid (e.g., D-amino acid), a synthetic amino acid, modified amino acid, amino acid derivative, precursor amino acids and conservative replacement. The specialist knows that the choice of amino acids included in the peptide, partly depends on the physical, chemical or biological properties required for antiviral peptide. So, for example, from the description of the invention the specialist will be obvious that the amino acids in the synthetic peptide may constitute one or more natural (L)-amino acids and non-natural (D)-amino acids. Amino acids that are not preferred, may be replaced by the preferred amino acids.

The term "amino acid substitution", referring to the amino acid sequence synthetic peptide derived in accordance with the present invention, and used in the description and in the claims means one or more amino acid substitutions in the sequence synthetic peptide, such substitutions, in which the ability of the peptide to contact the HR area HIV gp41 and inhibit gp41-mediated fusion remains largely unchanged (as may be determined by antiviral activity, expressed in the IC50within units ng/ml or mcg/ml, as illustrated in more detail below). Typically, the number of amino acid substitutions is approximately 1 to 10 amino acids of amino acids in the synthetic peptide, and more preferably from 1 amino acid to 5 amino acids in the synthetic peptide. As known in the art, amino acid substitutions may include conservative for the ENU", which is determined by the above-mentioned functions, and provides for replacement of amino acids with basically the same charge and size and the same hydrophilicity and/or aromaticity, as the deleted amino acid. Such conservative substitutions are known in the art, include, but are not limited to, replacement of glycine-alanine-valine; isoleucine-leucine; tryptophan-tyrosine; aspartic acid-glutamic acid, arginine-lysine; asparagine-glutamine and serine-threonine. Amino acid substitutions may include polymorphism in different positions of amino acids in the region of HR1 or HR2 region depending on the area, from which the synthetic peptide is available in the laboratory and/or clinical isolates of HIV. Such sequence polymorphism are available in the database of genes, such as GenBank, and other available databases of amino acid sequences of HIV.

The term "reactive functional group"used in the description and in the claims, means a chemical group or chemical molecule that can form a covalent bond or connection for the functional attachment of polymer to the synthetic peptide. With regard to chemical groups, the reactive functional groups known in the art, include, but are not limited to,maleimide group, thiol group, carboxypropyl, phosphoryl group, acyl group, hydroxyl group, acetyl group, a hydrophobic group, aminogroup, Danilina group, alphagraph, operations group, a group that reacts with a thiol group reacts with the amine group reacts with carboxyla etc. Chemical molecule may contain a linker. It is known that the linker is a compound or group that acts as a molecular bridge, functionally linking the two different molecules (e.g., one portion of the linker binds to the synthetic peptide, and another portion of the linker binds to the polymer with the formation of the conjugate of the present invention). These two different molecules can be attached to the linker Paladino. The specific size or composition of the linker is not limited provided that it can be used as a molecular bridge. The linkers known to specialists include, but are not limited to, chemical circuit chemical compounds (e.g., reagents), amino acids, etc. Such linkers can be, but are not limited to, homobifunctional linkers, heterobifunctional linkers, biologically stable linkers and biodegradable linkers, also known in the art. If you are using a linker, it is preferable nepl the stationary linker (for example, such a linker, which acts to functionally attached synthetic peptide was not rigidly bound to the polymer). Heterobifunctional linkers are well known to the experts, include one end having a first reactive functional group, specifically associated with the first molecule, and the opposite end having a second reactive functional group, specifically associated with the second molecule. For professionals it is obvious that a variety of bifunctional or polyfunctional reagents, such as Homo - and heterofunctional reagents (for example, the reagents described in the catalog of the Pierce Chemical Co., Rockford, III), can be used as a linker according to the invention. To optimize properties such as the stability of biological functions, resistance to certain chemical and/or temperature parameters and sufficient stereoselectivity or size, length and composition of the linker can vary depending on such factors as associated molecules and the conditions in which they are linking. For example, the linker should not have a significant impact on the ability of a synthetic peptide (with which it is associated) to function as an inhibitor of the fusion of HIV with the cell-target and the transmission of HIV to the target cell, or both. In accordance the present invention a reactive functional group, which is not preferred, may be substituted for the preferred reactive functional group.

The term "polymer"used in the description and in the claims, includes homopolymers and copolymers, and polymers, which can have a structure including a branched or linear structure, known in the art. The preferred polymer is a water-soluble polymer, and more preferably a water-soluble polymer, which is basically non-toxic when its in vivo introduction of individuals. Illustrative examples of such water-soluble polymers include, but are not limited to, polyols, polyethylene glycol ("PEG"), polypropylenglycol ("BCP"), dextran, carboxymethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, polyaminoamide (homopolymers, for example, polylysine or heteropolymer, for example, poly(D-L-alanine)-poly(L-lysine)), poly(alkylenes), copolymers of ethylene glycol and BCPs, copolymers of PEG and amino acids, copolymers of PEG and diablocody acid, and copolymers of polypropyleneoxide and ethylene oxide. It is preferable that the polymer used was a discrete molecule, i.e. a molecule consisting of an equal number of polymer chains (for example, 6 of ethylene units in the PEG), but not a mixture of polymer molecules, g is e, each of these molecules by their size are different from each other. Used in the present invention the term "polymer" may also include alkyl branched or straight chain (as will be seen below, for example, from table 5). The polymer used to obtain a conjugate according to the invention may have a molecular weight in a very wide interval, in the preferred embodiment of the invention the specified polymer has a molecular weight in the range from about 200 daltons to 100,000 daltons, and in a more preferred embodiment of the invention the specified polymer has a molecular weight in the range from about 300 daltons to 20,000 daltons. Many reactive functional groups can be attached to the polymer or to form part of the polymer, which is functionally attached to a synthetic peptide and the polymer may be present reactive functional group, more than one type for selective binding to synthetic peptides (for example, the reactive functional group of each type is available for the functional binding molecule of synthetic peptide, where each molecule of synthetic peptide can have a sequence that is similar or different from another sequence(s) of the molecule (molecules) attached synthetic peptide). Example and reactive functional groups, described above, and other groups can be, but are not limited to, ketones, esters, carboxylic acids, aldehydes, alcohols, amines, etc. In this connection, to create or view the reactive functional groups of the polymer, which can be used for functional binding of two or more molecules of synthetic peptide with the formation of a conjugate according to the invention, this polymer can be suitably constructed, modified, or appropriately functionalized by standard methods of organic chemical synthesis. Since in the present invention can be used in many water-soluble polymers and a variety of reactive functional groups, the method of chemical synthesis to create or join a reactive functional group will depend on the polymer and a reactive functional group, which is desirable to attach to the polymer. The polymer used in the present invention preferably has the following characteristics: (a) it is water soluble and preferably soluble in water systems that are normally present in vivo; (b) he has more than one reactive functional group (either the same type or different types, for example, the chemical with which tawu), where two or more molecules of synthetic peptide can be functionally attached to the polymer by more than one reactive functional group of the polymer (preferably, each of the reactive functional group of the polymer, suitable for functional binding to a synthetic peptide, was functionally attached to a reactive functional group of one molecule of synthetic peptide); and (C) for functional binding to a synthetic peptide with the formation of a conjugate according to the invention it has no significant effect on biological activity (e.g., antiviral activity) synthetic peptide, as you can define methods for assessing antiviral activity in vitro and/or in vivo and as will be described in more detail below. The polymer preferred for use in the present invention include polyol, and more preferably PEG. In accordance with the present invention the polymer, which is not preferred, may be substituted for the preferred polymer.

The term "increased activity"related to the conjugate according to the invention and used in the description and in the claims, means that this conjugate has a stronger antiviral activity against strains of HIV-1 that are resistant to one or n is how many individual synthetic peptides (monomer, which is not bound to the polymer) compared to the antiviral activity of individual synthetic peptide (as will be more apparent from the following descriptions). Preferably, such "enhanced action" conjugate meant antiviral activity against drug-resistant HIV-1, measured as IC50 or AS component and less (for example, in ng/ml) or equal to 1 µg/ml (in relation to the activity of synthetic peptide) (see, for example, table 5).

The term "synthetic peptide"related peptide according to the invention and used in the description and in the claims, means a peptide (a) obtained by chemical synthesis, recombinant expression, biochemical or enzymatic fragmentation of larger molecules, chemical cleavage of larger molecules, or their combination, or, in General, obtained by other known methods and selected; (b) having an amino acid sequence that includes not less than about 16 amino acid residues and no more than about 60 amino acid residues and comprising at least 14 contiguous amino acids belonging or region HR1 or HR2 region gp41 of HIV (which may include one or more amino acid substitutions); and (C) are able to inhibit HIV transmission in the target cell (preferably formed by the I complex with any of the areas of HR gp41 of HIV and/or prevent fusion between HIV-1 and cell-target), as may be determined by evaluating the antiviral activity in vitro and/or in vivo, and as will be described in more detail below. The term "isolated"related peptide, means that a synthetic peptide mainly contains no components that are not part of the whole structure of the peptide, for example, generally does not contain cellular material or culture medium used for the production of recombinant techniques, or substantially does not contain chemical precursors or other chemicals used for chemical synthesis or produced in biochemical or chemical reactions. Amino acid sequence synthetic peptide may include one or more amino acid substitutions and/or one or more modifications resulting from polymorphism and present in the sequence of the relevant region of gp41 of HIV, or it may include one or more amino acid substitutions, which is injected to stabilize the helical structure and/or to influence the oligomerization so that was the self-Assembly of peptides in the trimer. In addition, the amino acid sequence besides the fact that it has a core peptide derived from gp41 of HIV, may contain one or more enhancer peptides attached to korovou the peptide NAP is emer, at N-end, near-end, or at both the N - and C-ends, or it may contain a core peptide, derived from one or more HIV-1, HIV-2 and SIV (see, for example, U.S. patent No. 6258782; see also synthetic peptide containing the amino acid sequence represented in SEQ ID NO:5 and 46-59). Depending on the synthetic peptide synthetic peptide functionally incorporated in the polymer, may be present in the form of a monomer or oligomeric form, such as a trimer. In the example where the synthetic peptide is present as a trimer, only one molecule can be functionally attached to the polymer, while the remainder of the molecule of synthetic peptide containing the trimer, undergoes self-Assembly around functionally attached molecules of synthetic peptide. For example, illustrative synthetic peptides containing the HR1 peptides having amino acid substitutions compared to SEQ ID NO:1) and preferably subjected to the self-Assembly into trimers (e.g., a trimer consisting of three molecules of synthetic peptide), contain the amino acid sequence represented in SEQ ID NN:61-74.

Preferably the synthetic peptide according to the invention may contain a sequence of length not less than about 16 amino acid residues and not more than about 60 amino acid of Titkov, preferably at least 36 amino acids and not more than about 52 amino acids, and more preferably not less than about 41 amino acids and not more than about 51 amino acids. Preferably, a synthetic peptide containing the sequence originating from the region of gp41 HR1 HIV, consisted of a continuous sequence of at least contiguous amino acid residues 18-54 SEQ ID NO:1 (indicated by single-letter codes, NNLLRAIEAQQHLLQL TVWGIKQLQARILAVERYL KD), or its variant, formed as a result of polymorphism, because it was found that the key determinants in this part of the region HR1 affect biochemical and antiviral properties described in this invention. Representative synthetic peptides derived from the HR1 region are, but are not limited to, peptides having amino acid sequences shown in SEQ ID NO:3 and 6-31. Preferred synthetic peptide derived from the HR1 region, can be used for producing the conjugate according to the invention instead of the HR1 peptide, which is not preferred synthetic peptide. Preferably, the sequence synthetic peptide derived from the HR2 region gp41 of HIV, consisted of a continuous sequence of at least amino acid residues 43-51 SEQ ID NO:2 (e.g., QQEKNEQEL), p is since it was discovered, what are the key determinants in this part of the region HR2 affect biochemical and antiviral properties described in this invention. Representative synthetic peptides derived from the HR2 region are, but are not limited to, peptides having amino acid sequences shown in SEQ ID NO:4, 32, 75-99 and 114. Preferred synthetic peptide derived from the HR2 region, can be used for producing the conjugate according to the invention instead of the HR2 peptide, which is not preferred synthetic peptide. A large number of these synthetic peptides, which can be used in the present invention, have been described previously (for example, in U.S. patent No. 5656480, 6133418 and 6258782, which in its entirety are introduced in the present description by reference). The term "individual synthetic peptide", as used in the description and in the claims, means a synthetic peptide which is functionally not associated with the polymer, i.e. a peptide which has an unconjugated form, not containing polymer.

The present invention is illustrated in the following examples, which should not be construed as limiting the present invention.

Example 1

In one of the embodiments of the invention illustrates the method of obtaining the conjugate is in according to the invention. One of these methods involves the following stages: (a) the reaction of the first molecule of synthetic peptide to the polymer to obtain an intermediate, comprising the first intermediate, where the first molecule of synthetic peptide is functionally attached to the first reactive functional group of the polymer; (b) the reaction of the specified intermediate comprising the first intermediate with a second molecule of synthetic peptide with obtaining conjugate, where the second molecule of synthetic peptide is functionally attached to the intermediate comprising the first intermediate, through the second reactive functional groups of the polymer. For professionals it is obvious that this method may also allow for simultaneous connection of multiple molecules of synthetic peptide to the polymer, where more than one molecule of synthetic peptide functionally associated with the polymer with the formation of the conjugate and where each molecule of synthetic peptide which is functionally connected, functionally connected with the reactive functional group of the polymer.

Peptides were synthesized on a peptide synthesizer by standard methods of solid-phase synthesis and standard methods of peptide chemistry using FMOC (see also U.S. patent No. 6015881, p is reoccupancy the present assignee). In this example, synthetic peptides, in addition, include reactive functional groups, i.e., were blocked at the N end of the acetyl group and the C-end - of amide group. After cleavage from the resin, the peptides were besieged and the precipitate was liofilizovane. Then the peptides were purified by reversed-phase high-performance liquid chromatography and the identity of the peptides was confirmed using mass spectrometry by elektrorazpredelenie. In this example, T20 (SEQ ID NO:4) was used for functional attach to the polymer, resulting in a received conjugate according to the invention. As was described previously, the conjugates consisting of synthetic peptides, non-T20 (SEQ ID NO:4), regardless of whether they come from the region of HR1 or HR2 region gp41 of HIV-1, should be functionally similar with different conjugates, consisting of the illustrated sequence T20 (SEQ ID NO:4), because they have basically the same mechanism of action (inhibition of the merger), constructed mainly from similar units (heptad and motives of the type "latinboy lightning") and have similar conformational structure (alpha-helix and superspies). Other synthetic peptides include, but are not limited to, the amino acid sequence containing SEQ ID NO:3, 5-99 and 114. Similarly, the same itself is e or similar methods can be used for functional accession by any of the synthetic peptide to the polymer. Although this example shows only one type of synthetic peptide (for example, with the same amino acid sequence), derived from the HR2 region gp41 of HIV, however, from the following description it will be obvious that with the same polymer molecule may be functionally linked synthetic peptide more than one type. For example, when receiving a conjugate containing not less than two molecules of synthetic peptide is functionally attached to the polymer molecule can be used in various other combinations, which include, but are not limited to, combinations, where each molecule of synthetic peptide derived from the region of gp41 HR1 HIV and contains the same (identical) amino acid sequence, in contrast to other synthetic peptide contained in the conjugate; each molecule of synthetic peptide derived from the region of gp41 HR1 HIV and at least one of the molecules of synthetic peptide differs in its amino acid sequence compared another synthetic peptide contained in the conjugate (e.g., SEQ ID NO:3 and SEQ ID NO:27, if the conjugate contains two molecules of synthetic peptide is functionally attached to the polymer); each molecule of synthetic peptide derived from the HR2 region gp41 of HIV and contains the same (dentico) amino acid sequence unlike other synthetic peptide contained in the conjugate; each molecule of synthetic peptide derived from the HR2 region gp41 of HIV, and at least one of the molecules of synthetic peptide differs in its amino acid sequence unlike other synthetic peptide contained in the conjugate (e.g., SEQ ID NO:4 and SEQ ID NO:5, if the conjugate contains two molecules of synthetic peptide is functionally attached to the polymer); or at least one of the molecules of synthetic peptide contains the amino acid sequence derived from the HR1 region, which undergoes self-Assembly in solution with the formation of trimers (e.g., SEQ ID NO:63), and at least one of the molecules of synthetic peptide contains the amino acid sequence derived from the HR2 region gp41 of HIV (for example, SEQ ID NO:4) (because a small number of trimers mainly associated only with certain synthetic peptides derived from the HR2, and less preimushestvennogo with other synthetic peptides derived from the HR2). As is obvious from the description of the present invention, various combinations of synthetic peptides can also be used if more than two molecules of synthetic peptide is functionally attached to the polymer molecule. The number of molecules of synthetic peptide is functionally attached to the polymer molecule, depends on several factors, which may include, but are not limited to, the size of the polymer, the polymer composition, the composition of the synthetic peptide and the number of reactive functional groups of the polymer to the functional connection to the synthetic peptide. Preferably, the number of molecules of synthetic peptide is functionally attached to the polymer molecule was approximately from 2 to 20, and more preferably from about 2 to 5. In addition, as is obvious to the specialist, and depending on the reactive functional groups used for the functional merger of the synthetic peptide to the polymer, not less than two molecules of synthetic peptide can be functionally attached to the polymer through a part of the synthetic peptide selected from the group consisting of carboxy-late (C-end), amino end (N-Terminus), internal lysine and combinations thereof (for example, where one molecule of synthetic peptide functionally linked through its C-end and one molecule of synthetic peptide is functionally connected via its N-Terminus and so on).

In one illustrative embodiment of the invention, and as shown in figure 2, the conjugate according to the invention was obtained by functional binding of two molecules T20 (SEQ ID NO:4) via the N-end of sinteticheskogo peptide reactive with functional groups of the polymer, The PEG. More specifically, the molecule T20 (SEQ ID NO:4) was functionally linked by an amide bond with PEG-acid, whereas the other molecule T20 (SEQ ID NO:4) was functionally linked by an amide bond with a second PEG-acid, which produced a conjugate containing dimer T20-PEG. So, for example, dibasic PEG-acid (approximately 300 daltons, 11.4 mg, 0,0339 mmol), 7-Aza-1-hydroxybenzotriazole (NOUT, 10 mg, 0,0746 mmol) and N,N-diisopropylethylamine (DIEA, 13,1 mg, is 0.102 mmol) was dissolved in dimethylformamide (DMF) (5 ml)and then added tetrafluoroborate O-benzotriazol-1-yl-N,N,N, N'-tetramethylurea (TBTU, to 21.8 mg, 0,0678 mmol). The solution was stirred for 5 minutes at room temperature and was added T20, protected on the side chain (500 mg, 0,0678 mmol). The resulting solution was stirred at room temperature for 24 hours. For the deposition of peptide was added water (15 ml). The solid was collected by vacuum filtration, washed with water (2 × 5 ml) and dried, resulting in a received dimer "is protected on the side chain T20 - PEG" (487 mg) with a yield of 96%. This dimer "is protected on the side chain T20 - PEG" (485 mg) was dissolved in triperoxonane acid (TFA, 6.3 ml)containing water (0.35 ml) and dithiothreitol (DTT, 0.35 g) as acceptors cations. The solution was stirred at room temperature under nitrogen atmosphere for 4 hours. To precipitate the crude dimer T20-PEG is obavljale methyl tert-butyl ether (MTBE). The solid is centrifuged in a centrifuge and MTBE decantation in waste. Cycle MTBE-leaching was repeated twice. The solid was dissolved in a mixture of water/ACN, 3:1 (acetonitrile, 40 ml)and the pH is brought to 6-7 by the addition of ammonium hydroxide. Then the pH of the solution was reduced to 4-5 by addition of acetic acid (0.5 ml). The obtained turbid solution was left overnight at room temperature to remove the protection from the side chain. The obtained suspension was brought to pH 7-8, and all solids were again introduced into the solution, and then froze and was liofilizovane with obtaining the crude dimer T20-PEG (375 mg). The crude dimer T20-PEG was purified by high-performance liquid chromatography (HPLC) using a reversed phase C18, 5 microns, Packed as stationary phase, and using a mixture of acetonitrile/water with 0.1% TFA as mobile phase (40-50% organic substances within 90 minutes). The fractions containing pure product were collected, frozen and liofilizovane to obtain 40 mg of the conjugate (89.8% of the area, HPLC, MS: found 9196,545 calculated 9196,489).

Although in this illustrative example describes functional connection N-Terminus of the synthetic peptide to the polymer, however, the specialist it is obvious that for the functional merger of the synthetic peptide to the polymer can be used and other methods. So, for example, ka is Boxing-end of the synthetic peptide may be functionally attached to the polymer by standard methods, known in the art (for example, carboxy-end of the synthetic peptide having a terminal amine, may be functionally associated with PEG-carboxylic acid). In another example, the internal lysine synthetic peptide functionally attached (via amine reactive functional group to the polymer well known methods (for example, by activating the PEG using active of ester and N-hydroxysuccinimide, which leads to modification of the amino side chain of lysine residue located within the amino acid sequence synthetic peptide).

So, for example, a synthetic peptide was functionally attached to the polymer, its With-end with the formation of a conjugate according to the invention. More specifically, two molecules of synthetic peptide (T20, SEQ ID NO:4) were functionally connected through their corresponding reactive functional groups to PEG6-diamine in several successive stages of the synthesis.

Stage 1. Receiving PEG6-(NH-Phe-Z)2

In a 50-ml round-bottom flask equipped with a magnetic stirrer, was loaded Z-Phe-HE (0,43 g of 1.43 mmol, 2 equiv.) 6-CL-HOBT (0.27 g, 1.57 mmol, 2.2 equiv.) acetonitrile (6 ml) and DIEA (0,37 ml, 2.14 mmol, 3 EQ.). The obtained yellow solution was cooled in an ice bath at 0-5°and then added HBTU (0,60 g,1.57 mmol, 2.2 EQ.). To the resulting solution was added PEG6-diamine (0.20 g, 0,714 mmol, 1.0 EQ.), and then added CH2Cl2(2 ml × 2). The reaction mixture was stirred at 0-5°C for 15 minutes and then heated to room temperature and stirred for further 1.5 hours. The reaction mixture was transferred into a 125 ml separating funnel, and then added CH2Cl2(10 ml × 3) and washed with a solution of NaHCO3(0,33M, 15 ml × 3) and NaCl (15 ml). The organic phase was dried over MgSO4and filtered. The residue obtained after removal of solvent, was applied on a column of silica gel, which were decontaminated 10%Et3N in hexane. The desired product was suirable mixture of CH2Cl2-Meon (20:1). After removal of solvent received connection 1, PEG6-(NH-Phe-Z)2in the form of a colorless oil (0,23 g, 40%).

Stage 2. Receiving PEG6-(NH-Phe-H)2

In a 50-ml round-bottom flask equipped with a magnetic stirrer, was loaded connection 1, PEG6-(NH-Phe-Z)2(0.20 g, 0.24 mmol), 10% Pd-C (dry, 50 mg) and Meon (10 ml). The reactor was degirolami by double treatment with a mixture of N2/H2and then was first made at room temperature balloon N2throughout the night. Pd-C was filtered and the solvent was removed to obtain compound 2, PEG6-(NH-Phe-H)2in the form of colourless what about the oil (0,13 g, 95%).

Stage 3. Receiving PEG6-(NH-AA(36-27)-Fmoc)2

It should be noted that AA(36-27) indicates amino acids 36-27 SEQ ID NO:4. In a 100-ml round-bottom flask equipped with a magnetic stirrer, was loaded connection 2, PEG6-(NH-Phe-H)2(0,44 g, from 0.76 mmol, 1 EQ.), Fmoc-AA(27-35)-OH (3,34 g, 1.53 mmol, 2 equiv.) HOAT (0.31 g, 2.3 mmol, 3.0 equiv.) DIEA (of 0.53 ml, 3.1 mmol, 4 equiv.) and DMF (20 ml). The obtained yellow solution was cooled in an ice bath at 0-5°and then added HBTU (0,61 g of 1.61 mmol, 2.1 EQ.). The reaction mixture was stirred at 0-5°C for 10 minutes and then heated to room temperature and was stirred for 3 hours. In the reaction mixture was placed in an ice bath, was added H2O (30 ml)and the resulting white suspension was stirred for 30 minutes and then subjected to vacuum filtration. White solid was washed H2O (30 ml) and dried in a vacuum oven (35° (C) during the night (of 3.85 g of the crude compound 3, PEG6-(NH-AA(36-27)-Fmoc)2).

Stage 4. Receiving PEG6-(NH-AA(36-27)-H)2

To a solution of crude compound 3, PEG6-(NH-AA(36-27)-Fmoc)2(3,30 g, 0.67 mmol) in DMF (10 ml) was added piperidine (of 0.53 ml, 5.4 mmol, 8 EQ.). After stirring at room temperature for 4 hours the reaction mixture was cooled in an ice bath and added H2O (15 ml). The obtained white suspension per massively 20 minutes and then was filtered. White solid was washed with a mixture of MTBE-heptane (1:1, 25 ml × 2), and then was transferred into a 100-ml flask and triturated with EtOH-H2O (1:1, 10 ml). The solid was collected by vacuum filtration and washed with EtOH-H2O (1:1, 20 ml). Filtering and washing EtOH-H2O repeated again. The crude compound 4, PEG6-(NH-AA(36-27)-N)2was dried in a vacuum oven (35° (C) during the night (2,53 g).

Stage 5. Receiving PEG6-(NH-AA(36-17)-Fmoc)2

It should be noted that at this stage were added amino acids 26-17 SEQ ID NO:4. In a 100-ml round-bottom flask equipped with a magnetic stirrer, was loaded connection 4, PEG6-(NH-AA(36-27)-N)2(1,82 g, 0.41 mmol, 1 EQ.), Fmoc-AA(17-26)-OH (1,87 g, 0.82 mmol, 2 equiv.) HOAT (0.17 g, of 1.23 mmol, 3.0 equiv.) DIEA (0.31 in ml, of 1.64 mmol, 4 equiv.) and DMF (25 ml). The obtained yellow solution was cooled in an ice bath at 0-5°and then added HBTU (0.33 g, 0.86 mmol, 2.1 EQ.). The reaction mixture was stirred at 0-5°C for 15 minutes and then heated to room temperature and was stirred for 3 hours. In the reaction mixture was placed in an ice bath, was added H2O (30 ml) and the resulting white suspension was stirred for 30 minutes and then subjected to vacuum filtration. White solid was washed H2O (30 ml), and then returned to 100-ml flask, download the military IPA-H 2O (95:5, 20 ml). The mixture was heated to 60°C for 5 minutes and gradually cooled to room temperature. The solid was collected by vacuum filtration and washed with IPA (5 ml × 4). The crude compound 5, PEG6-(NH-AA(36-27)-Fmoc)2, was dried on the funnel under vacuum (4.4 g is not fully dried product).

Stage 6. Receiving PEG6-(NH-AA(36-17)-H)2

To a solution of crude compound 5, PEG6-(NH-AA(36-17)-Fmoc)2(3.0 g, wet) in NMP (10 ml) was added DBU (100 μl). After stirring at room temperature for 1 hour was added to the resin PL-SO3H (150 mg) and the mixture continued to stir for 40 minutes. The resin was filtered and washed with NMP (10 ml). After processing solution NMP-water (H2O) (30 ml) was received molokoobraznuyu emulsion. To this emulsion was added H2O (100 ml), after which the mixture was liofilizovane. The yellow sticky oil suspended in EtOH (10 ml)was heated to 50°C for 5 minutes, and then cooled to room temperature. After adding H2O (20 ml) was obtained white suspension, and this suspension was stirred for 30 minutes. White solid was collected by vacuum filtration and washed with EtOH-H2O (1:1, 10 ml × 2). The crude compound 6, PEG6-(NH-AA(36-17)-N)2, was dried in a vacuum oven (35° (C) during the night.

Stage 7. Receiving PEG6 -(NH-AA(36-1)-Ac)2

It should be noted that at this stage were added amino acids 16-1 SEQ ID NO:4. In a 50-ml round-bottom flask equipped with a magnetic stirrer, was loaded connection 6, PEG6-(NH-AA(36-17)-N)2(0,48 g, 0,056 mmol, 1 EQ.), Fmoc-AA(1-16)-AU (0,37 g, 0,112 mmol, 2 equiv.) HOAT (0,023 g, 0,169 mmol, 3.0 equiv.) DIEA (0,040 ml, 0,225 mmol, 4 equiv.) and DMF (5 ml). The obtained yellow solution was cooled in an ice bath at 0-5°and then added HBTU (0,047 g, 0,124 mmol, 2.2 EQ.). The reaction mixture was stirred at 0-5°C for 15 minutes and then heated to room temperature and was stirred for 3 hours. In the reaction mixture in an ice bath was added H2O (30 ml) and the resulting white suspension was stirred for 30 minutes and then subjected to vacuum filtration. White solid was washed H2O (30 ml), and then returned in a 50-ml flask and triturated with MeCN-H2O (9:1). The solid was collected by vacuum filtration and washed with H2O (10 ml×2). The crude compound 7, PEG6-(NH-AA(36-1)-Ac)2, was dried on the funnel under vacuum (0.73 g is not fully dried product).

Stage 8. Receiving PEG6-(T20)2

A 25-ml flask with the crude compound 7, PEG6-(NH-AA(36-1)-Ac)2(0.20 g) was loaded TFA/DTT/H2O (90:5:5, 2.5 ml). The obtained yellow solution was stirred at room is the temperature for 2 hours. To the reaction mixture, cooled in an ice bath, was added MTBE (8 ml) and the resulting suspension was stirred for 20 minutes and then filtered under vacuum. The yellow solid is washed with MTBE (5 ml × 2) and dried on the funnel under vacuum for 30 minutes to obtain is not fully dry matter). The yellow solid was dissolved in MeCN/H2O (1:1, 1 ml). The obtained yellow suspension was filtered through cotton wool and washed with MeCN/H2O (1:1, 1 ml × 2). the pH of the combined solution was brought to 4 by adding a solution of NaHCO3(0,n.). To this solution was added SPLA (60 μl) and the light yellow solution was stirred at room temperature overnight. the pH of the reaction mixture was brought to 8 by adding the solution To a2CO3(1M). The solution was diluted with MeCN-H2O (15:85, 3 ml) and was applied to the HPLC column for separation (PLRP-XL, 300A, 10 μm, 20 × 300 mm; buffer A, 100 mm NH4SLA in H2O, the pH is brought to 8.5 by addition of NH4HE; buffer B, MeCN: gradient: 20%-40%, 60 minutes; flow rate: 15 ml/min). The collected fractions was estimated using HPLC respectively, and pure fractions were combined for lyophilization. The final product containing the conjugate of the present invention (PEG6-(T20)2), was obtained as a white powder, 5,5 mg

Example 2

This example illustrates the enhanced biological available is th (for example, the length of time half-life in blood flow in vivo) conjugate according to the invention compared with the half-life of synthetic peptide taken separately. Synthetic peptide and conjugate were obtained using the methods and compositions described in example 1 above. It is important to note that the standard animal model used to determine the biological availability correlated with bioavailability of synthetic peptide in the body (in vivo) human (as described in more detail in U.S. patent No. 6258782). Briefly, kanalirovaniem mice intravenously injected dose or synthetic peptide or conjugate according to the invention, where the dose concentration in solution was determined by the method of Adelkhah, and corrected for the weight of the animal, the dose was 10 mg/kg and blood Samples were taken at predetermined intervals of time (0, 15, 30 min and 1, 2, 4, 6 and 8 hours) and was inserted in the tube-collections containing anticoagulant (EDTA). From each tube collector collected plasma, which was then analyzed using fluorescent liquid chromatography high pressure (ghvd). In addition to the dilution of the sample was done by serial dilution disasteradio solution in buffer and in plasma and this dilution was used to plot a standard curve, p is Asadi peak from the known concentration of the peptide. Then this curve was used to calculate the concentration of peptide in the plasma taking into account all the dilutions and amounts entered in column. The half-life (t 1/2) and the total AUC (area under the curve) individual synthetic peptide and conjugate containing a synthetic peptide according to the invention, are presented in table 1.

Table 1
The test agentAUC (ug·h/ml)t 1/2 (hours)
Peptide T201581,6
Conjugate5004,3

As illustrated in table 1, the conjugate according to the invention can be considerably longer (more than twice) the half-life in the bloodstream compared to the half-life of individual synthetic peptide in the bloodstream.

Example 3

This example illustrates the unexpected results obtained for antiviral activity using the conjugates according to the invention. It is important to note that in vitro-analysis of antiviral activity the antiviral effect of the synthetic peptide, demonstrated in in vitro analysis, correlated with anti-viral effect of the synthetic peptide in vivo. To determine protivovirusny the activity (for example, assess the ability to inhibit HIV transmission in the target cell) conjugates according to the invention was carried out in vitro analysis, which showed that based on the data obtained using synthetic peptides derived from any of the areas of HR gp41 of HIV, can be determined antiviral activity observed in vivo. More specifically, it was shown that the antiviral activity observed using in vitro analysis of infectivity (analysis of infectivity Magi-CCR5", see, for example, U.S. patent No. 6258782), correlates with antiviral activity observed in vivo for the same peptides derived from gp41 of HIV (see, for example, Kilby et al., 1998, Nature Med. 4:1302-1307). In addition, it should be emphasized that each of T20 (SEQ ID NO:4) and T (SEQ ID NO:5) showed strong antiviral activity against HIV in vitro analysis of infectivity, and clinical trials involving human subjects.

Results of the analysis to reduce the infectivity titers of the virus used indicator cell line MAGI or CCR5-expressing derivative cMAGI. Both cell lines used, the ability of tat in HIV-1 TRANS-activation of the expression of the reporter gene β-galactosidase, which is regulated by the HIV-LTR. Reporter gene β-gal was modified so that it was present in the nucleus and could be detection the n using the substrate X-gal as intense nuclear staining agent, acting for several days after infection. Thus, the number of stained nuclei can be interpreted as a number equal to the number of infectious virions when the infection inoculum, if staining was performed only a single cycle of infection. Infected cells were counted using a CCD-Visualizer and both, both primary and laboratory-adapted isolates was found a linear relationship between the level of entry of virus into the cell and the number of infected cells, visualized by using the imaging unit. In analyses using MAGI and cMAGI, 50% reduction of infectivity titer is significant (Vn/Vo=0.5) and gives the initial threshold value for the evaluation of antiviral activity (IC50 was defined as the dilution required to achieve a 50% reduction of the titer of infectious virus). Evaluated and also the second threshold value Vn/Vo=0.1, the corresponding 90%reduction in the titer of infectivity ("IC90"). The peptides tested for antiviral activity, was diluted in different concentrations and tested against the adjusted HIV inoculum with two or three repetitions, resulting in received approximately 1500-2000 infected cells per well in 48-hole microtiter tablet. The peptide (with appropriate dilution) was added to the cMAGI cells or MAGI, the Le which was added to the viral inoculum, and after 24 hours was added to the inhibitor of infection and fusion of cells (e.g., T20) to prevent passage of the second cycles of HIV infection and the spread of the virus to other cells. Cells were cultured for 2 more days, and then were fixed and stained with the substrate X-gal for detection of HIV-infected cells. The number of infected cells for each control and peptide dilution was determined using the CCD-Visualizer, and then to calculate the IC50 and IC90 (expressed in mcg/ml).

In this first example, the antiviral activity of two clinical isolates of HIV was received from the same HIV-infected individual. The first isolate, which for convenience are designated "20S HIV-1 was sensitive to the antiviral action of T20 (SEQ ID NO:4) both in vitro and in vivo. The second isolate, which for convenience are designated "20R HIV-1 was resistant to the antiviral action of T20 (SEQ ID NO:4) both in vitro and in vivo. Two clinical isolates, 20S HIV-1 and 20R HIV-1, were used in the in vitro analysis of infectivity in which the antiviral effect T20 (SEQ ID NO:4; individual synthetic peptide; table 2, peptide T20") was compared with the antiviral effect of the conjugate according to the invention (e.g., conjugate, containing two molecules T20 (SEQ ID NO:4), functionally attached to the PEG; table 2, "conjugate"), and polymer (such as PEG), having one is a molecule of synthetic peptide (T20), functionally attached to the polymer (table 2, PEG-T20-monomer"). The results, expressed as IC50 values in ng/ml, illustrated in table 2.

Table 2
Tested agent20S HIV-1: IC5020R HIV-1: IC50
Peptide T20101211
Conjugate3249
PEG-T20-monomer1149>20000

Based on the results presented in table 2, can be made several conclusions. First, as shown by the results obtained using T20-sensitive HIV isolate the conjugate according to the invention (in which at least two molecules of synthetic peptide is functionally attached to the polymer molecule) remained significant biological (antiviral) activity compared with the activity of individual synthetic peptide (for example, these activities differed only 3 times, and this difference was much smaller than the logarithmic difference). In contrast, as shown by the results obtained using T20-sensitive isolate, polymer with only one molecule of synthetic peptide, functional connec is nnuu to this polymer (for example, table 2, "PEG-T20-monomer"), found a significant change in (log reduction) of biological activity compared to the activity of individual synthetic peptide. It was unexpectedly found that the biological activity of the conjugate was stable at a higher level in comparison with the biological activity of PEG-T20-monomer, it could be observed when the additive effect of several molecules of synthetic peptide on one connection (for example, the effect of two molecules of synthetic peptide functionally related to the polymer compared with the effect of one molecule of synthetic peptide operatively linked to the polymer).

Secondly, as shown by the results obtained using T20-resistant HIV isolates, it was unexpectedly discovered that the conjugate according to the invention had a stronger antiviral activity (at least in the logarithmic difference) against HIV isolate that was resistant to the actions of individual synthetic peptide (and which was also resistant to PEG-T20-monomer). More specifically, quite unexpectedly, it was found that the conjugate having two functionally attached molecules T20, had significant antiviral activity against T20-resistant HIV isolates (enhanced effect). The results, systematizer the bathrooms in table 2, in General showed that the conjugate according to the invention retains substantial biological activity, i.e. antiviral activity against HIV, and has enhanced activity against HIV resistant strains compared with individual synthetic peptide. This biological activity has been demonstrated in in vitro analysis, and found that it correlated with antiviral effect observed in vivo.

To further illustrate the unexpected results, which indicated that the conjugate according to the invention had a strong antiviral activity against HIV isolates that are resistant to individual synthetic peptide were tested some other T20-sensitive isolates and T20-resistant isolates (isolates "A-D") using in vitro analysis of infectivity, as shown in table 3. IC50 and IC90 were expressed in µg/ml.

The results are systematized in table 3, in General, showed that the conjugate according to the invention not only retain significant biological activity, i.e. antiviral activity against HIV (compared with individual synthetic peptide), but also unexpectedly found increased action, then there is a strong antiviral activity against HIV isolates, which were the resist is ntname specific synthetic peptide.

Table 3
VirusPeptide T20

IC50
Peptide T20

IC90
Conjugate

IC50
Conjugate

IC90
HIV-1 T20S-A0,0060,0780,0340,200
HIV-1 T20R-A>5>50,3302,524
HIV-1 T20S-B0,0290,1170,0130,087
HIV-1 T20R-B1,8987,1790,0440,257
HIV-1 T20S-C0,0060,0730,0240,151
HIV-1 T20R-C1,5446,2850,1050,610
HIV-1 T20S-D0,0180,2080,0630,457
HIV-1 T20R-D2,039>50,0300,210

To further illustrate the fact that the conjugate according to the invention retains substantial biological activity compared to the activity of individual synthetic peptides were tested for more HIV strains in vitro analysis of infectivity. As can be seen in Table 4, IC50 and IC90 were you the awives in µg/ml.

Table 4
VirusPeptide T20

IC50
Peptide T20

IC90
Conjugate

IC50
Conjugate

IC90
IIIB/CEM40,0070,0520,0350,206
3'GIV/CEM40,0080,0500,0340,194
SIM/CEM40,5843,3980,0380,216

The results are systematized in table 4, in General, showed that the conjugate according to the invention not only retain significant biological activity, i.e. antiviral activity against HIV (compared with individual synthetic peptide), but also in some cases unexpectedly found a stronger antiviral activity than the activity of individual synthetic peptide.

The methods described in example 1 were produced additional conjugates using different polymers, different ends of the synthetic peptide, which were functionally attached to the polymer, and various reactive functional groups used for the functional merger of the synthetic peptide to the polymer. the ve molecules of synthetic peptide (for example, SEQ ID NO:4) were functionally associated with the polymer. Then these conjugates were tested for antiviral activity against HIV isolates IIIB, 20S HIV-1 and 20R HIV-1 (see table 2 for HIV-resistant strains), where specified antiviral activity (IC50) was expressed in µg/ml. the results obtained for the different conjugates (table 5, A-Q), are presented in table 5, which shows the ends of the synthetic peptide used for functional attach to the polymer (table 5, With the end marked "C", N-end labeled "N")or internal lysine, if it is used (table 5, "Lys 18" means the lysine amino acid residue 18 in the amino acid sequence synthetic peptide)and antiviral activity (IC50) against strain IIIB HIV-1 (table 5, "IIIB"), antiviral activity (IC50) against strain 20S HIV-1 (table 5, "20S"),and antiviral activity (IC50) against strain 20R HIV-1 (table 5, "20R").

tr>
Table 5
The test conjugateEndPolymerIIIB20S20R
Individual synthetic peptide--0,0070,0101,2
ANPEG-6/td> 0,0370,0290,050
BCPEG 60,1170,0530,070
CCPEG 60,0650,0460,050
DNalkyl C50,039being 0.0360,049
ENPEG-80,0180,0260,020
FNalkyl C80,0890,0490,087
GNalkyl C110,1740,1760,218
HNAt0,0460,0470,124
INPEG 4-6-40,0290,0120,029
JNPEG 3being 0.0360,0280,049
KNPEG 40,0330,0260,067
LCGLY-PEG-30,1070,061of 0.066
MCGLY-C20,1390,0850,114
NNPEG 100,0390,0170,023
OCGLY-C60,1400,0830,044
PCGLY-PEG-60,0880,0310,051
QLys 18PEG 60,0570,0550,120

In table 5 the number specified after the "PEG"means the number of ethylene units contained in the PEG (multiples of numbers that correspond to the multiple PEG-chains, where discrete molecule functionally linked to each other); alkyl with specified after the number refers to the alkyl chain, with the corresponding number of carbon atoms contained in the alkyl chain; means GLY amino acid glycine, which was used as a linker between the polymer and the synthetic peptide; and means at trehosnovnoy acid (for example, triamterenetriamterene). The results are systematized in table 5, showed that each of the various illustrated conjugates according to the invention not only retains substantial biological activity against HIV-1 (compared with the activity of individual synthetic peptide), but, as it was unexpectedly discovered, also possess the AET intensified action against HIV-resistant isolates compared with individual synthetic peptide.

Example 4

In another illustrative embodiment of the invention the conjugate according to the invention was obtained by functional binding of two molecules SEQ ID NO:114 through N-Terminus of the synthetic peptide with the reactive functional groups of the polymer PEG. More specifically, the molecule of SEQ ID NO:114 was functionally connected with the PEG-acid, whereas the other molecule of SEQ ID NO:114 was functionally connected with the second molecule of PEG-acid, resulting in the obtained conjugate containing dimer of SEQ ID NO:114-PEG. So, for example, dibasic PEG-6-acid (5.4 mg, to 0.016 mmol) and tetrafluoroborate O-(1H-6-chlorobenzotriazol-1-yl)-N,N,N, N'-tetramethylurea (TCTU, 11.4 mg, to 0.032 mmol) was dissolved in 1.5 ml of a mixture DHM:DMF, 2:1 (dichloromethane:dimethylformamide). Then was added N,N-diisopropylethylamine (DIEA, 11,1 μl, 0,064 mmol), and then immediately added a protected side chain peptide "H (hydrogen)-SEQ ID NO:114 (250 mg, to 0.032 mmol). The solution was stirred at room temperature for 4 hours. DHM was removed on a rotary evaporator, and then to precipitate the peptide was added water (5 ml). The solid was collected by vacuum filtration, washed with water (3 × 5 ml) and dried with getting protected on the side chain dimer "SEQ ID NO:114 - PEG-6" (225 mg) with a yield of 90%. Protected side-chain dimer "SEQ ID NO:114 - PEG-6" (225 mg) was dissolved in triperoxonane acid (TFA, and 4.5 ml), sod is Rasa water (0.25 ml) and dithiothreitol (DTT, 0.25 g) as acceptors cations. The solution was stirred at room temperature for 6 hours. To precipitate the crude dimer "SEQ ID NO:114 - PEG-6" was added tert-butyl ether (MTBE). The solution decantation and the solid is washed several times MTBE, and then filtered and air-dried. The solid was dissolved in 10 ml of a mixture water/acetonitrile, 1:1, and the pH is brought to 6-7 by the addition of dilute ammonium hydroxide. Then the pH of the solution was reduced to 4-5 by addition of acetic acid (0.2 ml). The obtained clear solution was left stirring overnight at room temperature to remove the protection from the side chain. The solution was frozen, and then liofilizovane with obtaining the crude dimer "SEQ ID NO:114 - PEG-6" (150 mg). The crude dimer "SEQ ID NO:114 - PEG-6 was purified by high-performance liquid chromatography (HPLC) using a reversed phase C18, 5 microns, Packed as stationary phase, and using a mixture of acetonitrile/water with 0.1% TFA as mobile phase (40 to 70% of organic substances within 90 minutes). The fractions containing pure product were collected, frozen and liofilizovane to obtain 8 mg of the conjugate according to the invention (89,2%, HPLC, MS: found 9268,43 calculated 9268,35).

In the methods described in example 3, to determine the antiviral activity of the HIV-1 strain, which was sensitive is to the antiviral activity of the peptide, containing SEQ ID NO:114 (table 6, "114-S"), and the HIV-1 strain, which was resistant to treatment with a peptide containing SEQ ID NO:114 (table 6, "114-R"), used in vitro analysis of infectivity in which the antiviral activity of a peptide containing SEQ ID NO:114 (table 6, the individual synthetic peptide") was compared with the antiviral activity of the conjugate according to the invention (e.g., conjugate, containing two molecules of SEQ ID NO:114, functionally attached to PEG-6; table 6, "conjugate"). The results, expressed in values of IC50 and IC90, in µg/ml, illustrated in table 6.

Table 6
Tested agent114S:IC50IC90114R:IC50IC90
Individual synthetic peptide0,0050,0342,40>20
Conjugate0,0160,1690,1512,46

The results are systematized in table 6, unexpectedly showed that the conjugate according to the invention not only retain significant biological activity against HIV (compared to the activity selected is taken on synthetic peptide), but also had intensified action against HIV isolates that are resistant to individual synthetic peptide. In addition, the conjugate is illustrated in this example, confirms the General properties of synthetic peptides originating either from the region of HR1 or HR2 region gp41 of HIV-1, and used to obtain the conjugate according to the invention.

Example 5

The present invention relates to conjugates that possess antiviral activity, as indicated by their ability to inhibit HIV transmission in the target cell; and to a method of inhibiting transmission of HIV to the target cell, where the method involves the introduction of the virus into the cell conjugate according to the invention in amounts effective to inhibit infection of the cell by the HIV virus, and more preferably for inhibiting the fusion of virus and target cells. This method can be used for treatment (therapeutic) of HIV-infected individuals or for treatment (preventive) individuals already exposed to the virus, or individuals with a high risk of HIV infection (for example, individuals who use drugs, or have promiscuous sex). For example, in the case of HIV-1-infected individual an effective amount of the conjugate must be entered in a dose sufficient to reduce the agrusti HIV from this individual. As we all know there are several standard methods of measuring the load of the HIV virus, and such methods include, but are not limited to, quantifying cultures of mononuclear cells in peripheral blood HIV RNA in plasma. The conjugates according to the invention can be entered one at a time, intermittently, periodically or continuously, as may be determined by the attending physician, for example, by monitoring viral load. Depending on the composition containing the conjugate, and such factors as the composition of the polymer and the synthetic peptide used to obtain the conjugate, and the availability of this conjugate pharmaceutically acceptable carrier and from nature pharmaceutically acceptable carrier conjugate according to the invention can be introduced frequency component from several days to several weeks or more, if possible. In addition, the conjugate according to the invention can provide a synergistic effect on inhibition of HIV transmission to the target cell when used in combination with other antiviral drugs (for example, by simultaneous administration or administration of the individual cycles, first with one drug and then with another drug, usually used to treat HIV infection (e.g., including, but not ogranichivayas what they other inhibitors of HIV transmission cell (e.g., CCR5 inhibitors, retrocyclin etc.), inhibitors of HIV integrase, reverse transcriptase inhibitors (e.g., nucleoside or non-nucleoside), protease inhibitors, and the like, well known to experts).

The effective dose of the conjugate according to the invention can be determined by methods well known in the art, for example, by determining the effectiveness of biological time half-life and toxicity. In a preferred embodiment of the invention, the interval of effective doses of the conjugate can be defined using the results of routine in vitro and in vivo studies, is well known to specialists. For example, in in vitro assays for antiviral activity described in this application, the specialist can determine the average inhibitory concentration (C) conjugate is necessary to block a certain level of viral infectivity (e.g., 50%inhibition, IC50; or 90%inhibition, IC90). Then, the appropriate dose may be selected by the technician using the pharmacokinetic data obtained from one or more animal models, which may be received a minimum concentration ([min]) of the given conjugate, which is equal to or exceeds a pre-determined is n, the number of IC. Although the doses usually depends on the chosen route of administration and from disasterarea composition, however the approximate range of doses of conjugate according to the invention may be in the range from a value not lower than 0.1 µg/kg body weight and to a value not higher than 10 mg/kg of body weight; preferably in the range of about 0.1 to 100 μg/kg of body weight; and preferably from about 10 mg to 250 mg of the conjugate.

The conjugate according to the invention can be administered to the individual in any way, delivering an active agent to target cells (cells that can be infected with HIV). For example, the conjugates according to the invention can be introduced by any suitable means, including oral, parenteral (e.g. intramuscular, intraperitoneal, intravenous or subcutaneous injection, or injection, percutaneous introduction or insertion of an implant), intranasal, pulmonary, vaginal, rectal, sublingual, or local injection, and these conjugates can be prepared in dosage forms appropriate for each of these techniques. The specific route of administration depends on the medical history of the individual, including any expected or anticipated adverse effects associated with such introduction, and on the composition of the injected conjugate (e.g., from the nature of the polymer and a synthetic peptide containing the I in this conjugate). The most preferred introduction is an injection (e.g. intravenous or subcutaneous injection), but may also be undertaken and continuous infusion (using, for example, drugs for sustained release or mini-pumps, such as osmotic pumps, etc.). The conjugate according to the invention may also contain one or more pharmaceutically acceptable carriers, the choice of which depends on the desired composition, parcel delivery, route of administration, schemes, and other factors known to the attending physician. In addition, as will be described in more detail below, the conjugate can contain a nucleotide sequence encoding a polymer and a synthetic peptide, which, after their introduction, using appropriate techniques and well-known expressing vectors expressed in interest in the cells.

Example 6

From the description given in this application specialist obviously, if the polymer used to obtain a conjugate according to the invention is a polymer obtained on the basis of polyaminoamide, can be synthesized or constructed polynucleotide encoding such a conjugate, and that this conjugate may be produced by the methods of recombinant DNA used as a means for production (for example, PR is dotirovanija in vivo) and/or as a method of inhibiting transmission of HIV to the target cell. For professionals it is also clear that the conjugate according to the invention can be encoded by more than one polynucleotide and that such polynucleotide can be synthesized based on the choice of codon-triplets known that they encode amino acids amino acid sequence of this conjugate, and on the basis of the degeneracy of the third base of the codon and the preference of this codon-triplet in the cell-the host (e.g., prokaryotic or eukaryotic cell, and the like), in which the desirable expression. For example, the polymer may contain polylysine, and lysine can be encoded by any of the codons AAA or AAG. In another example, can be used heterodimer consisting of lysine and alanine, while alanine is encoded by any one of the codons GCA, GCG, GCC or GCU. In another example, in which one molecule of synthetic peptide attached to the amino-end of the polymer, and the second molecule synthetic peptides attached to the carboxy-end of the polymer, it may be desirable in the presence of a flexible linker, which is functionally binds molecules of synthetic peptide to the polymer. Specialists are well aware that the flexible linker, which can be used for functional linkage of two amino acid sequences can consist of glycine or glycine, United the other amino acids, such as serine. It is known that glycine is encoded by any one or more codons, such as GGU, GGC, GGA or GGG, and serine, as you know, is encoded by any one or more codons, such as AGU, AGC, UCU, UCC, UCA, or UCG. Representative examples include, but are not limited to, (the number indicates the number of molecules): Gly(3), GlySerGly, Gly(4)Ser(3), GlySer, etc. Preferred flexible linker can be determined by standard methods known to the expert. For example, the conjugate may contain: synthetic peptide-flexible linker-polymer-flexible linker-synthetic peptide.

Illustrative, but non-limiting examples of polynucleotides encoding a synthetic peptide, which can be used to obtain a conjugate according to the invention include SEQ ID NO: 100-106 (for synthetic peptides containing SEQ ID NO: 63, 65, 66, 61, 62, 4 and 5, respectively); however, it is clear that different codons can be replaced with codons that encode the same amino acid as the original codons. In addition, based on the preference of these codons specialist can easily determine the codon preference for the synthetic peptide has the same sequence and/or sequence of similar origin (for example, originating from regions HR1 or HR2, such as, but not limited to, SEQ ID NO:3, 6-60,64 and 67-99). In addition to this example can be noted that SEQ ID NO:107-113 encode the same respective peptides as SEQ ID NO: 100-106. However SEQ ID NO: 100-106 are polynucleotide containing the codon preference for bacterial expression, whereas SEQ ID NO:107-113 are polynucleotide containing codons preferred for expression in the expression systems of mammals.

In one of the embodiments the present invention relates to a prokaryotic expression vector containing polynucleotide encoding a conjugate according to the invention and to its use for the recombinant production of the conjugate. In one example, polynucleotide can be located in the prokaryotic expression vector so that, when producing the conjugate in bacterial cells-the masters he was producyrovtsa in the form of a hybrid protein with sequences that facilitate purification of the conjugate. For example, specialists known sequences that are expressed as part of a hybrid protein with the desired polypeptide and facilitate its production in inclusion bodies present in the cytoplasm of prokaryotic cells used for expression (see, for example, Tokatlidis et al., 1993, Protein Eng. 6:947-952). Taurus inclusion can be separated from other prokaryotic cellular components shall new methods known in the art, for example using denaturing agents and fractionation (e.g., by centrifugation, column chromatography and the like). In another example, presents commercially available vectors, which give interest to the sequence of a nucleic acid expressed in the form of a protein or peptide, so that after the expression of the gene product also contained numerous terminal his-tag residues (His-tags), which can be used for purification of the gene product by standard methods.

For professionals obviously, the sequence of a nucleic acid encoding a conjugate according to the invention can be incorporated into a plasmid or in replasment vectors, or in other expressing the system, which can also be used, including, but not limited to, bacteria transformed bacteriophagous vector, or kosmidou DNA; yeast containing yeast vectors; fungi containing vectors on the basis of fungi; cell lines insects infected with virus (e.g. baculovirus); and cell lines, transfetsirovannyh a plasmid or viral expression vectors, or infected with recombinant virus (e.g. vaccinia virus, adenovirus, adeno-associated virus, retrovirus, and the like). For successful EC is pressie conjugate needed to a recombinant DNA molecule comprising a sequence encoding a conjugate, or the vector contains the necessary elements regulating transcription and translation, which are compatible with the system owner, specifically used for expression, and recognized them. Using methods known to experts in the field of molecular biology, including methods described above, various promoters and enhancers can be incorporated into the vector or recombinant DNA molecule containing the coding sequence in order to increase the level of expression of the conjugate, provided that such a high level of expression of the conjugate is compatible (for example, non-toxic) specifically used by the system host cell. As obvious to the expert, the choice of promoter depends on the expression system. Promoters can vary in length, that is, the ability to stimulate transcription. Generally speaking, for the expression of cloned gene, it is desirable to use strong promoters in order to achieve a high degree of transcription of the gene and its expression with the formation of the gene product. For example, a known bacterial, fagbemi or plasmid promoters that provide a high level of transcription in the system host cells E. coli are promote the lac, the trp promoter, recA promoter, ribosomal RNA promoter, the promoters .sub.R and .sub.L, lacUV5, ompF, bla, lpp and the like, which can be used for transcription built nucleotide sequence that encodes a synthetic peptide. Promoters mammals that are commonly used in expressing vectors for expression in systems of mammals are promoters derived from the genes of mammalian viruses. As examples can serve as an early SV40 promoter, LTR promoter of the mouse virus, tumors of the mammary gland, the major late promoter of adenovirus, the promoter of the herpes simplex virus and CMV.

In the case when the expression of the conjugate may cause loss or damage to host cells the cell strain/cell line host and expressing vectors can be chosen so that the action of the promoter inhibited until then, until you need its specific induction. For example, for some operons adding specific inducers is necessary for efficient transcription of the integrated DNA (for example, the lac operon is induced by the addition of lactose or isopropylthio-beta-D-galactoside; the trp operon is induced in the absence of tryptophan in the culture medium; and tetracycline can be used in expressing the vectors mammals with tet-sensitivity is entrusted promoter). Thus, expression of the conjugate can be adjusted by culturing the transformed or transfected cells under conditions in which the promoter regulating expression of the coding sequence, not induced, and when the cells reach a suitable density in the culture medium, this promoter can be induced for expression of the coding sequence. Well known to experts and other regulatory elements used for efficient gene transcription or translation of the transcript, and these elements are enhancers, signals the initiation of transcription or translation, the sequence of the transcription termination and polyadenylation sequence, etc.

The above detailed description of specific embodiments of the invention are presented only for illustrative purposes. On the basis of the present description and graphical material and available scientific information specialist can easily modify and/or adapt the present invention for use in a variety of ways, not leaving, however, beyond the basic concept of the present invention; and, therefore, such modifications and/or adaptations should not go beyond the scope of the appended claims.

1. Conjugate consisting of a water-soluble polymer, and EET molecular weight of from 200 to 20,000 daltons, and is a polyethylene glycol or an alkyl chain, to which by means of the reactive functional groups of functionally attached to at least two molecules of synthetic peptides, where each synthetic peptide contains the amino acid sequence originating from the region of HR1 or HR2 gp41 of human immunodeficiency virus (HIV), where each synthetic peptide contains the amino acid sequence consisting of at least 16 amino acids and no more than 60 amino acids, and where specified conjugate possesses antiviral activity against HIV strains that are resistant to individual synthetic peptide.

2. The conjugate according to claim 1, where the specified polymer includes polyethylene glycol, containing a certain number of ethylene units.

3. The conjugate according to claim 1, where each synthetic peptide specified conjugate contains an amino acid sequence derived from the HR1 region gp41 of HIV.

4. The conjugate according to claim 3, where each synthetic peptide specified conjugate contains the identical amino acid sequence.

5. The conjugate according to claim 1, where each synthetic peptide specified conjugate contains an amino acid sequence derived from the HR2 region gp41 of HIV.

6. The conjugate according to claim 5, where each synthetic peptide specified conjugate contains the identical amino acid sequence.

7. To jugat according to claim 1, where at least one molecule of synthetic peptide specified conjugate contains an amino acid sequence derived from the HR1 region gp41 of HIV, and where at least one molecule of synthetic peptide specified conjugate contains an amino acid sequence derived from the HR2 region gp41 of HIV.

8. The conjugate according to claim 1, where the said molecule of synthetic peptide is functionally attached to the polymer by means of each synthetic peptide selected from the group consisting of N-end C-end and an internal lysine.

9. A method of obtaining a conjugate according to claim 1, comprising the stage of: (a) the reaction of the first molecule of synthetic peptide to the polymer with formation of an intermediate product, where this first molecule of synthetic peptide is functionally attached to the first reactive functional group of the polymer; and (b) the reaction of the specified intermediate product with a second molecule of synthetic peptide with the formation of the conjugate according to claim 1, where the specified second molecule of synthetic peptide is functionally attached to the intermediate product through a second reactive functional group of the polymer.

10. The method according to claim 9, where the specified polymer includes polyethylene glycol, containing a certain number of ethylene jingle the century

11. The method according to claim 9, where each synthetic peptide specified conjugate contains an amino acid sequence derived from the HR1 region gp41 of HIV.

12. The method according to claim 11, where each synthetic peptide specified conjugate contains the identical amino acid sequence.

13. The method according to claim 9, where each synthetic peptide specified conjugate contains an amino acid sequence derived from the HR2 region gp41 of HIV.

14. The method according to item 13, where each synthetic peptide specified conjugate contains the identical amino acid sequence.

15. The method according to claim 9, where at least one molecule of synthetic peptide specified conjugate contains an amino acid sequence derived from the HR1 region gp41 of HIV, and where at least one molecule of synthetic peptide specified conjugate contains an amino acid sequence derived from the HR2 region gp41 of HIV.

16. The method according to claim 9, where these molecules of synthetic peptide is functionally attached to the polymer by means of each synthetic peptide selected from the group consisting of N-end C-end and an internal lysine.

17. A method of inhibiting transmission of HIV to the target cell, where the method includes the introduction of the virus into the cell conjugate according to claim 1 in an amount effective for Engibarov the Oia infection of the cell specified by a virus.

18. A method of inhibiting transmission of HIV to the target cell, where the method includes the introduction of the virus into the cell conjugate according to claim 3 in an amount effective to inhibit infection of the cell specified by a virus.

19. A method of inhibiting transmission of HIV to the target cell, where the method includes the introduction of the virus into the cell conjugate according to claim 4 in an amount effective to inhibit infection of the cell specified by a virus.

20. A method of inhibiting transmission of HIV to the target cell, where the method includes the introduction of the virus into the cell conjugate according to claim 5 in an amount effective to inhibit infection of the cell specified by a virus.

21. A method of inhibiting transmission of HIV to the target cell, where the method includes the introduction of the virus into the cell conjugate according to claim 6 in an amount effective to inhibit infection of the cell specified by a virus.

22. A method of inhibiting transmission of HIV to the target cell, where the method includes the introduction of the virus into the cell conjugate according to claim 7 in an amount effective to inhibit infection of the cell specified by a virus.

23. A method of inhibiting transmission of HIV to the target cell, where the method includes the introduction of the virus into the cell conjugate of claim 8 in an amount effective to inhibit infection of the cell specified by a virus.

24. The way Inga the financing of HIV transmission to the target cell, where this method includes the introduction of the virus into the cell conjugate according to claim 9 in an amount effective to inhibit infection of the cell specified by a virus.

25. The method according to 17, where the specified conjugate inhibits the fusion of virus and target cells, preventing infection of cells specified by a virus.

26. The method according to 17, where specified, the conjugate further comprises a pharmaceutically acceptable carrier.

27. The method according to p where specified conjugate is administered to HIV-infected individual.



 

Same patents:

FIELD: medicine, virology, molecular biology, genetic engineering.

SUBSTANCE: invention relates to nucleic acid constructions and to vaccine preparations containing such construction and to using such preparations in medicine. Nucleotide sequence that encodes HIV-1 gag protein or its fragment containing gag epitope and HIV-1 Nef protein or its fragment containing nef epitope, and RT protein or its fragment containing RT epitope is coupled operatively with heterologous promoter wherein gag sequence is optimized by cordons. This sequence is a component of pharmaceutical composition and used in treatment and/or prophylaxis of HIV-infection and AIDS.

EFFECT: valuable medicinal properties of constructions, protein and pharmaceutical composition.

15 cl, 50 dwg, 22 ex

FIELD: biotechnology, immunology, virology.

SUBSTANCE: invention relates to CR3 chimera gene having sequence of SEQ ID NO:1 encoding chimera polyprotein. Said gene contains fragments, encoding fragments enriched with cyrotoxic T-cell epitope (CTL). Such fragments are selected from internal conserved and regulatory proteins, HIV epitopes of T-helper cells (Th), and B-cell epitope. Chimera polyprotein contains fragments of various HIV proteins inducing anti-HIV-1 immune response.

EFFECT: invention useful in HIV treatment and prevention.

7 cl, 10 dwg, 2 tbl, 11 ex

FIELD: peptides, pharmacy.

SUBSTANCE: invention relates to compounds of pegylated polypeptides T20. Also, invention describes pharmaceutical compositions comprising pegylated polypeptide compounds T20, methods for their preparing and using such compounds and compositions.

EFFECT: valuable medicinal properties of compounds.

13 cl, 1 tbl, 5 ex

FIELD: chemistry of peptides, medicine, virology, pharmacy.

SUBSTANCE: invention relates to compounds of PEG-containing polypeptide T1249. Also, invention describes pharmaceutical compositions comprising compounds of PEG-containing polypeptide T1249, methods for their preparing and using in pharmaceutical composition for preparing a medicinal agent used for inhibition of HIV-infection.

EFFECT: valuable medicinal properties of polypeptide, improved preparing method.

15 cl, 8 tbl, 6 dwg, 12 ex

FIELD: virology.

SUBSTANCE: invention proposes different compositions able to induce production of antibodies against Tat HIV-1 that can inhibit multiplication of HIV-1. Also, invention proposes a method for induction of antibodies raised against Tat HIV-1, in vitro method for assay of the presence of antibodies and their titer values, a method for reducing HIV-1 virus levels, sequence of synthetic nucleic acid and synthetic molecule. Proposed group of inventions can be used for inhibition of multiplication of HIV-1 in infected patients and for attenuation of HIV-1 multiplication after the primary infection in early infected persons.

EFFECT: valuable methods and compositions.

39 cl, 7 dwg, 9 tbl, 5 ex

The invention relates to molecular biology and genetic engineering, specifically to the creation of synthetic polyepitope vaccines against HIV-1

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The invention relates to the field of medicine and biotechnology and relates to immunogenic compositions containing the chimeric polypeptide of HIV, method of induction of an immune response to VIC and VIC/2, chimeric polypeptide VIC and VIC/2, a DNA molecule that encodes a chimeric polypeptide, the plasmid vector for expression of a chimeric polypeptide, polyclonal antibodies to VIC or VIC/2 and diagnostic test systems for the detection of antibodies to VIC and VIC

The invention relates to biotechnology and immunology, and can be used to produce vaccines against HIV infection

The invention relates to new synthetic peptides Monomeric type from 13 to 33 amino acids or dimeric type 26-66 amino acids with linear or cyklinowanie intersectionality disulfide bridges form corresponding to the formula (I)

-Z-Trp Gly Cys-Cys Tyr Thr Ser(I)

inwhichoznachaeteti,biocytin,Alamogordo,acetyl(CH3The CO-), an aliphatic chain, which may contain one or more thiol, aldehyde or amine functions, with aliphatic chain is a predominantly alkyl chain with 1-6 carbon atoms, or alkenylphenol chain with 2 to 6 carbon atoms, or aminoalkylindole chain with 2 to 6 carbon atoms; Z is a peptide sequence of one of the formulas

< / BR>
in which1means a peptide sequence of from 2 to 9 amino acids;2is a peptide sequence with 0-5 amino acids,is a peptide sequence of formula (is Nina; (AA2- the residue of glycine, (AA3- the residue of lysine or arginine, (AA4- residue leucine or alanine, (AA5) - valine residue, provided that (AA1), (AA2), (AA3), (AA4), (AA5never together form a peptide sequence Lys Gly Lys Leu Ile and Lys Gly Lys Leu Val-;recorded at the group FROM serine, means: hydroxyl, a peptide sequence of formula (XII) -Val--wheresequence of formulas (XIII) -(AA6)-Thr-Asn-(AA7)-(AA8), in which (AA6) - glutamine or arginine, (AA7) - glutamine or serine, (AA8) - threonine in whichrecorded on the balance-WITH - free amino acids (AA8that means HE, the peptide sequence of formula (HU) -Val-wherehas the above meaning; compositions for the detection of infections caused by HIV-1 group 0, the methods of diagnostics of infections caused by HIV-1 group 0 and diagnostic set

FIELD: organic chemistry of natural compounds.

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EFFECT: valuable medicinal properties of compound.

4 tbl, 9 ex

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to derivatives of piperidine of the general formula (I): or their pharmaceutically acceptable salts or isomers wherein Q means nitrogen atom (N); X and Z are chosen independently from group consisting of -CH and N under condition that one or both groups among Q and Z mean N; R, R4, R5, R and R are chosen independently from group consisting of hydrogen atom (H) and (C1-C6)-alkyl; R1 means H, (C1-C6)-alkyl, R9-aryl-(C1-C6)-alkyl-, (C1-C6)-alkyl-SO2-, (C3-C6)-cycloalkyl-SO2-, fluoro-(C1-C6)-alkyl-SO2-, R9-aryl-SO2-, R9-heteroaryl-SO2-, -N(R22)(R23)-SO2-, (C1-C6)-alkyl-C(O)-, (C3-C6)-cycloalkyl-C(O)-, fluoro-(C1-C6)-alkyl-C(O)-, R9-aryl-C(O)-, CH3CH2-NH-C(O)- or R9-aryl-NH-C(O)-; R2 means H or (C1-C6)-alkyl, and R3 means H, (C1-C6)-alkyl, (C1-C6)-alkoxy-(C1-C6)-alkyl-, (C3-C10)-cycloalkyl-, (C3-C10)-cycloalkyl-(C1-C6)-alkyl-, R9-aryl, R9-aryl-(C1-C6)-alkyl- or R9-heteroaryl under condition that each X and X doesn't mean N, or R2 and R3 in common mean =NOR10. Proposed compounds can be used as selective CCR5 antagonists. Compounds are useful in HIV treatment. Also, invention describes a pharmaceutical composition based on compounds thereof and combination with antiviral or anti-inflammatory agent.

EFFECT: valuable medicinal properties of compounds and pharmaceutical composition.

16 cl, 4 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: invention relates to method for reducing of LDL-cholesterol and/or triglyceride level increased due to therapy by HIV protease inhibitors in HIV-infected subjects. According to invention Atazanavir is administered in combination with other HIV protease inhibitor, metabolized cytochrom P450 monooxygenase in therapeutically effective amounts.

EFFECT: effective treatment due to Atazanavir ability to cytochrom P450 monooxygenase inhibition; increased concentration of HIV protease inhibitors without preparation dose.

3 cl

FIELD: medicine, biology, virology.

SUBSTANCE: invention involves creature of complex of membranotropic compounds providing target delivery of antiviral preparation to HIV-1/2 damaged focus and suppression of viral infection at initial and later steps of its development. Invention provides effect on more expanded targets of human immunodeficiency virus and blocking HIV-infection at the early steps of interaction virus/cell based on synergism of components in the proposed complex. Modifying agents of polyanionic matrix - norbornene or adamantine and peptide-simulators of HIV-1/2 co-receptor bind with different sites of gp120 of HIV-1 that excludes the competition possibility and reciprocal steric hindering in virus-specific pharmacophore-modifying agents. Invention can be used for prophylaxis of HIV-infection and AIDS treatment, and in research works for study of ligand-receptor interaction (of type surface viral proteins - cellular receptors).

EFFECT: valuable biological and medicinal properties of complex.

3 cl, 5 tbl, 2 dwg, 3 ex

FIELD: chemistry of peptides, virology.

SUBSTANCE: invention relates to novel chemical compounds, namely, to glycyrrhizic acid (GA) glycopeptide with glycyl-L-phenylalanine: 3-O-{2-O-[N-(β-D-glucopyranosyluronoyl)-glycyl-L-phenylalanine-N-(β-D-glucopyranosyluronoyl)-glycyl-L-phenylalanine)]}-(3β,20β)-11-oxo-olean-12-ene-30-oic acid possessing anti-HIV-1 activity. This compound shows less toxicity (CD50 = 250 mcg/ml) as compared with the known anti-HIV preparation azidothymidine (CD50 = 3.5 mcM) and elicits the expressed anti-HIV-1 activity of high effectiveness and inhibits accumulation of virus-specific protein p24 (ID50 = 0.73 mcg/ml) that by 170-fold lower as compared with GA. This compound exceeds GA by the selectivity index (IS = 342.5) by 30-fold in culture cells MT-4 infected with the strain HIV-1/EVK. Glycopeptide in the concentration 0.80 mcg/ml (ID90 = 0.80 mcg/ml) inhibits reproduction of virus by 90%. Anti-HIV-1 activity (inhibition of p24) of the proposed compound in the concentration 10 mcg/ml is similar with activity of azidothymidine in the dose 0.05 mcg/ml.

EFFECT: enhanced and valuable antiviral properties of compound.

3 tbl, 2 ex

FIELD: virology, biotechnology, medicine, biochemistry.

SUBSTANCE: invention relates to creature agents used in AIDS treatment. Invention proposes pentapeptide of the following structure: Tyr-Pro-Ile-Glu-MeHis of molecular mass 672 Da. Peptide is prepared from sea worm Polychaeta, Eunicidae viscera homogenate extract. Method involves the successive purification by hydrophobic chromatography and reversed-phase high-performance liquid chromatography. Invention expands assortment of natural non-nucleoside inhibitors of HIV reverse transcriptase.

EFFECT: valuable medicinal properties of agent.

3 tbl, 10 dwg

Antigenic peptides // 2312941

FIELD: peptides, medicine, immunology.

SUBSTANCE: invention relates to novel methods for producing family of antigenic peptides possessing antiviral effect, and to peptide libraries obtained by using indicated methods, and to compositions based on thereof. Method for producing antigenic peptides involves detection of a great number of variable positions in protein site of pathogenic organism; selection of peptide sequence in protein of pathogenic organism comprising a great number of variable positions; selection of one or more substituting amino acid residues for one of variable positions or sequences possessing antigenic affinity with amino acid residues in variable position of this protein in natural conditions by using a matrix of antigenic similarity of amino acids showing possibility for substitution of one amino acid residue with another residue and with retaining interaction of antibody with antigen and corrected in correspondence with amino acids frequencies in hypervariable sites; incorporation of selected substituting amino acid residues into peptide sequence; synthesis of family of antigenic peptides. Also, invention relates to a method for calling the immune response and to a method for diagnosis of HIV-infection by using family of peptides prepared according to methods of the invention.

EFFECT: valuable biological and medicinal properties of peptides, improved preparing method.

233 cl, 43 tbl, 1 dwg, 5 ex

FIELD: organic chemistry.

SUBSTANCE: invention relates to new pyrasole derivatives of formula I wherein R5 represents phenyl or heteroaryl ring of formulae IIIa-IIIh meanings of the rest substituents are as defined in specification. Also disclosed are pharmaceutical composition based on said derivatives of formula I and uses thereof.

EFFECT: new biologically active compounds and pharmaceutical compositions based on the same for HIV inhibition.

13 cl, 54 ex, 2 tbl

FIELD: bioactive substances, in particular glycyrrhizic acid derivatives.

SUBSTANCE: invention relates to glycyrrhizic acid derivatives of general formula wherein R is formula or hydrogen atom, with the proviso that at least two of said R are . Also disclosed is inhibitor of human immunodeficiency virus reproduction, including di- and/or trinicotinates of glycyrrhizic acid.

EFFECT: new glycyrrhizic acid derivatives with high bioactive activity; anti-HIV inhibitor of improved activity.

2 cl, 14 ex, 3 tbl

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to azaindoleoxoacetic derivatives of piperazine of the general formula (I): wherein Q is chosen from the group consisting of the following compounds: ; -W- represents compound of the formula: . Proposed compounds possess antiviral activity both in separate using and in combination with other antiviral, anti-infectious agents, immunomodulating agents or inhibitors HIV entering. Also, invention describes a pharmaceutical composition based on compounds of the formula (I).

EFFECT: valuable medicinal properties of compounds and pharmaceutical composition.

55 cl, 10 tbl, 169 ex

FIELD: chemical and pharmaceutical industry.

SUBSTANCE: invention relates to lyophilized pharmaceutical immunocytokine composition including immunocytokine and containing as cytokine component interleukin-2 (IL-2), sugar or aminosugar, amino acid and surfactant, wherein said composition contains: immunocytokines from 0.1 to 25 mg/ml; sugar or aminosugar 1-200 mg/ml; amino acid 1-200 mMol/l; and surfactant 0.001-1 mass %.

EFFECT: composition for parantheral administering with prolonged storage time even at increased temperatures.

13 cl, 8 ex, 4 tbl, 2 dwg

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